Microphysiological Systems for the Evaluation of Biomaterials in Regenerative Therapies

Abstract

[eng] The design of bioresponsive materials capable of stimulating the body’s innate regenerative potential is opening unprecedented possibilities to treat tissue and organ failure, which is one of the most important burdens of healthcare systems worldwide. Unfortunately, their development is hampered by the lack of adequate preclinical models, which are essential in the successful transition of a biomaterial to the clinical trials phase. Most of the experiments rely on animal models, which usually fail to predict the material interactions with the human body, as they are unable to recapitulate the complexities of our physiology. During the last decades, the advancements in the field of microtechnology have allowed to create advanced cell culture systems capable of replicating tissue and organ-level physiology by mimicking relevant conditions such as cell organization or microenvironmental cues. These platforms, known as microphysiological systems (MPS), have shown in different studies their great potential in predicting mechanisms of action, safety, and efficacy of different drugs, attracting a lot of attention from the pharmaceutical industry and regulatory agencies. However, few studies have explored the possibility of using microphysiological systems for the preclinical testing of biomaterials. The goal of this thesis is to fill this knowledge gap by developing microfluidic cell culture systems that allow to reliably predict the actual in vivo response of different materials. One of the proposed platforms is aimed at assessing the potential of a biomaterial to stimulate endothelial progenitor cell recruitment in a bone tissue microenvironment. This is a critical step in the neovascularization and bone regeneration process that has not been properly studied due to the lack of adequate models. The proposed device allowed to identify the role of calcium ions in stimulating the recruitment of rat endothelial progenitor cells (rEPC) to the site of injury, which is mediated by an increase in the release of osteopontin, a chemotactic and mitogenic protein produced by rat bone-marrow mesenchymal stromal cells (BM-rMSC). The platform was also used to evaluate a calcium-releasing biomaterial based on electrospun polylactic acid (PLA) fibers with calcium-phosphate (CaP) nanoparticles. The results show a significant increase in terms of rEPC recruitment and the release of osteopontin and other pro-angiogenic and inflammatory proteins by BM-rMSC with respect to a regular PLA control, which is in close agreement with previous experiments performed in a murine in vivo model. The other platform proposed in this thesis is aimed at providing a physiologically relevant model of cardiac tissue to study a myocardial ischemia-reperfusion injury. There are currently no reliable in vitro models to mimic this disease, making these contributions extremely relevant for cardiac regeneration studies. A first prototype of the platform based on the combination of aligned electrospun PLA fibers with

a user-friendly electrical stimulation setup in a microfluidic cell culture platform produced a biomimetic cardiac tissue in 2D. This was confirmed by the high anisotropy of the tissue constructs, based on the co- culture of neonatal mouse cardiomyocytes with cardiac fibroblasts, as well as the upregulation of several key cardiac markers such as contractile and structural proteins. In order to make the model more physiologically relevant, a second device was developed to obtain human-derived 3D tissues. This platform is based on the self-assembling of primary cardiac fibroblasts (hCF) co-cultured with human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) in a fibrin-based hydrogel around two microposts structures, which exert a passive mechanical tension that stimulates tissue maturation and cell alignment. We first performed a screening using 2D assays based on hPSC-CM monolayers to select the best environmental conditions to mimic an ischemia-reperfusion injury. We then characterized the response of the human- derived cardiac organoids to an ischemia-reperfusion injury, consisting of an 8 h culture period at 0 % oxygen in an ischemic solution that replicates the acidic and hyperkalemic conditions observed in vivo, followed by a refreshment with fully supplemented cell media and recovery of 21 % environmental oxygen concentrations. We observed a drastic increase in cell death by necrosis and apoptosis as well as a strong fibrotic response, characterized by an increase in hCF proliferation, differentiation towards myofibroblasts and collagen I deposition. Taken together, we believe that the platforms developed in this thesis constitute an extremely valuable and versatile tool to perform preclinical studies, offering a promising alternative to animal studies for the development of new biomaterials and drug discovery.

[spa] El diseño de biomateriales capaces de estimular la capacidad innata del cuerpo de regenerarse está abriendo una oportunidad sin precedentes para el tratamiento y reemplazamiento de órganos y tejidos, una de las principales cargas en los sistemas de salud a nivel mundial. Desafortunadamente, el desarrollo de estas terapias se ve lastrado por la falta de modelos preclínicos adecuados, que son esenciales en la transición exitosa de un biomaterial a la aplicación clínica. La mayoría de estos experimentos se basan en el uso de modelos animales, que habitualmente fallan en la predicción de las interacciones que ocurren en el cuerpo humano, debido a las diferencias inherentes que existen en términos de fisiología. Durante las últimas décadas, los avances en el campo de la microtecnología han permitido crear plataformas de cultivo celular capaces de replicar elementos fisiológicos a nivel de tejidos y órganos denominados sistemas microfisiológicos. Estos sistemas han demostrado su gran utilidad en la predicción de mecanismos de acción, seguridad y eficacia de diferentes fármacos, atrayendo una gran atención por parte de las agencias regulatorias. Sin embargo, pocos estudios han explorado la posibilidad de usar este tipo de sistemas para la evaluación preclínica de biomateriales. El objetivo de esta tesis es realizar contribuciones en este campo mediante el desarrollo de sistemas microfluídicos de cultivo celular capaces de predecir la respuesta in vivo de diferentes materiales. En esta tesis se presentan principalmente dos modelos diferentes de sistemas microfisiológicos. El primer está relacionado con el reclutamiento de células progenitoras endoteliales en un entorno de regeneración ósea para el estudio de la vascularización de biomateriales, mientras que el segundo busca generar un modelo de tejido cardíaco fisiológicamente relevante para estudiar una lesión por isquemia-reperfusión y posibles terapias regenerativas.