The thesis is focused on material development based on hyaluronic acid usable in regenerative medicine, especially for heart tissue regeneration after myocardial infarction. The object of the study is the oxidized form of hyaluronic acid (HA-Ox) and hydroxyphenyl derivative of HA (HA-TA). HA-Ox can be crosslinked with a bifunctional alkoxyamine POA and HA-TA undergoes an enzymatic reaction in the presence of hydrogen peroxide catalysed by horseradish peroxidase leading to gel formation. To describe the materials, chemical and physical properties, gelation kinetics and conditions of crosslinking reactions were studied. Hydrogels were characterized by mechanical and viscoelastic properties, degradability or stability in simulated body fluids. These hydrogels serve as scaffolds for the selected cell type. To promote cell adhesion and viability, an RGD sequence has been bonded to the structure of HA-TA. This resulting material is also compatible with selected applicators. Its viscosity and extrusion force are low enough to allow application with a catheter with a very small internal diameter. The applicability of the material through the supply tube to the hydrogel reservoir of the second SPREADS device showed good homogeneity, cell distribution and viability. Finally, the material was applied in vivo using these devices during a preclinical study.

The main aim of doctoral thesis was the study on reactivity, transport and barrier properties of biocolloidal and synthetic polymeric substances by simple diffusion techniques. It was studied mainly the influence of basic physic-chemical parameters (temperature, concentration, pH and modification of material) on the reactivity and barrier ability of chosen compounds. Further substances were chosen as a model compounds: biocolloids (humic acids, alginate, chitosan, hyaluronate) and synthetic polymer (polystyrenesulfonate). Reactivity, barrier and transport properties of chosen substances were studied by interactions with oppositely charged basic organic dyes (methylene blue, rhodamine 6G, amido black 10B respectively) in hydrogels medium based on linear polysaccharide (agarose). The attention was also paid to basic physic-chemical characterisation (infrared spectroscopy, rheology, elemental analysis, thermogravimetry and scanning electron microscopy) of chosen materials and also hydrogels. Key part of the whole doctoral thesis was the optimization of selected diffusion techniques (diffusion cell technique and non-stationary diffusion in cuvettes) designated for the study on reactivity and barrier properties of wide range compounds (optimized method should be used as an universal method for simple and fast determination of reactivity of different compounds at given or changing conditions). The rate of reactivity, transport and barrier properties was determined based on fundamental diffusion parameters such as diffusion coefficients, break-through time so called lag time, interfacial concentration of chosen organic dye, apparent equilibrium constant, tortuosity factor, partition coefficient.

The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.

Title: Microstructure, swelling and deformation behavior of methacrylate hydrogels with interpenetrating network structure Author: Zhansaya Sadakbayeva Department: Department of Macromolecular Physics, Faculty of Mathematics and Physics, Charles University in Prague Supervisor: Ing. Miroslava Dušková-Smrčková, Dr., Institute of Macromolecular Chemistry, Czech Academy of Sciences Consultant: Prof. Ing. Karel Dušek, DrSc., Institute of Macromolecular Chemistry, Czech Academy of Sciences Abstract: This work is devoted to interpenetrating polymer network (IPN) hydrogels prepared by sequential processes of redox-initiated radical polymerization of the first network prepared from 2-hydroxyethyl methacrylate (HEMA), and UV- initiated radical polymerization of the second network prepared from 2-hydroxyethyl methacrylate (HEMA) or glycerol methacrylate (GMA). Microstructure, swelling and deformation responses of the IPN hydrogels and their constituent network hydrogels were tested by various techniques. The microstructure of the first poly(HEMA) network was found sensitive to polymerization conditions. A novel route for one-step synthesis of double-porous poly(HEMA) cryogel was proposed. The formation of the second poly(GMA) network in the environment of non-porous and macroporous poly(HEMA) parent networks was...

This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization. Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance. Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23602号 / 医博第4789号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Osteoarthritis (OA), a degenerative disease of articular joints, is the leading cause of chronic disability in the US and affects more than a third of adults over 65 years old. Due to the obesity epidemic and an aging population, the prevalence of OA is expected to rise in both young and old adults. There are no disease modifying OA drugs. Therefore, providing any treatment options that delay the onset or progression of OA is highly desirable. The scope of this dissertation examines two different strategies to promote translational therapies for OA. The first approach investigated whether Smad ubiquitin regulatory factor 2 (Smurf2), an E3 ubiquitin ligase, could be a potential therapeutic target for OA. The second approach examined the incorporation of small chemical residues to enhance the physical and bioactivity of a bioinert scaffold for cartilage tissue repair. Overexpression of Smurf2 in chondrocytes was shown to accelerate spontaneous OA development in mice. We hypothesized that reduced Smurf2 expression could slow the progression of OA and enhance the performance of cells for cartilage repair. By performing surgical destabilization of the medial meniscus (DMM) on Smurf2-deficient mice, loss of Smurf2 was shown to mitigate OA changes in young mice but this protection diminished in older mice. Assessment of Smurf2-deficient chondrocytes in vitro revealed an upregulation of chondrogenic genes compared to wild-type; however, these differences were not seen at the protein level, deterring its potential use for cell-based therapies. During the course of this study, new insights about how age and sex affects different joint compartments in response to DMM surgery were also uncovered. These results broadened existing understanding of DMM-induced OA in mice but also questioned the validity of such a model to identify disease modifying targets that are translatable to OA in humans with advanced age. Due to a lack of innate repair mechanisms in cartilage, damage to cartilage increases the risk of developing OA early. Tissue engineering provides a unique strategy for repairing damaged cartilage by delivering cells in a well-controlled environment that can promote the formation of neotissue. We hypothesized that synthetic chemical residues could enhance the mechanical properties of a bioinert scaffold and promote matrix production of encapsulated chondrocytes. Covalent incorporation of small anionic or zwitterionic chemical residues in a polyethylene glycol-based hydrogel improved its stiffness and resistance to fluid flow, however, the resulting physical environment can also exert a dominant negative effect on matrix production of encapsulated chondrocytes. These results suggest that modulating the biosynthesis of chondrocytes with biochemical signals requires a concurrent reduction in any conflicting mechanotransduction signaling, emphasizing the importance of a degradable system to promote new cartilage formation. In summary, this dissertation establishes Smurf2 as a modulator of OA progression but implies that other factors such as age or protein(s) with redundant Smurf2 functions may play a role in limiting its effect as a therapeutic target. This work also reveals fundamental biology about how chondrocytes behave in response to physical and chemical cues in their microenvironment, which will aid in the design of better scaffolds for cartilage tissue engineering.

Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies. / Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling.

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