Development and validation of a personalizable model of the hepatic arterial tree and particle transport

Liver cancers are increasingly common and require improved treatment methods. Radioembolization inhibits tumor growth by inserting small spheres into the hepatic artery that are carried into smaller vessels near tumors, where they deposit radiation. Predicting toxicity for this treatment is challenging due to the microanatomical liver structure and the tendency of microspheres to cluster when trapped. This work describes and validates a novel method to generate personalized hepatic arterial tree models and simulate realistic glass microsphere distributions. It describes methods to generate realistic arterial trees using physically constrained macrocell growth models that can incorporate measurable parameters such as initial and terminal vessel diameters and blood pressure. It also describes models of microsphere infusion that simulate different embolic effects and trapping behavior. Microsphere cluster-size histograms and images at PET resolution were simulated for several infusion methods in a hepatic arterial tree model generated from liver and proper hepatic artery segmentations taken from the XCAT phantom. To verify the distributions created through microsphere infusion simulation, a characterization of the three-dimensional clustering of glass microspheres in tissue was necessary. However, traditional methods to find microsphere locations requires creating thin slices of treated tissue that are stained and examined microscopically, which is a very time-consuming process. This thesis describes how to use micro-CT and image processing to detect glass microspheres in whole tissue samples. The detection method was verified by processing phantoms composed of glass microspheres embedded in agar that could also be imaged microscopically to determine true microsphere locations. Using this, >93% of microspheres in the phantom were correctly detected and the percentage of erroneously detected microspheres was <5%. The mean absolute error between the true and estimated dose maps was 4.2%. The detection method was then applied to a treated porcine liver sample to characterize microsphere clustering behavior in normal liver tissue. A histogram of these cluster sizes was compared with histograms simulated using the tree and transport models. The most realistic clustering behavior was produced by having microspheres trap in vessels when their diameters exceeded the vessel diameters, and when embolic effects did not affect later microsphere traversal

Retinal fluorotachometry : a clinically applicable method of retinal flow measurements

A considerable percentage of diseases in humans is related to disturbances of capillary perfusion. Measurements of capillary flow are of scientific as well as of practical clinical interest. Their scientific value lies in the possibility to gain more insight into pathophysiological processes which are related to capillary malperfusion {e.g. in hypertension, diabetes mellitus, senile vasculopathy). Their clinical value lies in the possibility of an early assessment of capillary malperfusion and the measurement of the effect of treatment on the disturbed capillary flow.Blood is a highly dynamic organ: it is continuously in motion and it continuously exchanges substances with the living tissues of the organism. By these two dynamic processes at capillary level, the "milieu interieur" is maintained.Retinal fluorotachometry {RFT) is a new clinical method for measurements of retinal blood flow and in particular retinal capillary perfusion. "Retinal fluoro" refers to: a fluorescent dye front in the retinal vessels, and "tachometry" [Gr. taches: speed, metrein: to measure] means: measurement of the speed. So RFT is: measurement of the speed of a fluorescent dye front in the retinal vessels. The development and application of this method is the subject of this thesis

Spectral and Temporal Interrogation of Cerebral Hemodynamics Via High Speed Laser Speckle Contrast Imaging

Laser Speckle Contrast Imaging (LSCI) is a non-scanning wide field-of-view optical imaging technique specifically developed for cerebral blood flow (CBF) monitoring. In this project, a versatile Laser speckle contrast imaging system has been designed and developed to monitor CBF changes and examine the physical properties of cerebral vasculature during functional brain activation experiments. The hardware of the system consists of a high speed CMOS camera, a coherent light source, a trinocular microscope, and a PC that does camera controlling and data storage. The simplicity of the system’s hardware makes it suitable for biological experiments. In controlled flow experiments using a custom made microfluidic channel, the linearity of the CBF estimates was evaluated under high speed imaging settings. Under the camera exposure time setting in the range of tens of micro-seconds, results show a linear relationship between the CBF estimates and the flow rates within the microchannel. This validation permitted LSCI to be used in high frame rate imaging and the method is only limited by the camera speed. In an in vivo experiment, the amount of oxygen intake via breathing by a rat was reduced to 12% to induce the dilation of the vessels. Results demonstrated a positive correlation between the system’s CBF estimates and the pulse wave velocity derived from aortic blood pressure. To exemplify the instantaneous pulsatility flow study acquired at high sampling rate, a pulsatile cerebral blood flow analysis was conducted on two vessels, an arteriole and a venule. The pulsatile waveform results, captured under sampling rate close to 2000 Hz. The pulse of the arteriole rises 13ms faster than the pulse of the venule, and it takes 6ms longer for the pulse of the arteriole to fall below the lower fall-time boundary. By using the second order derivative (accelerated) CBF estimates, the vascular stiffness was evaluated. Results show the arteriole and the venule have increased-vascular-stiffness indices of 0.95 and 0.74. On the other side, the arteriole and the venule have decreased-vascular-stiffness indices of 0.125 and 0.35. Both vascular stiffness indices suggested that the wall of arteriole is more rigid than the venule. The proposed LSCI system can monitor the mean flow over function activation experiment, and the interrogation of blood flow in terms of physiological oscillations. The proposed vascular stiffness metrics for estimating the stroke preliminary symptom, may eventually lead to insights of stroke and its causes

Targeting human synovium using homing peptides identified by in vivo phage display

PhDRheumatoid arthritis (RA) is a chronic inflammatory condition affecting diarthrodial synovial joints. Non-random patterns of inflammatory cell recruitment suggest the presence of synovial-specific vascular determinants which enable recruitment of specific inflammatory cell subsets. Using a model whereby human synovial and skin tissue is transplanted into SCID mice, we have previously used in vivo peptide phage display to identify novel peptide sequences which confer synovial homing specificity to human synovium. This synovial localisation was blocked by co-administration of free peptide thus confirming its specificity. In this project the in vivo homing properties of the peptide were further explored. The synovial localization of the synovial-specific phage was shown to be specifically increased after intragraft injection of TNFα. Sequence homology was shown between the expressed CKSTHDRLC (3.1) peptide and an extracellular domain of the leucocyte integrin mac-1. The homing properties of the free peptide were investigated by conjugation to the radioisotopes 111In and 99mTc. No significant differences were found in vivo between homing of the 3.1 monomeric peptide to transplanted human skin and synovium. The influence of valency and size of the molecules were investigated through the development of novel techniques: polymerization of the peptide was achieved by conjugation to radiolabelled streptavidin and fluorescent microspheres. In vivo experiments found no significant difference between localization of polymerised 3.1 or scrambled control peptide to either transplanted skin or synovium with either construct. Despite the negative results reported here, the techniques described have potential for the investigation of other targeted short-peptide sequences. Finally, the model was further developed as a tool for the pre-clinical imaging of human synovium in vivo using an 111In- conjugated anti-E-selectin antibody. It was shown that this could be used to resolve specific from non-specific uptake and hence represents, potentially, a powerful new tool for the development of human tissue-specific targeting strategies

Doctor of Philosophy

dissertationGlobally, hepatocellular carcinoma (HCC) of the liver is diagnosed in over 700,000 people annually and trends indicate increasing prevalence. The majority of cases, >80%, are detected at advanced stages where systemic chemotherapies have little efficacy. The primary curative treatment is liver transplant, but if a donor liver is not available, only palliative care such as transarterial chemoembolization (TACE) is possible. TACE targets the tumor blood supply. An embolic containing a chemotherapeutic agent is injected into the tumor's vasculature via an endovascular catheter, subsequently shutting down blood flow while delivering localized chemotherapy. A presently approved product, Lipiodol, is an oily emulsion mixed with a chemotherapeutic used in conjunction with gelatin particles or synthetic polymer beads that act as emboli. Calibrated spherical drug eluting beads are now gaining favor for this procedure, replacing the multistep oil emulsion system. These beads, however, have shortcomings: aggregation of smaller diameter beads, fracturing of beads while under strain in the catheter, off target embolization particularly in pulmonary circulation, elution of only charged small molecule therapeutics, nondegradability, limited tumor depth penetration, and revascularization induced by a hypoxic state. To address these limitations, a genetically engineered silk-elastinlike protein polymer (SELP) system was developed to create a liquid-to-solid embolic agent capable of retaining and releasing a wider range of therapeutics, controlled degradation into nontoxic amino acids, and soluble until injected into the body where they transition irreversibly to a solid hydrogel network. This provides potential for ideal injectability as a low viscosity fluid at room temperature followed by optimal embolization by a highly stable hydrogel at body temperature. The proposed research involved engineering a SELP formulation with suitable viscosity for injection into the tumor vasculature via a microcatheter and a suitable gelation rate and gel strength for stable embolization. The drug release properties of the polymer matrix were determined for small molecule chemotherapeutics such as doxorubicin and anti-angiogenic sorafenib. Preliminary in vivo performance of the novel system for TACE was evaluated using a rodent model. Future directions include expansion of in vivo studies, particularly in an animal model for HCC and TACE to study therapeutic efficacy and longterm biocompatibility

Tissue engineered micro and macrovasculature utilizing stromal vascular fraction.

This dissertation describes the use of stromal vascular fraction to tissue engineer 3D microvasculature and macrovasculature. Stromal vascular fraction is an easily isolatable cell source from adipose tissue depots. It has demonstrated remarkable potential both in vitro and in vivo for forming microcirculation capable of perfusion upon implantation. SVF is clinically utilized as a therapeutic cell source for anti-inflammation for osteoarthritis and is being studied for ischemic tissue application to stimulate revascularization. The work described herein is divided within four chapters. Chapter I provides an introductory overview and lists the aims and hypothesis for the dissertation. Chapter II describes experiments towards elucidating specific aim 1: determine the mechanism by which SVF forms neovascular networks in 3D fibrin gels in vitro. This was accomplished through a multitude of experiments describing SVF undergoing vasculogenesis and angiogenesis in a 2D automated in vitro assay, and the ability to inhibit these processes via NOTCH and PDGF-B/PDGFR-b interruption. These mechanisms, as well as integrin dependent mechanisms, were analyzed within 3D fibrin and 3D collagen I culture systems as well. It is believed that the activation of the fibrin specific integrin aVb3 plays a role in hyper-stimulating fibrin-embedded endothelial cells in a VEGF dependent manner. Chapter II describes experiments towards understanding specific aim 2: create deliverable tissue units of SVF-derived microvasculature or macrovasculature utilizing bioprinting, and electrospinning technologies. This was accomplished through bioprinting spheroids containing cells embedded in collagen I or fibrin using superhydrophobic surface technology or electrospinning varying porosities of PCL and pressure sodding SVF cells into the material. It is possible to automate and create dosable units of microvascular tissue in spheroid format using SVF cells, ECM such as fibrin or collagen I, and bioprinting technologies. Additionally, it is possible to create blood vessel mimics of multiple porosities in order to retain and allow cellular infiltration within the biomaterial. Chapter IV is an overall summary and conclusion of the dissertation. These studies could hopefully generate more knowledge on the creation of tissue engineered microvasculature and microvasculature for use in treating ischemic cardiomyopathies