Abnormalities in the vascular pattern of a retina, such as morphologic changes in vessel shape, branching pattern, width, tortuosity, or the appearance of retinal lesions, may be associated with the occurrence of retinopathies or cardiovascular diseases. Thus, an automated quantitative analysis of changes in vessel morphology may help indicating the clinical signs of aforementioned retinopathies, describing their early occurrence or severity. The responses obtained from different types of retinal vessels, i.e., arteries and veins, may be variable to retinopathies and their measurement may lead to a more precise diagnosis compared to that by the average response accounted for the entire vessel network. I propose a set of automated methods in order to analyze the retinal vessel network and to quantify its morphologic properties with respect to arteries and veins, in two-dimensional color fundus images. The analytical methods include; 1) Forma- tion of a well connected vessel network, 2) Structural mapping of a vessel network, 3) Artery-venous classification, and 4) Blood vessel hemorrhage detection. The quan- tification methods include vessel morphology analysis based on the measurement of tortuosity, width, branching angle, branching coefficient, and fractal dimension. The aforementioned morphologic parameters are measured with respect to arteries and veins separately in a vessel network. The methods are validated with the manually annotated retinal fundus images as a ground truth. The major contribution of this thesis includes the development of automated methods for; 1) Identification and separation of retinal vessel trees for individual vessel analysis, 2) Automated quantification of morphologic characteristics of retinal vessels for quick and precise measurement, 3) Automated quantification of vessel morphology with respect to arteries and veins, and 4) Analysis of two datasets, a) malarial retinopathy subject dataset, b) longitudinal study dataset. The ability of the automated methods to quantify the retinal vessel specific properties may enable the individual vessel analysis as an alternative to a time- consuming and subjective clinical evaluation, or to a quantitative morphology char- acterization averaged over the entire vessel network. The objective evaluation may indicate the progression of retinopathies precisely and may help characterizing nor- mal and abnormal vascular patterns with respect to arteries and veins. This may enable a quick diagnosis, treatment availability, prognosis, and facilitation of clinical health-care procedures in remote areas.

Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.

The engineering of human vascular grafts is an intense area of study since there is crucial need for alternatives to native vein or artery for vascular surgery. This current study sought to prove that a tissue engineered blood vessel (TEBV) 1mm in diameter could be developed from human smooth muscle cells and that endothelial progenitor cells (EPCs) could be cultured and used to endothelialize these grafts. This project had four specific aims: the isolation and characterization of EPCs, the seeding of a novel scaffold with EPCs and exposure to physiologic shear stress in vitro, the development of TEBV from human smooth muscle cells that are strong enough to implant in vivo, and the in vivo implantation of TEBV into the rat aortic model with a comparison of EPC seeded TEBVs pretreated with shear stress and unseeded TEBVs. The results yielded isolation of four EPC lines and a flow system design capable of seeding EPCs onto a novel scaffold with preliminary studies indicating that it is capable of exposing the EPCs to physiologic shear stress, although further studies require more optimization. The development of mechanically strong TEBV was highly successful, yielding TEBVs comparable to native vessels in collagen density and burst pressure, but with much lower compliance. Current implantation studies indicated that unseeded TEBV grafts implanted into the rat aorta without anticoagulation is highly thrombogenic. However, anticoagulation using Plavix may be capable of maintaining graft patency. These TEBVs did not rupture or form aneurysm in vivo and the future completion of the in vivo studies are likely to demonstrate the high potential of these grafts.

Optical coherence tomography (OCT) is a technology that enables 2D cross-sectional images of tissue microstructure. This interferometric technique provides resolutions of approximately 10-20 um with a penetration depth of 1-2 mm in highly scattering tissues. With the use of fiber optics, OCT systems have been developed for intravascular imaging with a demonstrated improvement in both resolution and dynamic range compared to commercial intravascular ultrasound systems. OCT studies of normal, atherosclerotic, and stented arteries indicate the ability of OCT to visualize arterial structures. These results suggest OCT may be a valuable tool for studying luminal structures in tissue engineered constructs.In the present study, new endoscopic OCT systems and analysis techniques were developed to visualize the growth and response of the cellular lining within a tissue engineered blood vessel mimic (BVM). The BVM consists of two primary components. A biocompatible polymeric scaffold is used to form the tubular structure. Human microvessel cells from adipose tissue are sodded on to the inner surface of the scaffold. These constructs are then developed and imaged within a sterile bioreactor.Three specific aims were defined for the present study. First, an OCT longitudinal scanning endoscope was developed. With this endoscope, a study of 16 BVMs was performed comparing images from OCT and corresponding histological sections. The study demonstrated that endoscopic imaging did not visually damage the mimic cellular lining. OCT images showed excellent correlation with corresponding histologicalsections. Second, a concentric three element endoscope was developed to provide radial cross-sections of the BVM. OCT images using this endoscope monitored lining development on three types of polymeric scaffolds. In the third specific aim, automated algorithms were developed to assess the percent cellular coverage of a stent using volumetric OCT images.The results of the present study suggest that OCT endoscopic systems may be a valuable tool for assessing and optimizing the development of tissue engineered constructs. Conversely, the BVMs modeled the arterial response to deployed stents allowing the development of automated OCT analysis software. These results suggest that blood vessel mimics may be used to advance OCT technology and techniques.

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In this thesis a recently developed energy resolving x-ray detector (Medipix) is used to investigate potential medical applications of spectral x-ray imaging. Computed Tomography (CT) is one of the most important medical imaging modalities. Recent developments in CT techniques include dual-energy CT, where images are taken with two different x-ray spectra by either using two x-ray tubes operated at different voltages, or modulating the operating voltage of a single tube. These techniques provide spectral information in the CT dataset but are limited to what can be achieved by manipulating the x-ray source, since the detectors used in current CT systems are unable to provide spectral information about the detected x-rays. A preliminary investigation of the use of the Medipix detectors for two different medical applications is presented. The first, applications is imaging of blood vessels for diagnosis of vascular diseases, and the second, characterising and measuring the energy dependence of x-ray attenuation in fat and liver tissue using the Medipix2 detector. This second investigation is part of work towards (eventually) quantifying the fat content of liver tissue in vivo, which is important for the early diagnosis of fatty liver disease. While an early attempt to identify iron fluorescence x-rays in a Monte-Carlo simulation of blood vessel x-ray image was not successful, the potential for improving image contrast using the changes in x-ray attenuation at the iodine k-edge iodine have been investigated in a series of further simulations and appears to be feasible. The potential use of spectral imaging to differentiate and quantify tissues without the need for added contrast material has been investigated by using a Medipix2 detector to measure the energy dependence of x-ray absorption in fat and liver tissue. The results of this experimental work show significant differences in x-ray attenuation between these two tissues that suggest this form of spectral imaging may be useful in practice.

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

To be successful a tissue engineered small diameter blood vessel must be non-immunogenic, non-thrombogenic, have mechanical properties similar to native vessel and be vasoactive. The vascular media is responsible for the mechanical properties and the vasoactivity of the vessel. The collagen hydrogel approach has been long used and has many advantages, but has not yet achieved the mechanical integrity needed for implantation. No collagen-based tissue engineered vascular media has been shown to be vasoactive using culture techniques required to achieve the cell numbers needed to make a vascular graft. To study collagen synthesis, two model systems were used. Cells were seeded on top of an adsorbed collagen I or fibrin layer. Alternatively the cells were encapsulated in a collagen or fibrin hydrogel. Collagen I, decorin and biglycan synthesis was affected by both matrix type and presentation. After two weeks in culture the smooth muscle cells produce more type I collagen in the collagen based hydrogels then in the fibrin hydrogels and was used for further studies. The collagen based tissue engineered vascular media produced a consistent vasoactive response between two and eight weeks of culture. The smooth muscle cells have functional endothelin, kinin, adrenergic, serotonergic and purinergic receptors. The application of cyclic strain improves both the tissue strength and the contractile response. Use of transforming growth factor-β improved tissue strength, but reduced the contractile response. Transforming growth factor- β actually promoted a more contractile cell phenotype, but a stronger contractile force was required to overcome the thick compact collagen hydrogel and elicit a measurable contraction. This work adds to what is known about collagen-based tissue engineered vascular medias by identifying means of improving not only strength but vasoactivity. The trade-offs found between these two important characteristics are relevant to all tissue engineered medias.

Thesis (doctoral)--Lund University, 1993. / Added t.p. with thesis statement inserted.