Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.

Title: Methodological detection solution scaffolds response to mechanical stress, depending on the degree of hydration Objectives: Determining the extent of lateral deformation u scaffolds made of PVA polymer electrospinning technique. Identify the extent of differences in transverse deformation for different groups of nanofiber scaffolds made of PVA polymer electrospinning technique. Methods: Research scaffolds, we used a measuring device μ-tester, which has two jaws. For the measurement, we chose uniaxial tension test in -tester and record the fluorescence microscope was used with HD camera Olympus 320 for online video recording. Results: The results of this study showed that the ratio of the samples U: L and crosslinking time affects the degree of lateral deformation of the samples scaffolds. Samples scaffolds are compressible, some groups even reached the limits of incompressibility 0.5 Poisson's ratio. Keywords: Poisson, Poisson's ratio, scaffold, nanofiber scaffold, scaffold hydrated, electrospun scaffold, lateral deformation

<p>This investigation delineates the optimal synthesis and characterization of innovative epoxy-carbon nanotube (CNT) nanocomposite filaments via electrospinning. Electrospinning thermosetting materials such as epoxy resins presents significant challenges due to the polycationic behavior arising from intermolecular noncovalent interactions between epoxide and hydroxyl groups, resulting in a substantial increase in solution surface tension. In this study, electrospinning submicron epoxy filaments was achieved through partial curing of epoxy via a thermal treatment process in an organic polar solvent, circumventing the necessity for plasticizers or thermoplastic binders. The filament diameter can be modulated to as low as 100 nm by adjusting electrospinning parameters.</p> <p><br></p> <p>Integrating a minimal amount of CNT into the epoxy matrix yielded enhanced structural, electrical, and thermal stability. The CNTs were aligned within the epoxy filaments due to the electrostatic field present during electrospinning. The modulus of the epoxy and epoxy-CNT filaments were determined to be 3.24 and 4.84 GPa, respectively, resulting in a 49% improvement. Epoxy-CNT nanofibers were directly deposited onto carbon fiber reinforced polymer (CFRP) prepreg layers, yielding augmented adhesion, interfacial bonding, and significant mechanical property enhancements. The interlaminar shear strength (ILSS) and fatigue resistance demonstrated a 29% and 27% increase, respectively, under intense stress conditions. Up to 45% of the Barely Visible Impact Damage (BVID) energy absorption was increased. In addition, the strategic incorporation of CNT (multi-walled) networks between the layers of CFRP resulted in a significant increase in thermal and electrical conductivities.</p> <p>This study also introduces a scalable fabrication procedure to address large volume processing, reproducibility, accuracy, and electrospinning safety. Electric fields of the experimental multi-nozzle setups were simulated to elucidate the induced surface charges responsible for the Taylor cone formation of the epoxy-CNT solution droplet on the nozzle tips. Electrospinning parameters were subsequently optimized for the multi-nozzle system and analyzed alongside simulated data to improve stability and synthesize fibers with smaller diameters.</p> <p><br></p> <p>Smaller diameter epoxy-CNT nanofibers proved critical as CNTs maintained alignment within the nanofibers when compared to larger diameter nanofibers. This research examines the impact of effective parameters on the diameter of electrospun epoxy-CNT nanofibers using artificial neural networks (ANNs). Consequently, employing a genetic algorithm (GA) and Bayesian optimization (BO) methods enable accurate prediction of epoxy-CNT nanofiber diameters prior to electrospinning. The presented models could aid researchers in fabricating electrospun thermosetting and thermoplastic scaffolds with specified fiber diameters, thereby tailoring these scaffolds for specific applications.</p>

The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....

La medicina regenerativa és una disciplina que ha guanyat reconeixement en les últimes dècades pel fet que moltes malalties no són tractables amb fàrmacs convencionals. Molts grups de recerca i empreses inverteixen temps i diners en la producció de nous paradigmes per curar malalties com el Parkinson, l'artrosi o la regeneració de medul·la espinal. Aquestes propostes es basen en l'ús de teixits biomimètics per reparar òrgans danyats. En aquesta tesi es presenta un nou model experimental per estudiar la formació d'os i cartílag i eventualment la reparació d'aquests teixits. Hem utilitzat Fibroblasts Embriònics de Ratolí (MEFs) combinats amb diferents materials biomimètics per estudiar os i cartílag in vitro i in vivo. Aquests MEFs es van cultivar in vitro i in vivo en RAD16-I, un pèptid auto-ensamblable amb estructura similar a matrius extracel·lulars genèriques, amb l'objectiu d'estudiar l'evolució dels fibroblasts en aquestes dues condicions. També s'han recobert superficialment micropartícules de hidroxiapatita obtenint càrregues inorgàniques similars a l'os i biològicament actives per a utilitzar-les com a substituts d'os o cartílag. Amb la idea de millorar els recobriments superficials, hem desenvolupat una plataforma que permet dur a terme proves combinatòries amb factors de creixement i altres compostos biològicament actius. Cultius in vitro de MEFs han mostrat que quan fibroblasts embrionaris primaris de ratolí es cultiven en RAD16-I, estableixen una xarxa intercel·lular que causa una contracció cel·lular organitzada, proliferació i migració cel·lulars i culmina amb la formació d'una estructura bilateral i simètrica amb un eix central distingible. Durant aquest procés morfològic, augmenta l'expressió d'un grup de gens mesodèrmics (brachyury, Sox9, Sox5, Sox6, Runx2). L'expressió de brachyury està localitzada primer en l'eix de simetria central i després s'extén als dos costats de l'estructura. Per acabar, la formació espontània d'un teixit similar al del cartílag acompanya l'expressió de Sox9 i Runx2.L'estudi in vivo de MEFs es va fer gràcies a la tècnica de presa d'imatges no invasiva basada en bioluminiscència (BLI) que ha desenvolupat en el grup de recerca del dr. Jerónimo Blanco. Aquests experiments han mostrat que el RAD16-I és una matriu molt permissiva per a la supervivència i proliferació cel·lulars in vivo. A més, sembla que les pobres propietats mecàniques que té el RAD16-I no li suposen cap desavantatge en termes de creixement cel·lular in vivo. Finalment, hem desenvolupat diferents tipus de micropartícules de hidroxiapatita (HA) no recobertes, i recobertes mitjançant polimerització assistida per plasma. Els recobriments permeten afinar les propietats de la HA i produir partícules que satisfacin les necessitats de diferents aplicacions mèdiques en reparació d'os i cartílag. També hem desenvolupat un mètode per produir plataformes basades en plaques de 96 pous que permetin fer proves combinatòries amb compostos biològicament actius per vàries aplicacions en medicina regenerativa. En conclusió, hem aportat noves idees i eines que permetran trobar teixits regeneratius basats en l'ús de fibroblasts i materials biomimètics. / La medicina regenerativa es una disciplina que ha ganado reconocimiento en las últimas décadas porque muchas enfermedades no son tratables con fármacos convencionales. Muchos grupos de investigación y empresas invierten tiempo y dinero en la producción de nuevos paradigmas para curar enfermedades como el Parkinson, la artrosis o la regeneración de médula espinal. Estas propuestas se basan en el uso de tejidos biomiméticos para reparar órganos dañados. En esta tesis se presenta un nuevo modelo experimental para estudiar la formación de hueso y cartílago y tal vez la reparación de estos tejidos. Hemos utilizado Fibroblastos Embrionarios de Ratón (MEFs) combinados con diferentes materiales biomiméticos para estudiar hueso y cartílago in vitro e in vivo. Estos MEFs se cultivaron in vitro e in vivo en RAD16-I, un péptido auto-ensamblable con estructura similar a matrices extracelulares genéricas, con el objetivo de estudiar la evolución de los fibroblastos en estas dos condiciones. También se han recubierto superficialmente micropartículas de hidroxiapatita obteniendo cargas inorgánicas similares al hueso y biológicamente activas para utilizarlas como sustitutos de hueso o cartílago. Con la idea de mejorar los recubrimientos superficiales, hemos desarrollado una plataforma que permite llevar a cabo pruebas combinatorias con factores de crecimiento y otros compuestos biológicamente activos. Cultivos in vitro de MEFs han mostrado que cuando fibroblastos embrionarios primarios de ratón se cultivan en RAD16-I, establecen una red intercelular que causa una contracción celular organizada, proliferación y migración celulares y culmina con la formación de una estructura bilateral y simétrica con un eje central distinguible. Durante este proceso morfológico, aumenta la expresión de un grupo de genes mesodérmicos (brachyury, Sox9, Sox5, Sox6, Runx2). La expresión de brachyury está localizada primero en el eje de simetría central y después se extiende a los dos lados de la estructura. Para terminar, la formación espontánea de un tejido similar al del cartílago acompaña a la expresión de Sox9 y Runx2.El estudio in vivo de MEFs se hizo gracias a la técnica de toma de imágenes no invasiva basada en bioluminiscencia (BLI) que han desarrollado en el grupo de investigación del dr. Jerónimo Blanco. Estos experimentos han mostrado que el RAD16-I es una matriz muy permisiva para a la supervivencia y proliferación celulares in vivo. Además, parece que las pobres propiedades mecánicas que tiene el RAD16-I no le suponen ninguna desventaja en términos de crecimiento celular in vivo. Finalmente, hemos desarrollado diferentes tipos de micropartículas de hidroxiapatita (HA) no recubiertas, y recubiertas mediante polimerización asistida por plasma. Los recubrimientos permiten afinar las propiedades de la HA y producir partículas que satisfagan las necesidades de diferentes aplicaciones médicas en reparación de hueso y cartílago. También hemos desarrollado un método para producir plataformas basadas en placas de 96 pozos que permitan hacer pruebas combinatorias con compuestos biológicamente activos para varias aplicaciones en medicina regenerativa. En conclusión, hemos aportado nuevas ideas y herramientas que permitirán hallar tejidos regenerativos basados en el uso de fibroblastos y materiales biomiméticos. / Regenerative medicine is a discipline that has gained recognition in the last decades because many diseases are not treatable with traditional drugs. Many research groups and companies invest time and money in the production of new paradigms to cure conditions such as Parkinson's, arthrosis or spinal cord injuries. These approaches are based in the use of biomimetic tissues to replace damaged organs. In this work we present a new experimental model to study the formation of bone and cartilage and eventually to repair these tissues. We have used Mouse Embryonic Fibroblasts (MEFs) combined with different biomimetic materials to study bone and cartilage formation in vitro and in vivo. MEFs have been cultured in vitro and in vivo in RAD16-I, a synthetic self-assembling peptide with structure similar to generic extracellular matrix milieu, to study the evolution of these fibroblasts in both conditions. Also, hydroxyapatite microparticles have been surface coated to produce biologically active bone-like inorganic charges for use in cartilage or bone substitutes. In order to improve the particles' coatings, we have developed a platform that allows us to perform combinatorial testing of growth factors and other biologically active compounds. In vitro cultures of MEFs has shown that when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction, proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Sox5, Sox6, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed the expression of Sox9 and Runx2.In vivo study of MEFs was facilitated by a non-invasive bioluminescence imaging (BLI) technique to detect luciferase-expressing cells, developed by Dr. Blanco's research group. These experiments showed that RAD16-I is a very permissive scaffold for cell survival and proliferation in vivo. Furthermore, it seems that the poor mechanical properties of RAD16-I are no disadvantage in terms of cell growth in vivo.Finally, we have developed different types of coated and uncoated hydroxyapatite (HA) microparticles by plasma polymerization. The coatings permit to tune the properties of HA and produce particles that suit the needs of different medical applications in bone and cartilage repair. Moreover, we have developed a method to produce platforms based on 96-well plates that allow the combinatorial testing of biologically active compounds for various applications in regenerative medicine. In conclusion, we have supplied new insights and tools that will enhance the finding of new regenerative tissues based on fibroblasts and biomimetic materials.