Endothelzellen bilden eine monozellulare Grenzschicht in Blutgefäßen. Ihre Migration ist ein kritischer Teilschritt bei der Gefäßbildung, zum Beispiel während der Wundheilung. Obwohl bereits eine Reihe der dafür relevanten Mediatoren und pathogenen Determinanten bekannt sind, fehlt bisher eine quantitative Analyse der molekularen Mechanismen der Gefäßbildung und Zellmigration. Voraussetzung dafür sind Verfahren zur automatisierten Bestimmung von Zelltrajektorien in Sequenzen von Mikroskopaufnahmen migrierender Zellverbände. Dazu wurde ein statistisches Modell entwickelt, welches die Segmentierung und Verfolgung von Zellen in Bildsequenzen ermöglicht. Im vorliegenden Beitrag stellen wir dieses Modell vor, diskutieren die sich daraus ergebenden Lern- und Erkennungsalgorithmen und präsentieren erste Resultate. / Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials.

No description available.

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

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

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Vito, Raymond, Committee Member ; Nerem, Robert, Committee Chair ; Eskin, Suzanne, Committee Member ; Hanson, Stephen, Committee Member ; Gibbons, Gary, Committee Member.

The Tissue Engineering Research Lab at California Polytechnic State University, San Luis Obispo focuses on creating tissue-engineered blood vessel mimics (BVMs) for use in preclinical testing of vascular devices. These BVMs are composed of electrospun scaffolds made of an assortment of polymers that are seeded with different cell types. This integration of polymers with cells leads to the need for biocompatibility testing of the polymer scaffolds. Many of the lab’s newest scaffolds have not been fully characterized for biologic interactions. Therefore, the first aim of this thesis developed methods for in vitro cytotoxicity testing of polymers used in the fabrication of BVMs. This included cytotoxicity testing using direct contact and elution-based methods, along with fluorescent staining to visualize the scaffold effects on cells. The second aim of this thesis implemented the newly developed cytotoxicity protocols to evaluate the biocompatibility of existing polymers, ePTFE and PLGA, used in the tissue engineering lab. The results demonstrated that ePTFE and PLGA were noncytotoxic to cells. The third aim of this thesis evaluated the biocompatibility of novel polymers used to fabricate BVMs: PLGA with salt, PLLA, and PCL. Elution-based methods concluded that PLGA with salt, PLLA, and PCL were noncytotoxic to cells; however, the direct contact method illustrated PLGA with salt and PCL were mildly cytotoxic at 24 and 48 hours. Potential causes of this variability include the addition of salt to PLGA, dissolving PCL in dichloromethane, inadequate sample sizing, and the inherent differences between the test methods. Overall, this thesis developed and implemented methods to evaluate the biocompatibility of polymer scaffolds used in the BVM model, and found that ePTFE, PLGA, and PLLA scaffold materials were biocompatible and could be implemented in future BVM setups without concerns. Meanwhile, PLGA with salt and PCL’s toxicity was mild enough to urge future cytotoxicity testing on PLGA with salt and PCL before further use in the lab.

In treating cancer with hyperthermia, an understanding of the heat losses associated with the presence of a large functioning blood vessel in or proximal to a treatment area is needed in order to optimize any protocol. A three-dimensional computer model based on the Bioheat transfer equation (BHTE) has been developed to account for temperature changes in and around functioning blood vessels during laser-induced hyperthermia. The light source is modelled using an approximation to the transport theory solution for an isotropic point source in an infinite homogeneous tissue medium with anistropic scattering. The derived BHTE's for tissue, vessel and blood are solved for temperature using the implicit finite differences method. The validity of the model was tested by comparing predicted temperatures to measured temperatures from a series of dynamic phantom studies using two vessel diameters and three flow rates. Large experimental temperature variations were observed and increased proportionally with increasing thermal gradients. The model consistently over-estimates (~ 1-2C) absolute temperatures close to the source and under-estimates (~ 1-2C) them far from the source. This could be due to uncertainties associated with the estimated thermal conductivity and measured optical properties of the tissue material. Both model and experiments show a small convective heat loss due to the presence of a blood vessel. The model predicts that at high flow rates, temperature reductions of 2C or greater are limited to distances less than 0.3 cm from the surface of a 0.144 cm (outer diameter) vessel and less than 0.8 cm from the surface of a 0.40 cm vessel. The vessel has a negligible effect on temperatures at distances greater than ~ 1.75 cm. The predicted temperature change due to blood flow and the measured change agree to within experimental errors. There was better agreement with the larger diameter vessel. / Thesis / Master of Science (MS)

A major limitation in tissue engineering is the lack of nondestructive methods to assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a scanning fiber imaging method capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque vascular scaffold, contained in a bioreactor. This imaging modality is based on local excitation of fluorescent cells, acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells, stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled endothelial cells on the luminal surface through a ~500 µm thick tubular scaffold at cell-level resolutions and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution of the order of 20-30 µm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable nondestructive monitoring of endothelial cells seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future. / Ph. D.

Tissue engineered blood vessels (TEBVs) have great potential as tools for disease modeling and drug screening. However, existing methods for fabricating TEBVs create homogenous tissue tubes, which may not be conducive to modeling focal vascular diseases such as intimal hyperplasia or aneurysm. In contrast, our lab has a unique modular system for fabricating TEBVs. Smooth muscle cells (SMCs) are seeded into an annular agarose mold, where they aggregate into vascular tissue rings, which can be stacked and fused into small diameter TEBVs. Our goal is to create a platform technology that may be used for fabricating focal vascular disease models, such as intimal hyperplasia. Because tubes are fabricated from individual ring units, each ring can potentially be customized, enabling the creation of focal changes or regions of disease along the tube length. In these studies, we first demonstrated our ability to modulate cell phenotype within individual SMC ring units using incorporated growth factor-loaded degradable gelatin microspheres. Next, we evaluated fusion of ring subunits to form composite tissue tubes, and demonstrated that cells retain their spatial positioning within individual rings during fusion. By incorporating electrospun polycaprolactone cannulation cuffs at each end, tubes were mounted on bioreactors after only 7 days of fusion to impart luminal medium flow for 7 days at a physiological shear stress of 12 dyne/cm2. We then created focal heterogeneities along the tube length by fusing microsphere-containing rings in the central region of the tube between rings without microspheres. In the future, microspheres may be used to deliver growth factors to this localized region of microsphere incorporation and induce disease phenotypes. Due to the challenges of working with primary human SMCs, we next evaluated human mesenchymal stem cells (hMSCs) as an alternative cell source to generate vascular SMCs. We evaluated the effects of microsphere-mediated platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and transforming growth factor beta-1 (TGF-β1) delivery on ring thickness, proliferation, and contractile protein expression over a 14 day period. Finally, we created a structurally distinct region of smooth muscle within tissue tubes by fusing human aortic SMCs in a central region between hMSC rings. In summary, we developed a platform technology for creating modular tubular tissues that may be further developed into an in vitro intimal hyperplasia model. It may also be modified to model other focal vascular diseases, such as aneurysm, or to create other types of multi-tissue tubular structures, such as trachea.

歯科基礎医学会, 日下部 豊寿 = Toyohisa Kusakabe, 実験的歯の移動時における圧迫側歯槽骨に生じる背部骨吸収と血管分布 = Rear resorption at the pressure side incident in orthodontic tooth movement, 歯科基礎医学会雑誌, 39(6), DEC 1997, pp.623-640 / Hokkaido University (北海道大学) / 博士 / 歯学