During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.

Gelatin methacrylate (GelMA) is a ubiquitous biocompatible photopolymer used in tissue engineering and regenerative medicine due to its cost-effective synthesis, tunable mechanical properties, and cellular response. Biotechnology applications utilizing GelMA have ranged from developing cell-laden hydrogel networks to cell encapsulation and additive manufacturing (3D printing). However, extrusion based 3D printing is the most common technique used with GelMA. Mask projection microstereolithography (MPµSL or µSL) is an advanced 3D printing technique that can produce geometries with high resolution, high complexity, and feature sizes unlike extrusion based printing. There are few biomaterials available for µSL applications, so 3D printing GelMA using µSL would not only add to the repertoire materials, but also demonstrate the advantages of µSL over other 3D printing techniques. A novel GelMA resin was tested with µSL to create a porous scaffold with a height and print time that has not been displayed in the literature before for a scaffold of this size. The resin consists of GelMA, deionized water, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, photoinitiator), and 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid (sulisobenzone, UV blocker) and can be processed at room temperature. Four resins were tested (w/w %) and characterized for µSL printing: 20% GelMA 0.5% UV blocker, 20% GelMA 1.0% UV blocker, 30% GelMA 0.5% UV Blocker, and 30% GelMA 1.0% UV blocker. Swell testing, working curve, photo-rheology, photo-DSC (dynamic scanning calorimetry), 3D printing, and cell culture tests were performed and results showed that 30% GelMA 1.0% UV blocker had the best 3D print fidelity among resin compositions. / Master of Science / Three dimensional (3D) printing is a widely used technology to rapidly produce structures with varying degrees of complexity. 3D printing of biological components is of interest because as the world population increases, there is a lack of donors available to compensate for organ loss and tissue replacement. 3D printing offers a solution to great custom scaffolds and structures that mimic physiological geometry and properties. One printing technique is known as microstereolithography, or µSL, which uses a projector-like system to pattern ultraviolet (UV) light in specific arrangements to generate complex geometries and 3D parts. Gelatin is a material of interest for this technology because gelatin is derived from collagen, which is the most abundant protein found in the body. Gelatin can be modified so that it is reactive with UV light, and can be processed with µSL to generate 3D structures. In this work, gelatin was modified into the form of gelatin methacrylate (GelMA) in order to develop and test resin formulations for use with µSL. Four different resins were tested and characterized and the results indicated that one GelMA resin produced prints with greater fidelity and resolution than other formulations. This resin has been identified for potential applications in tissue engineering and 3D printed organ development.

abstract: Severe cases of congenital heart defect (CHD) require surgeries to fix the structural problem, in which artificial grafts are often used. Although outcome of surgeries has improved over the past decades, there remains to be patients who require re-operations due to graft-related complications and the growth of patients which results in a mismatch in size between the patient’s anatomy and the implanted graft. A graft in which cells of the patient could infiltrate, facilitating transformation of the graft to a native-like tissue, and allow the graft to grow with the patient heart would be ideal. Cardiac tissue engineering (CTE) technologies, including extracellular matrix (ECM)-based hydrogels has emerged as a promising approach for the repair of cardiac damage. However, most of the previous studies have mainly focused on treatments for ischemic heart disease and related heart failure in adults, therefore the potential of CTE for CHD treatment is underexplored. In this study, a hybrid hydrogel was developed by combining the ECM derived from cardiac tissue of pediatric CHD patients and gelatin methacrylate (GelMA). In addition, the influence of incorporating gold nanorods (GNRs) within the hybrid hydrogels was studied. The functionalities of the ECM-GelMA-GNR hydrogels as a CTE scaffold were assessed by culturing neonatal rat cardiomyocytes on the hydrogel. After 8 days of cell culture, highly organized sarcomeric alpha-actinin structures and connexin 43 expression were evident in ECM- and GNR-incorporated hydrogels compared to pristine GelMA hydrogel, indicating cell maturation and formation of cardiac tissue. The findings of this study indicate the promising potential of ECM-GelMA-GNR hybrid hydrogels as a CTE approach for CHD treatment. As another approach to improve CHD treatment, this study sought the possibility of performing a proteomic analysis on cardiac ECM of pediatric CHD patient tissue. As the ECM play important roles in regulating cell signaling, there is an increasing interest in studying the ECM proteome and the influences caused by diseases. Proteomics on ECM is challenging due to the insoluble nature of ECM proteins which makes protein extraction and digestion difficult. In this study, as a first step to perform proteomics, optimization on sample preparation procedure was attempted. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

abstract: Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli. These complexities make it difficult to isolate and assess the effects of specific parameters including matrix stiffness and tumor architecture on disease progression. In this regard, morphologically accurate tumor models are becoming instrumental to perform fundamental studies on cancer cell invasion within well-controlled conditions. In this study, the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique was explored to microengineer a 3D breast tumor model. The microfabrication process presented herein enabled precise localization of the cells and creation of high stiffness constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and normal mammary epithelial cells (MCF10A) were embedded separately within the tumor model and cellular proliferation, migration and cytoskeletal organization were assessed. Proliferation of metastatic MDA-MB-231 cells was significantly higher than tumorigenic MCF7 and normal mammary MCF10A cells. MDA-MB-231 exhibited highly migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that were confined within the micropatterned circular features. F-actin staining revealed unique 3D protrusions in MDA-MB-231 cells as they migrated throughout the surrounding matrix. Alternatively, there were abundance of 3D clusters formed by MCF7 and MCF10A cells. The results revealed that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in creating 3D tumor models with well-defined features and tunable stiffness for detailed studies on cancer cell invasion and drug responsiveness. / Dissertation/Thesis / Supplementary Movie 3 / Supplementary Movie 1 / Supplementary Movie 2 / Supplementary Movie 5 / Supplementary Movie 4 / Masters Thesis Bioengineering 2015

Les maladies cardiovasculaires sont une des causes de mortalités les plus élevées mondialement. Parmi celles-ci, on retrouve l’infarctus du myocarde, qui n’a pour traitement que la transplantation cardiaque. Or, dû à la faible quantité de donneur, une solution alternative est recherchée. De ce fait, l’ingénierie tissulaire permet le développement de tissus et d’implants thérapeutiques tels les patchs cardiaques, qui peuvent être bioimprimés. Or, une des limitations actuelles de l’utilisation d’une telle stratégie est la vascularisation de tissu bioimprimés. Dans cette étude, la bioimpression 3D a été utilisée afin de bioimprimer des patchs perfusables de gélatine méthacrylate (GelMA) à utiliser potentiellement pour le tissu cardiaque. Il a été possible de développer une bioencre pouvant être utilisée pour une application dans le tissu cardiaque, d’évaluer l’imprimabilité de l’encre et de bioimprimer de patchs standards et perfusables. Pour ce faire, GelMA a été synthétisé et les propriétés mécaniques ont été évaluées pour finalement sélectionner une encre de 10 % GelMA, ayant un module de Young approprié pour le tissu cardiaque, de 23,7±5,1 kPa. Par la suite, les processus d’impression, standard et coaxial, de patchs standards et perfusables ont pu être optimisés. Finalement, des patchs perfusables de GelMA 10% et gélatine 2% ont pu être imprimés avec une viabilité cellulaire élevée, jusqu’à 79,7±8,7 % et 83,5±5,7 % obtenue aux jours 1 et 7 de culture respectivement, avec des fibroblastes 3T3. La présence de canaux vides et la perfusabilité des patchs démontrent le potentiel de cette méthode pour éventuellement bioimprimer des patchs cardiaques vascularisés épais. / Cardiovascular diseases are a leading cause of death worldwide. Myocardial infarction captures a significant segment of this population, and the end-stage myocardial infarction can only be treated by heart transplantation. However, due to the scarcity donors, tissue engineering has been considered as an alternative solution. Tissue engineering allows the development of tissues and therapeutic implants such as cardiac patches. However, one of the main hurdles in the use of such a strategy is the vascularization of bioprinted tissue. In this study, 3D bioprinting was used to bioprint perfusable gelatin methacrylate (GelMA) patches for a potential use in cardiac tissue. This work consists in the development of a bioink that can be used for the cardiac tissue, the evaluation of the printability of the ink, and the final bioprinting of standard and perfusable patches. For this purpose, GelMA was synthesized and a final concentration of 10 % was selected as it showed an appropriate Young's modulus for cardiac tissue, of 23.7±5.1 kPa, while maintaining high biocompatibility. Subsequently, the printing process of standard and perfusable patches could be optimized with the use of GelMA and gelatin inks. Finally, 10% GelMA and 2% gelatin vascularized patches could be printed with high cell viability, of up to 79,7±8,7 % and 83,5±5,7 % on days 1 and 7 of culture respectively for 3T3 fibroblasts. Additionally, the presence of hollow channels of the perfusable patches demonstrates the potential of this method to be eventually applied to the bioprinting of thick vascularized cardiac patches.