The resurgence, and eventual rise to prominence in the field of tissue engineering, that electrospinning has experienced over the last decade speaks to the simplicity and adaptability of the process. Electrospinning has been used for the fabrication of tissue engineering scaffolds intended for use in nearly every part of the human body: blood vessel, cartilage, bone, skin, nerve, connective tissue, etc. Diverse as the aforementioned tissues are in both form and function, electrospinning has found a niche in the repair of each due to its capacity to consistently create non-woven structures of fibers ranging from nano-to-micron size in diameter. These structures have had success in tissue engineering applications because of their ability to mimic the body’s natural structural framework, the extracellular matrix. In this study we examine a number of different techniques for altering scaffold properties (i.e. mechanical strength, degradation rate, permeability, and bioactivity) to create electrospun structures tailored to unique tissue specific applications; the end goal being the creation of a cellularized tissue engineering ligament analogue. To alter the mechanical properties of electrospun structures while maintaining high levels of bioactivity, synthetic polymers such as polydioxanone were blended in solution with naturally occurring proteins like elastin and fibrinogen prior to electrospinning. Cross-linking of electrospun structures, using glutaraldehyde, carbodiimide hydrochloride, and genipin, was also investigated as a means to both improve the mechanical stability and slow the rate of degradation of the structures. Fiber orientation and scaffold anisotropy were controlled through varying fabrication parameters, and proved effective in altering the mechanical properties of the structures. Finally, major changes in the structure of electrospun scaffolds were achieved through the implementation of air-gap electrospinning. Scaffolds created through air-gap electrospinning exhibited higher porosity’s than their traditionally fabricated counterparts, allowing for greater cell penetration into the scaffold. Overall, this collection of results provides insight into the diversity of electrospinning and reveals innumerous options, both pre and post fabrication, for the tissue engineer to create site-specific engineering scaffolds capable of mimicking both the form and function of native tissue.

The purpose of this work was to develop an automated method of measuring fiber diameters of electrospun scaffolds from scanning electron microscopy images of these scaffolds. Several automated methods were developed and evaluated by comparison to known values and data obtained via the standard manual method. Simulated images with known diameters were used as test images to evaluate the accuracy of each measurement technique. Eight scanning electron microscopy images were also used for the evaluation of the automated methods compared to the standard manual method. All diameter measurements were made in pixels. Five new automated methods coded in MATLAB were developed. The five methods varied the approach of identifying edges of fibers as well as assigning edges to single fibers and calculating the distance between edges assigned to the same fiber. One-way analysis of variance and the Tukey-Kramer tests were performed for comparison of all methods per image. The Custom Canny Slopes automated method was shown to accurately approximate the mean diameters in ten simulated images as well as microscopy image of real scaffolds (p<0.05).

The purpose of this study was to construct a flowmeter that could accurately measure the hydraulic permeability of electrospun fibrinogen scaffolds, providing insight into the transport properties of electrospun scaffolds while making the measurement of their topographical features (fiber and pore size) more accurate. Three different concentrations of fibrinogen were used (100, 120, and 150mg/ml) to create scaffolds with three different fiber diameters and pore sizes. The fiber diameters and pore sizes of the electrospun scaffolds were analyzed through scanning electron microscopy and image analysis software. The permeability of each scaffold was measured and used to calculate permeability-based fiber diameters and pore sizes, which were compared to values obtained through image analysis. Permeability measurement revealed scaffold permeability to increase linearly with fibrinogen concentration, much like average fiber diameter and pore size. Comparison between the two measurement methods proved the efficacy of the flowmeter as a way to measure scaffold features.

The process of electrospinning has given the field of tissue engineering insight into many aspects of tissue engineered scaffolds, including how factors such as fiber diameter and porosity are affected by polymer concentration. However, the affects of fiber alignment upon the material properties of electrospun scaffolds remains unclear. The purpose of this study is to determine how the material properties of electrospun gelatin scaffolds are affected by changes in fiber alignment and starting gelatin concentration. Gelatin scaffolds, with starting concentrations of 80, 100, and 130mg/m1, were electrospun onto a target mandrel rotating at various speeds. Samples of each scaffold were taken parallel and perpendicular to the axis of mandrel rotation. Fast Fourier Transform (FFT) analysis was performed on these samples, to determine how fiber alignment is affected by starting polymer concentration and the rotational speed of the target mandrel. Mechanical tests were aiso performed on these samples. Results were analyzed by Three-way ANOVA. It was determined that starting gelatin concentration, mandrel speed, and direction of fiber alignment interact together to produce effects on the mechanical properties of electrospun gelatin scaffolds.

The goal of this research was to develop a system of injectable hydrogels to deliver stem cells to musculoskeletal defects, thereby allowing cells to remain at the treatment site and secrete soluble factors that will facilitate tissue regeneration. First, production parameters for encapsulating cells in microbeads were determined. This involved investigating the effects of osmolytes on alginate microbead properties, and the effects of alginate microbead cell density, alginate microbead density, and effects of osteogenic media on microencapsulated cells. Although cells remained viable in the microbeads, alginate does not readily degrade in vivo for six months. Therefore, a method to incorporate alginate lyase in microbeads was developed and optimized to achieve controlled release of viable cells. Effectiveness of this strategy was determined through cell release studies and measuring proteins and expression of genes that are characteristic of the cell’s phenotype. Lastly, in vivo studies were done to assess the ability of alginate microbeads to localize microencapsulated cells and support chondrogenesis and osteogenesis. This project will provide insight to the tissue engineering field regarding cell-based therapies and healing musculoskeletal defects.

Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. A hallmark of epithelial cells is strong cell-cell junctions. These junctions include E-Cadherin, a type of adherens junction that is critical for both barrier function and contact inhibition. Prior experiments by other groups have shown that adherens junctions are subject to mechanical tension. Externally applied forces (e.g. stretch) results in changes in E-Cadherin forces that coordinate proliferation. My current work tests the hypothesis that E-Cadherin forces mediate the spatial regulation of cell proliferation even in the absence of externally applied forces.

Diseases involving fibrosis cause tens of thousands of deaths per year in the US alone. These diseases are characterized by a large amount of extracellular matrix, causing stiff abnormal tissues that may not function correctly. To take steps towards curing these diseases, a fundamental understanding of how cells interact with their substrate and how mechanical forces alter signaling pathways is vital. Studying the mechanobiology of cells and the interaction between a cell and its extracellular matrix can help explain the mechanisms behind stem cell differentiation, cell migration, and metastasis. Due to the correlation between force, extracellular matrix assembly, and substrate stiffness, it is vital to make in vitro models that more accurately simulate biological stiffness as well as measure the amount of force and extracellular matrix assembly. To accomplish this, blends of two types of poly(dimethylsiloxane) (PDMS) were made and the material properties of these polymer blends were characterized. A field of 5µm or 7µm microscopic pillars (referred to as posts) with a diameter of 2.2µm were fabricated from these blends. Each combination of PDMS blend and post height were calibrated and the stiffness was recorded. Additionally, polymer attachment experiments were run to ensure cells survived and had a normal phenotype on the different blends of PDMS when compared to pure PDMS. Finally, cells were placed onto a field of posts and their forces were calculated using the new stiffness found for each blend of post. Varying the PDMS material stiffness using blends allow posts to be much more physiologically relevant and help to create more accurate in vitro models while still allowing easy and accurate force measurement. More biologically relevant in vitro models can help us acquire more accurate results when testing new drugs or examining new signaling pathways.

La création d'un réseau vasculaire fonctionnel est une préoccupation importante afin d'assurer la parfaite vitalité des produits d’ingénierie tissulaire (IT). La compréhension des mécanismes de l'angiogenèse est essentielle dans un objectif de synthèse de produits d’ingénierie tissulaire vascularisés. Dans ce travail, nous avons visé à caractériser le microenvironnement responsable de l'angiogenèse des cellules endothéliales (CEs). Pour cela, nous avons élaboré des biomatériaux bioactifs (polymères fonctionnalisés par des peptides, et contrôlé leur distribution à l'échelle micrométrique) afin de mimer une situation physiologique des CEs.Dans en premier temps, nous avons mis au point une stratégie de fonctionnalisation biochimique d’un matériau polymère (le polyéthylène téréphtalate, PET) en utilisant des peptides spécifiques des CEs. L'immobilisation de ces peptides a permis d’assurer une bioactivité de ces surfaces, et l’amélioration des fonctions des CEs comme l'adhésion, l’étalement et la migration cellulaire.Ensuite, notre travail s’est inscrit dans l’évaluation de l’impact d’une distribution contrôlée de peptides en surface de matériaux (acquise par photolithographie) sur le comportement des CEs et sur l’angiogenèse. Nos résultats ont montré que les CEs adhèrent et sont alignés sur les « micropatterns » peptidiques quelle que soit la taille de ces « micropatterns » (lignes de largeurs comprises entre 10 et 100 µm). Nous avons mis en évidence que la taille des « micropatterns » bioactifs a un réel impact sur le comportement des CEs (l’étalement, l'orientation et la migration cellulaire). La morphogenèse des CEs (la formation d’un « tube-like ») a été mise en évidence sur des matériaux microstructurés par des lignes peptidiques de 10 et 50 µm de largeur, quels que soient les peptides RGD ou SVVYGLR immobilisés en surface. Nous avons montré que la lumière de structures tubulaires peut être constituée d’une à quatre cellules selon la contrainte géométrique appliquée sur les « micropatterns ». Nos travaux ont montré que le « sprouting » ainsi que la formation du réseau vasculaire peuvent être induits seulement sur des surfaces « micropatternés » par des peptides SVVYGLR. Nos résultats démontrent que l'induction de l'angiogenèse est multiparamétrique. Celle-ci est dépendante de constituants biochimiques ainsi que de leur micro-distribution.Troisièmement, nous avons utilisé la modélisation mathématique pour comprendre l'impact de « micropatterns » bioactifs sur la migration des CEs. Un modèle de type continu Patlak-Keller-Segel a été utilisé, et les résultats numériques sont bien conformes avec nos résultats expérimentaux. Pour finir, nos travaux se sont focalisés sur l'étude de la stabilisation de ces structures tubulaires. Les résultats ont montré que les cocultures de CEs avec les péricytes, ainsi que le recrutement de composant de membrane basale (Matrigel) peuvent stabiliser ces structures vasculaires.En conclusion générale, le travail réalisé dans cette thèse a prouvé que le « micropatterning » des principes bioactifs sur polymères est efficace pour stimuler l'angiogenèse et pour construire une vascularisation fonctionnelle. Enfin, ce travail a permis de comprendre la biologie de l’angiogenèse et pourra aider indéniablement tous les travaux en cours s’inscrivant dans l’ingénierie tissulaire. / The creation of a functional vascular network is a major concern to ensure the vitality of perfect tissue engineered products. Understanding the mechanisms of angiogenesis is essential for the vascularization in tissue engineering. In this work, we aimed to characterize the microenvironment responsible for angiogenesis of endothelial cells. To achieve this request, we developed bioactive biomaterials (polymers functionalized mainly with peptides, and controlled their distribution at micrometer scale) to mimic a physiological microenvironment of endothelial cells. First, we developed the biochemical functionalized of polymer materials (polyethylene terephthalate, PET) using endothelial cell specific peptides. The peptide immobilization ensured the bioactivity onto material surfaces, and enhanced endothelial cell functions such as cell adhesion, spreading and migration. Second, we introduced photolithographical technique to control geometrical distribution of peptides on material surfaces, and studied the effects of peptide micropatterning onto endothelial cell (EC) angiogenesis. ECs were adhered and aligned onto peptides micropatterns whatever the size of peptide micropatterns. However, EC behaviors (cell spreading, orientation and migration) were significantly more regulated on smaller micropatterns (10 and 50 µm) than on larger stripes (100 µm). EC morphogenesis into tube formation can also switch onto the smaller micropatterns (10 and 50 µm) with either RGD or SVVYGLR peptides. The central lumen of tubular structures can be formed by single-to-four cells due to geometrical constraints applied on the micropatterns. Sprouting angiogenesis of ECs and vascular network formation can be induced on surfaces micropatterned with angiogenic SVVYGLR peptides. Our overall results revealed that the induction of angiogenesis is multi-parametric. This is dependent on biochemical constituents and their micro-distributions. Third, we employed mathematical modeling to understand the impact of bioactive micropatterns on endothelial cell migration. A continuous Patlak-Keller-Segel type model was used, and the numerical results were in well accordance with our experimental results.Furthermore, we also developed the study of stabilization of tubulogenenic structure in this thesis. The results showed that the co-cultures of endothelial cells with pericytes, as well as the recruitment of basement membrane components (Matrigel) can stabilize the vascular tube structures. In general conclusion, our work in this thesis proved that bioactive micropatterning of polymer is effective to stimulate angiogenesis and to construct functional vascularization. This work helps us to understand the fundamental biology of angiogenesis, and has great potential for application in tissue engineering.

There is a growing epidemic of chronic heart failure (CHF) in the developed world. The costs associated with providing care is profound and despite our best efforts, new, more effective treatments for CHF are needed; 50% of patients diagnosed with CHF are dead within 5 years. Current paradigms rely heavily on pharmacologic interventions, which merely help manage the disease. Surgical interventions may also be considered for late stage CHF patients such as heart transplant or left ventricular assist device (LVAD) but require burdensome and invasive surgical procedures. In addition they are costly, and require the need for life long immunosuppressive and anticoagulant therapies respectively. Despite our best intentions, the long-term prognosis for CHF patients remains poor. With over a decade of clinical investigation taken place, data from cell-based therapy trials remains inconsistent. While demonstrating safety, limited efficacy has been reported and to date, no stem cell therapy has been approved by the FDA. Despite these shortcomings important lessons have been learned that can be applied to future developments. Retrospective analysis of early cell-based clinical trial data has suggested that variations in isolated cell number, viability, and potency from donor to donor in autologous preparations yielded wide discrepancies in functional outcomes. In addition, sub culturing adult stem cells, even for short periods of time in 2D polystyrene environments void of complementary cell populations and extra cellular matrix protein interactions, may alter the therapeutic potential of a given cell. As a solution, allogeneic approaches where donor cell quality and potency can be assessed and optimized may help achieve functional benefits. Furthermore, co-dosing with multiple cell populations or developing 3D sub-culture environments that more closely mimic the in vivo milieu may ultimately yield more potent therapeutic cell populations. While these alterations may improve cell-based therapy outcomes, other solutions have been proposed such as tissue engineering. While the concept of tissue engineering is not new, advancements in biomaterials, bioreactor design and cell sources have greatly enhanced the reality of these preparations. Previously, one of the greatest limitations to tissue engineering is overcoming the cell requirements for developing and testing where millions if not billions of cells are required. Cell sourcing limitations appear to have been solved with the discovery and development of induced pluripotent stem cell (iPSC) derived cell populations. First reported in 2007, they have the ability to generate embryonic like pluripotent stem cells without the ethical concerns of embryonic stem cells. These iPSCs hold tremendous potential for drug toxicology / screening, personalized medicine and cell therapies. The body of work described in this dissertation looks at developing and testing a tissue engineered cardiac patch to treat heart failure. For which, an emphasis has been to provide 1) structural support for engrafted cells and 2) a rapidly inducible vascular supply once implanted in vivo. Biomaterials were sourced that facilitate infill by multiple cell populations in 3D culture and the establishment of extra cellular matrix deposits. Together, these patches enhanced cellular development in vitro and result in long term functional improvements in small animal models for CHF. Additional feasibility work was performed in large animal models to permit upscaling and development of surgical implantation techniques to demonstrate clinical applicability

Les lésions de cartilage ne cicatrisent pas spontanément et la réparation de ce tissu est un challenge. Les techniques chirurgicales restant insatisfaisantes, la thérapie cellulaire et l'ingénierie tissulaire sont maintenant envisagées. La transplantation de chondrocytes autologues (TCA) existe déjà mais cette procédure nécessite l'amplification des chondrocytes qui s'accompagne d'une perte du phénotype différencié (dont l'indicateur est le collagène de type II), au profit d'un phénotype fibroblastique (dont l'indicateur est le collagène de type I, retrouvé dans les tissus fibreux). La TCA conduit donc à une greffe de chondrocytes dédifférenciés produisant un fibrocartilage, dont les propriétés mécaniques sont différentes du cartilage hyalin natif. L'objectif de mes travaux était de développer un nouveau kit d'ingénierie tissulaire du cartilage par association de chondrocytes humains, de biomatériaux et d'une sélection de facteurs solubles. Nous avons utilisé le cocktail FGF-2/insuline (FI) pour l'amplification cellulaire et le cocktail BMP-2/insuline/T3 (BIT) pour redifférencier les chondrocytes dans des éponges de collagène. Nos résultats ont montré que cette combinaison permet la synthèse d'une matrice cartilagineuse dans les supports collagène. Cependant, cette synthèse s'est trouvée favorisée en périphérie des éponges cultivées en conditions statiques. Nous avons ensuite utilisé un bioréacteur pour perfuser les éponges et nos résultats ont révélé alors un dépôt plus homogène de cartilage dans ces supports. De manière très intéressante, nous avons aussi observé l'arrêt de l'expression du collagène de type I. Ainsi, notre approche multifactorielle combinant des chondrocytes humains, des biomatériaux collagène, une combinaison FI-BIT et une culture en perfusion permet la reconstruction d'un cartilage non fibrotique / Cartilage lesions are irreversible and cartilage repair is challenging. Actual surgical techniques remain unsatisfactory and therefore, cell therapy and tissue engineering approaches are now considered. The Autologous Chondrocytes Transplantation (ACT) already exists but this procedure requires chondrocytes amplification. During this amplification, a dedifferentiation process occurs: chondrocytes lose their differentiated phenotype (characterized by type II collagen) towards a fibroblastic phenotype (characterized by type I collagen, a component of fibrous tissues). ACT leads to the graft of dedifferentiated chondrocytes, hence provoking the production of a fibrocartilage that presents different mechanical properties than native hyaline cartilage. The aim of my work was to develop a new kit of tissue engineering for cartilage repair using human chondrocytes, biomaterials and a selection of soluble factors. We used a cocktail of FGF-2 and insulin (FI) for cell amplification and a cocktail of BMP-2, insulin and T3 (BIT) for chondrocyte redifferentiation in collagen sponges. Our results showed that the combination allows the synthesis of a cartilaginous matrix in collagen scaffolds. However, matrix production is favored in periphery of the sponges cultivated in static conditions. A perfusion bioreactor was then used to perfuse the sponges and our results revealed a more homogeneous deposition of cartilage in the scaffolds. Very interestingly, we also noticed a stop of type I collagen expression. Thus, our multifactorial approach combining human chondrocytes, collagen scaffold, the combination FI-BIT and culture under perfusion allows the reconstruction of a non-fibrotic cartilage