Die Versorgung mit Nährstoffen und Sauerstoff in artifiziellen, 3D Scaffolds für das Tissue Engineering ist Grundvoraussetzung für die Viabilität darin kultivierter Zellen. Die Kombination aus definierter Architektur und biomimetischer Zusammensetzung ist dabei entscheidend, sodass neben dem Stofftransport auch die Adhäsion, Proliferation, Migration und Morphologie von Zellen gesteuert werden können. Die Verwendung von Komponenten der nativen Extrazellulären Matrix, wie Collagen Typ I (COL I), als Scaffold-Material wird dafür als optimal angesehen. Jedoch stellt die Gestaltung der Architektur und Bereitstellung instruktiver Elemente zur Steuerung der Zellverhaltens bei COL I-basierten Scaffolds, aufgrund der mechanischen und biochemischen Eigenschaften des COL I, besondere Ansprüche an die Bearbeitungsverfahren. Ziel dieser Arbeit war es daher, Verfahren zur Ausstattung von modularisierbaren, COL I-basierten Membranen mit einer instruktiven biochemischen Zusammensetzung, Mikroarchitekturen als Basis für ein initiales vaskuläres System und Gradienten des Vascular Endothelial Growth Factors (VEGF) zu entwickeln. Diese Scaffolds sollten anschließend hinsichtlich der Beeinflussung des Wachstums, der Orientierung und der gerichteten Migration von humanen Endothelzellen aus der Nabelschnurvene (HUVEC) charakterisiert werden. COL I-Scaffolds konnten durch Plastic Compression in Membranform hergestellt und mit Fibronectin (FN) und Hyaluronsäure (HA, mit hohem Molekulargewicht) ausgestattet werden. Die Inkorporation von FN erhöhte die HUVEC-Proliferation, während die Proliferationsrate bei der verwendeten HA konstant blieb. Anisotrope Mikrostrukturen als Basis für ein vaskuläres System und zur HUVECOrientierung wurden durch einen 3-stufigen Prozess auf den COL I-Membranen erzeugt. Dabei wurde die Mikrostrukturübertragung durch mikrostrukturierte Polystyren-Stempel in einem Druckumformprozess realisiert. Die erhaltenen Mikrogräben mit Breiten von 10-40 µm beeinflussten die Orientierung der HUVEC deutlich. Für die Erzeugung von VEGF-Gradienten zur Steuerung der gerichteten Migration von HUVEC wurde ein Immersionsverfahren mit Hilfe der Modellproteine Bovines Serumalbumin und Hyaluronidase entwickelt. Dieses ermöglichte die Erzeugung eines linearen, graduellen Konzentrationsverlaufs der Modellproteine auf der Membranoberfläche. Der entwickelte Prozess konnte auf VEGF mit einem minimalen Gradientenprofil von 3,8-11,8 pg/mm² nach Immersion in 100 ng/mL VEGF-Lösung übertragen werden. Erste Versuche zur Induktion der HUVEC-Migration zeigten, dass ein erfolgreiches Gradientenprofil jedoch noch gefunden werden muss. Die erzeugten COL I-Membranen bilden eine gute Ausgangsposition für die Bereitstellung modularer, biomimetischer Tissue-Engineering-Scaffolds, mit initialer Vaskularisierung und zellinstruktiven Elementen. Außerdem besitzen sie das Potential, durch eine Vielzahl von Bio-Engineering-Methoden modifiziert und biochemisch den Anforderungen an das zelluläre Mikromilieu ausgestattet zu werden. / The supply of nutrients and oxygen in artificial, three-dimensional scaffolds for tissue engineering is a basic prerequisite for the viability of cells cultivated therein. The combination of defined architecture and biomimetic composition are crucial in this context, so that besides the metabolic transport, the adhesion, proliferation, migration and morphology of cells can also be controlled. The use of native extracellular matrix components, such as collagen type I (COL I), as scaffold material is considered optimal for this purpose. However, designing the architecture and providing instructive elements to control cell behavior in COL I-based scaffolds poses special challenges to processing methods due to the mechanical and biochemical properties of COL I. Therefore, the aim of this work was to develop basic procedures to equip modularizable COL I-based membranes with instructive biochemical composition, microarchitectures as initial vascular system, and vascular endothelial growth factor (VEGF) gradients and to characterize the obtained scaffolds with respect to influencing human umbilical vein endothelial cell (HUVEC) growth, orientation, and migration. It was shown that COL I scaffolds can be prepared by Plastic Compression in membrane form and equipped with fibronectin (FN) and hyaluronic acid (HA, with high molecular weight). Incorporation of FN increased HUVEC proliferation, whereas the proliferation rate remained constant with the HA used. Anisotropic microstructures as a basis for a vascular system and for HUVEC orientation were generated by a 3-step process on the COL I membranes. In this process, the microstructure transfer was realized by microstructured polystyrene stamps in a pressure forming process. The obtained microtrenches with widths of 10-40 µm significantly affected the orientation of HUVEC. For the generation of VEGF gradients to control the directional migration of HUVEC, an immersion technique was developed using the model proteins bovine serum albumin and hyaluronidase. This allowed the generation of a linear, gradual concentration gradient of the model proteins on the membrane surface. The developed process could be applied to VEGF with a minimum gradient profile of 3.8-11.8 pg/mm² after immersion in 100 ng/mL VEGF solution. Initial experiments to induce HUVEC migration showed that a successful gradient profile still needs to be found, however. The generated COL I membranes provide a good starting point for the provision of modular, biomimetic tissue engineering scaffolds, with initial vascularization and cell-instructive elements. They also possess the potential to be modified by a variety of bioengineering methods and biochemically equipped to meet the requirements of the cellular microenvironment.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23897号 / 工博第4984号 / 新制||工||1778(附属図書館) / 京都大学大学院工学研究科材料工学専攻 / (主査)教授 杉村 博之, 教授 山田 啓文, 教授 邑瀬 邦明 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

This dissertation aims to further the understanding of the complex communication that occurs as cells interact with topographical and chemical patterns on a biomaterial interface. The research accomplishes this through two aims fabricating cell substrate surface topography and chemical patterns independently using non-cleanroom approaches, and analyzing higher order cellular response to surface features. The work will impact biomaterial surface modification and fabrication which will apply to biomedical implanted devices, tissue engineering scaffolds, and biological analysis devices. The first aim seeks to apply non-traditional topographical and chemical patterning methods in order to create independent topographical and chemical patterns on cell culture substrates. Experiments use the resulting patterned substrates to quantify cellular alignment to surface topography and compare the relative influence of topographical and chemical patterns on cellular response. The combined patterning methods of imprint lithography and micro-contact printing result in a high-throughput technique applicable to a variety of materials and a range of feature sizes from nanoscale through microscale, thereby enabling future analysis of cell response to surface features. The second aim evaluates the impact of topographical and chemical features on cellular differentiation. Experiments use patterned topography overlaid with a characterized chemical model layer to evaluate the effects of topography on myoblast differentiation and alignment. Chemical patterns that independently control available cell spreading area and modulate cell-cell contact are used to investigate the impact of cell-cell contact on differentiation.

Les travaux présentés se sont intéressés à la régulation des forces produites par le cytosquelette d'actine. Le rôle primordial joué par le microenvironnement a été au centre de nos investigations. L'étude de ces phénomènes a nécessité le développement de techniques innovantes. La première permet le contrôle en temps réel de la forme de la cellule. Elle utilise un laser UV pulsé pour modifier le microenvironnement adhésif de la cellule et contrôler les zones disponibles pour son étalement. La seconde est une amélioration d'une technique existante au sein du laboratoire. Il s'agit de produire des îlots de protéines d'adhésions, de forme contrôlée, sur un substrat déformable d'acrylamide. Ces supports permettent le contrôle de la taille de la cellule et de son organisation interne. En outre, l'élasticité de l'acrylamide permet la mesure des forces générées par la cellule. La dernière technique a combiné le patterning sur acrylamide avec l'ablation laser. Les forces produites au sein d'une structure particulière du cytosquelette ont ainsi pu être estimées. Deux grands mécanismes de régulation des forces ont pu être mis en évidence. L'utilisation de techniques de spectrométrie de masse, de mesure de forces et de biologie moléculaire a permis de mettre en évidence la coopération entre les différents types d'intégrines au niveau de l'adhésion cellulaire. Cette coopération permet un couplage entre l'architecture du cytosquelette et la quantité de moteurs moléculaires mettant en tension ces structures. Ces mécanismes sont primordiaux pour l'adaptation de la cellule à la rigidité de son environnement. Ce sont les structures d'actine qui produisent les forces qui seront transmises au niveau des adhésions. La corrélation entre la taille de ces structures et les forces générées est encore mal caractérisée. La relation entre taille des fibres de stress et répartition des forces au sein de la cellule a pu être étudiée et suggère que la force produite par une fibre de stress augmente avec sa longueur. Une étude systématique de la contractilité des cellules, sur des patterns de différentes tailles, a permis de montrer la relation entre la taille des fibres de stress et la force générée. Une relation biphasique a ainsi été mise en évidence. Quand la taille de la cellule augmente, la force générée au sein des fibres de stress commence par augmenter avant de diminuer au delà d'une longueur critique. Cette longueur correspond également à la taille maximale observée sur des cellules libres de s'étaler sans contraintes. Les résultats obtenus suggèrent que cette chute de force est liée à une augmentation excessive du ratio myosine/actine qui ne permet plus une production de force efficace. Le mécanisme pourrait faire intervenir le désassemblage des structures d'actine par la myosine ou la quantité insuffisante d'actine pour permettre un travail efficace des moteurs moléculaires. La rencontre de ces deux mécanismes permet de définir le champ des possibles pour la cellule en terme de contractilité. Le mécanisme de chute de forces observé n'a pas pu être expliqué à ce jour mais nous travaillons activement pour qu'il le soit dans les mois à venir. Ce phénomène aura sans doute un grand rôle à jouer dans l'intégrité mécanique des tissus et les phénomènes de migration. La chute de force au delà de la longueur critique permet en effet de déstabiliser les adhésions et pourrait être à l'origine de la rétraction de la cellule dans la migration ou du détachement d'une cellule de ces voisines dans le cas d'un tissu sous forte contraintes. Ce détachement protégerait ainsi la cellule d'un déchirement sous l'effet de forces trop importantes. / Our work has been focused on the regulation of the forces generated by the actin cytoskeleton. We have more precisely studied the role of the cellular microenvironment in this process. It was necessary to overcome some technical challenges to study these mechanisms. We developed two new techniques. The first one allows for the dynamic control of cell shape. A pulsed UV laser is used to modify the adhesive microenvironment around the cell and to create new area available for cell spreading. The second technique is an improvement of an existing technique from the laboratory. It consists in producing ECM protein islands on a elastic acrylamide substrate. This substrate provides the control of cell shape and internal organization. Plus, the elasticity of the substrate is compatible with traction forces measurements. The last technique combines acrylamide micropatterning and laser ablation of intracellular actin structures. Thus, the forces produced by a particular intracellular structure can be estimated. Two keys mechanisms of force regulation were shown. The use of mass spectrometry, traction force microscopy and molecular biology made it possible to study the interaction between different integrins in the adhesion complex. Cooperation was shown. It allows for the coupling between the architecture of the cytoskeleton and the amount of molecular motors in action. This process is necessary for the adaptation of cell forces to substrate stiffness. Actin structures are the one responsible for force production. This force can then be transmitted to the environment through adhesions.. The link between the length of actin fibers and the force produced was more precisely studied. The results showed a correlation between stress fibers length and the force generated inside it. This was true only above a certain critical value. After that, the force was rather decreasing with increasing fiber length. This critical length corresponds to the maximal length of cell axis on infinite 2D substrate. Our main hypothesis is that a too high myosin/actin ratio will block the proper force production/transmission within the fiber. Disassembly of actin by myosin or limited pool of actin are the two explanations we are currently following. The combination of these two-regulation process put brakes on force production by the cell. Above a certain length, the force produced is decreasing. This decreases in turn the strength of the adhesions anchored to these fibers. This will destabilize the adhesions and causes cell retraction The interplay between the regulation by the adhesion and the production of forces within the fiber set some limits on the level of forces produced by the cell. These processes are likely to be modified in a pathological context and can lead to tumor formation. They also protect the cell from being destroyed by stretching. If the length/stretch is too high, the cell will decrease its forces and detach from neighboring cells. This provide a system protecting the cell from being destroyed by massive deformations within the body

No description available.

GEMs (Gas Electron Multiplier) and Thick-GEMs (Thick-Gas Electron Multiplier) are MPGDs (Micropattern Gas Detector) that make part of the new generation of gaseous detectors, allowing high counting rates, low cost when compared to solid state detectors, high radiation hardness and gain when using multiple structures. Besides that, the handling and maintenance of these detectors is relatively simple, being versatile to detect different types of radiation. Therefore, these detectors are an effective alternative to build imaging systems with large sensitive area. This work consists in the study and characterization of a set of gaseous detectors, more specifically the Thick-GEMs produced in the High Energy Physics and Instrumentation Center at IFUSP, which were tested showing promising results in terms of gain, energy resolution and operational stability. However, due to the low signal-to-noise ratio of the Thick-GEMs, the X-ray fluorescence imaging system was mounted using GEMs. During this work the necessary software tools for image processing and reconstruction were developed as a parallel study in computational simulations to better understand the operation of gaseous detectors. X-ray fluorescence techniques are essential in areas such as medicine and the study of historical and cultural heritage since they are non-invasive and non-destructive. Techniques to check the authenticity of masterpieces are required and museums are gradually becoming more interested in the Physics and instrumentation needed to characterize their patrimony. / Os GEMs (Gas Electron Multiplier) e Thick-GEMs (Thick-Gas Electron Multiplier) são estruturas do tipo MPGD (Micropattern Gas Detector) que fazem parte da nova geração de detectores de radiação a gás e permitem altas taxas de contagens, baixo custo quando comparados com os detectores de estado sólido, uma elevada resistência à radiação e ganhos elevados, quando utilizadas estruturas múltiplas para multiplicação. Além disso, o manuseio e manutenção desses detectores é relativamente simples, sendo versáteis em relação à montagem podendo detectar diferentes tipos de radiação. Sendo assim, a utilização desses detectores é uma alternativa eficiente para montar um sistema de imagem com grande área sensível. Este trabalho consiste no estudo e caracterização de um conjunto de detectores gasosos, mais especificamente os Thick-GEMs produzidos pelo grupo de Física de altas energias e Instrumentação do IFUSP, que foram testados para serem empregados em um sistema de imagem de fluorescência de raios-X. Os Thick-GEMs testados apresentaram resultados promissores em termos de ganho, resolução em energia e estabilidade operacional. No entanto, devido à baixa relação sinal-ruído, um sistema de imagem de fluorescência de raios-X foi montado utilizando GEMs. Durante o trabalho as ferramentas de software necessárias para processamento e reconstrução de imagens foram desenvolvidas, assim como um estudo paralelo de simulações computacionais para entender melhor o funcionamento de detectores gasosos. Técnicas como o imageamento por fluorescência de raios-X são de suma importância pois são consideradas não invasivas e não destrutivas. Sua utilização tem uma importância imprescindível nas áreas da medicina e na análise de patrimônios histórico e cultural. Atualmente, a verificação e validação de autenticidade de obras é um requisito obrigatório e alguns museus começam a se interessar cada vez mais em áreas da Física e da instrumentação necessária para caracterizar o seu patrimônio.

GEMs (Gas Electron Multiplier) and Thick-GEMs (Thick-Gas Electron Multiplier) are MPGDs (Micropattern Gas Detector) that make part of the new generation of gaseous detectors, allowing high counting rates, low cost when compared to solid state detectors, high radiation hardness and gain when using multiple structures. Besides that, the handling and maintenance of these detectors is relatively simple, being versatile to detect different types of radiation. Therefore, these detectors are an effective alternative to build imaging systems with large sensitive area. This work consists in the study and characterization of a set of gaseous detectors, more specifically the Thick-GEMs produced in the High Energy Physics and Instrumentation Center at IFUSP, which were tested showing promising results in terms of gain, energy resolution and operational stability. However, due to the low signal-to-noise ratio of the Thick-GEMs, the X-ray fluorescence imaging system was mounted using GEMs. During this work the necessary software tools for image processing and reconstruction were developed as a parallel study in computational simulations to better understand the operation of gaseous detectors. X-ray fluorescence techniques are essential in areas such as medicine and the study of historical and cultural heritage since they are non-invasive and non-destructive. Techniques to check the authenticity of masterpieces are required and museums are gradually becoming more interested in the Physics and instrumentation needed to characterize their patrimony. / Os GEMs (Gas Electron Multiplier) e Thick-GEMs (Thick-Gas Electron Multiplier) são estruturas do tipo MPGD (Micropattern Gas Detector) que fazem parte da nova geração de detectores de radiação a gás e permitem altas taxas de contagens, baixo custo quando comparados com os detectores de estado sólido, uma elevada resistência à radiação e ganhos elevados, quando utilizadas estruturas múltiplas para multiplicação. Além disso, o manuseio e manutenção desses detectores é relativamente simples, sendo versáteis em relação à montagem podendo detectar diferentes tipos de radiação. Sendo assim, a utilização desses detectores é uma alternativa eficiente para montar um sistema de imagem com grande área sensível. Este trabalho consiste no estudo e caracterização de um conjunto de detectores gasosos, mais especificamente os Thick-GEMs produzidos pelo grupo de Física de altas energias e Instrumentação do IFUSP, que foram testados para serem empregados em um sistema de imagem de fluorescência de raios-X. Os Thick-GEMs testados apresentaram resultados promissores em termos de ganho, resolução em energia e estabilidade operacional. No entanto, devido à baixa relação sinal-ruído, um sistema de imagem de fluorescência de raios-X foi montado utilizando GEMs. Durante o trabalho as ferramentas de software necessárias para processamento e reconstrução de imagens foram desenvolvidas, assim como um estudo paralelo de simulações computacionais para entender melhor o funcionamento de detectores gasosos. Técnicas como o imageamento por fluorescência de raios-X são de suma importância pois são consideradas não invasivas e não destrutivas. Sua utilização tem uma importância imprescindível nas áreas da medicina e na análise de patrimônios histórico e cultural. Atualmente, a verificação e validação de autenticidade de obras é um requisito obrigatório e alguns museus começam a se interessar cada vez mais em áreas da Física e da instrumentação necessária para caracterizar o seu patrimônio.