京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13265号 / 論医博第2179号 / 新制||医||1038(附属図書館) / (主査)教授 別所 和久, 教授 安達 泰治, 教授 戸口田 淳也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

A study was made of the effects of diphenylhydantoin (DPH) and the carrier solution on the spleen, lymph node, and thymus. DPH was injected i.p. at concentrations of 5 and 10 mg./100 gm. for 30 and 60 days. Hematologic effects observed were leucocytosis, neutrophilia, eosinophilia, and lymphopenia. Respiratory measurements of lymph node tissue slices were made using the oxygen electrode method. The carrier solution was found to cause a marked increase in oxygen consumption. A DPH effect on lymph tissue respiration was not observed. The carrier alone caused an atrophy of the lymph nodes and thymus, as well as an increase in the total body weight. Histological examination revealed that the 5 mg./100 gm. DPH injected for 60 days and the 10 mg./100 gm. DPH injected for 30 or 60 days produced a histiocytic cell type lymphoma, resembling Hodgkin's disease in the lymph node, thymus, and spleen in rats. The data indicated that DPH may not be a direct carcinogen, but it may interfere with the normal immune mechanism to produce the changes observed.

Cardiac disease causes approximately a third of the deaths in the United States. Furthermore, most of these deaths are due to a condition termed atherosclerosis, which is a buildup of plaque in the coronary arteries, leading to occlusion of normal blood flow to the cardiac muscle. Among the methods to treat the condition, stents are devices that are used to restore normal blood flow in the atherosclerotic arteries. Before advancement can be made to these devices and changes can be tested in live models, a reliable testing method that mimics the environment of the native blood vessel is needed. Dr. Kristen Cardinal developed a tissue engineered blood vessel mimic to test intravascular devices. Among the scaffolding material used, electrospun poly (lactide-co-glycolide) (PLGA) has been used as an economic option that can be made in house. PLGA is a biodegradable co-polymer, and when electrospun, creates a porous matrix with tailorable properties. Currently, the standard PLGA electrospinning protocol produces consistent fibrous scaffolds with a mean fiber diameter of 5-6 microns. Research indicates that cell adhesion is more successful in fibrous matrices with a mean fiber diameter at the nanometer level. However, because previous work in the Tissue Engineering Laboratory at Cal Poly sought to ensure a consistent fibrous, there was no model or equation to determine how to change the electrospinning parameter settings to create scaffolds with an optimal mean fiber diameter. To fill this need, biomedical engineering senior Steffi Wong created a design of experiment to systematically approach the electrospinning variables and determine how they interacted with each other, as well as their effect on fiber diameter. The aims of this thesis were to perform the said design of experiments and determine a model to predict the resulting mean fiber diameter of a scaffold based on the electrospinning parameters as well as to determine what combination of parameters would lead to a scaffold with an optimal mean fiber diameter between 100-200 nanometers. The variables tested were solution concentration, gap distance, flow rate, and applied voltage. Each scaffold was imaged and a mean fiber diameter was calculated and used as the predicted variable in a regression analysis, with the variables indicated above as the predictors. The goal of 100-200 nanometer mean fiber diameter was not reached. The smallest mean fiber diameter calculated was 2.74 microns—half of that of the standard protocol. The regression analysis did result in a model to describe how the voltage, gap distance, and flow rate affected the fiber diameter.

<p>An essential stage of repair for a healing wound is the proliferation of cells in the damaged space. Cells such as fibroblasts, grow and migrate to aid in construction of new tissue and to close the wound. Current methods of studying fibroblast proliferation in wound healing include a 2D wound healing assay in which a cell monolayer is scratched, and the cells migrate into the pseudo-wound. However, this lacks the 3D architecture of a physiological wound. Current 3D models of wound healing often rely on the use of a preexisting matrix for structural assistance, however an isolated system of cell growth without requirement of structural aid may gather new insights on intercellular behavior and mechanical properties. Additionally, we to desire to fabricate a high through-put and easy to use 3D wound healing model than currently offered. Our engineering objective is to create a novel 3D model of wound healing.</p> <p><br></p> <p>This project aims to optimize fibroblast adhesion and proliferation for 3D microtissue fabrication by altering surface and extracellular matrix (ECM) properties to SU-8 scaffolding. Additionally, we consider the effect of different geometries on cell proliferation and cellular stresses/strains, fibronectin production as pseudo-wounds close, and make comparisons to intercellular cancer behavior. Our results show around a 66% decrease in overall culture time required for the microtissues to reach full confluency. Varying geometries in the tessellated design have revealed structural changes in the actin cytoskeleton formation of fibroblasts, and increased fibronectin production along edges of tensioned cells preparing to “close” the wound. When compared to human breast cancer cells, the cancer cells lack the ability to make critical cell to cell junctions that we observe in fibroblasts, noting the characteristic that cancer is like a wound that never heals.</p>

This thesis investigates new processing methods and bone tissue engineering applications of cross-linked cellulose nanocrystal (CNC) aerogels. Aerogels are highly porous, low-density materials that have been praised for their high surface area and interconnected pores, but criticized for their brittleness. This prompted a search for new aerogel “building blocks” to produce more flexible materials; CNCs meet this need and chemically cross-linked CNC aerogels have good compressive strength and shape recovery properties in air and liquid environments. CNCs are high aspect ratio, non-toxic and renewably-sourced nanoparticles. Literature has demonstrated CNC aerogel production using cryo-templating with controlled drying. In this work, we produce aerogels using a new scalable process called pressurized gas expansion (PGX) and compare them to conventional cryo-templated aerogels. PGX aerogels were found to have more expanded fibrillar morphology, a range of mesopore sizes and smaller macropores, in contrast to cryo-templated aerogels that had a sheet-like morphology surrounding larger macropores. Additionally, PGX aerogels had higher specific surface area and porosity, but lower compressive strength due to a lower cross-link density. While neither CNC aerogel type dispersed in water, PGX aerogels partially shrank whereas cryo-templated aerogels did not; this is attributed to their morphological differences. This work shows that new aerogel processing methods can introduce new properties and thus broaden the potential applications of CNC aerogels. One specific biomedical application was evaluated for CNC aerogels – their use as bone tissue scaffolds. Cryo-templated aerogels comprised of CNCs with different surface chemistries, either sulfate or phosphate groups, were found to have attractive chemical, physical and mechanical properties for bone tissue engineering. This work shows that both types of CNC aerogels can facilitate cell proliferation, favorable differentiation, and can nucleate uniform hydroxyapatite growth. These positive in vitro results and the bimodal pore morphology of CNC aerogels make them promising bone scaffolds for in vivo studies. / Thesis / Master of Applied Science (MASc) / Aerogels are light, porous, sponge-like materials that are essentially 99% air by volume. In this work, the aerogels are made from non-toxic plant-based nanoparticles called cellulose nanocrystals (CNCs). This thesis investigates: 1) new ways to control CNC aerogel properties and pore size through different processing methods and 2) the use of CNC aerogels to aid in the repair of damaged bones. High-resolution microscopy and nano-characterization tools show that CNC aerogels have tunable properties, which may extend their possible applications. The internal structure, sponge-like mechanical properties and biocompatibility of CNC aerogels allowed them to be successfully utilized to support bone cells and grow bone-like mineral.

A knowledge of the fundamental optical properties of breast tissues is necessary in order to optimize transillumination imaging techniques for the diagnosis of breast disease. The optical absorption and scattering coefficients have been measured in normal and diseased breast tissues, over the range of wavelengths from 500 to 1100 nm. The tissues were obtained from surgical specimens, and consisted of normal glandular and adipose tissues, fibrocystic disease, fibroadenoma, and ductal carcinoma. Total attenuation coefficients were measured for thin slices of tissue obtained on a microtome. The diffuse reflectance and transmittance were measured for 1.0 mm thick samples of these tissues, using standard integrating sphere techniques. Monte Carlo simulations were performed to derive the scattering and absorption coefficients, as well as the mean cosine of the scattering angle. The results indicate that scatter exceeds absorption by at least two orders of magnitude. The absorption coefficients are strongly affected by the presence of blood, particularly at wavelengths below 600nm. The scattering coefficients lie in the range 30 mm to 90 mm-1 at 500 nm, and fall smoothly with increasing wavelength to between 10 mm and 50 mm at 1100 nm. The scatter coefficient for adipose tissue differs, in that it is invariant with wavelength over this spectral range. The scattered light, for all tissues examined, is highly forward peaked, with the mean cosine of the scattering angle in the range 0.945 to 0.985. This value remains constant with wavelength to within +/-0.01 for any given tissue. The absorption coefficients and scattering properties of each tissue type fall within distinct ranges at each wavelength. Fibrocystic disease and adipose tissue appear to be the most clearly distinguishable groups. The optical properties of carcinoma do not differ significantly from those of normal glandular tissues, although both groups differ from other tissue types. The implications of these results for imaging are yet to be determined. / Thesis / Master of Science (MS)

Due to the inability of intra-articular injuries to adequately self-heal, current therapies are largely focused on palliative care and restoration of joint function rather than true regeneration. Subsequently tissue engineering of chondral and osteochondral tissue constructs has emerged as a promising strategy for the repair of partial and full-thickness intra-articular defects. Unfortunately, the fabrication of large tissue constructs is plagued by poor nutrient transport to the interior of the tissue resulting in poor tissue growth and necrosis. Further, for the specific case of osteochondral grafts, the presence of two distinct tissue types offers additional challenges related to cell sourcing, scaffolding strategies, and bioprocessing. To overcome these constraints, this dissertation was focused on the development and validation of a microfluidic hydrogel platform which reduces nutrient transport limitations within an engineered tissue construct through a serpentine microfluidic network embedded within the developing tissue. To this end, a microfluidic hydrogel was designed to meet the nutrition requirements of a developing tissue and validated through the cultivation of chondral tissue constructs of clinically relevant thicknesses. Additionally, optimal bioprocessing conditions with respect to morphogen delivery and hydrodynamic loading were pursued for the production of bony and cartilaginous tissue from bone marrow derived mesenchymal stem cells. Finally, the optimal bioprocessing conditions were implemented within MSC laden microfluidic hydrogels to spatially engineer the matrix composition of a biphasic osteochondral graft through directed differentiation.

No description available.

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences