Abstract
The main objectives of the research presented in this thesis are to develop a representative in vitro model of human aortic valve disease, and to engineer a drug delivery platform to deliver non-coding RNA over an extended period of time. These are important next steps towards a better understanding of
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aortic valve disease, towards identification of therapeutic targets, and towards a therapeutic treatment that can slow or halt disease progression.
Chapter 1 introduces the anatomy of the aortic valve and pathobiology of calcific aortic valve disease. Known microRNAs involved in aortic valve disease are discussed, and the opportunities for microRNAs as therapeutic agents are identified. Additionally, requirements for better and more accurate in vitro models of aortic valve disease are proposed. The identified challenges and opportunities form the basis for the research presented in this thesis. Applying the previously outlined recommendations, Chapter 2, describes how the first human 3D bioprinted heart valve disease model is engineered. Nano-indentation is used to measure the biomechanical properties of the individual layers of the native aortic valve, which are in turn replicated in the model by fine-tuning the concentration and ratio of biopolymers used to mimic the aortic valve extracellular matrix. Non-diseased aortic valve interstitial cells are mixed with these gelatin- and hyaluronic acid-based biopolymers and bioprinted into 3D constructs with the same mechanical properties as the native valve. The cells inside the constructs are exposed to osteogenic factors to mimic disease progression. Chapter 3 is taking a first important step towards using the 3D aortic valve disease in vitro model in search of novel therapeutics by testing the effect of manipulating microRNA expression on the formation of microcalcifications in calcific aortic valve disease. Specifically, aortic valve interstitial cells are producing microcalcifications in the 3D constructs as a result of osteogenic stimulation and microRNA-214 expression is either increased or repressed via mimics or anti-miRs, respectively. Overexpressing miR-214 results in the formation of fewer microcalcifications compared to non-transfected controls.
In a broader perspective, Chapter 4 describes the role of non-coding RNAs in cardiac regeneration, the repair of cardiac tissue, which in turn enhances or restores the functional capabilities of the heart. Specifically, it studies the role of non-coding RNAs in species with inherent cardiac regenerative capacity to uncover and understand the mechanisms that drive cardiac regeneration, such as cardiomyocyte proliferation and neovascularization. Elaborating on the regenerative capacity in lower vertebrates and rodents and their role as scientific models aids in comprehending the role of non-coding RNAs in cardiomyocyte proliferation and neovascularization. In order to use these non-coding RNAs for therapeutic purposes, reliable delivery strategies need to be developed. Chapter 5 describes how a drug delivery system is engineered to deliver microRNA therapeutics locally that are released over an extended period of time. An injectable, self-healing hydrogel is designed to release microRNA therapeutics coupled to gold nanoparticles. The interaction of a polymer chain with a nanoparticle gives this gel shear-thinning and self-assembling properties, making it suitable for injection. The gold nanoparticles are functionalized with a polymer for stability, a peptide to facilitate cellular uptake, and microRNA therapeutics. The gold nanoparticle-coupled miRNAs were released from the hydrogel over the course of several days, proven functional in an in vitro assay, and shown to infiltrate the cells in the 3D valve disease model. Furthermore, biodistribution of the gold nanoparticle-coupled miRNA after subcutaneous injection of the loaded hydrogel in mice showed hepatic and renal clearance.
The studies presented in this thesis are summarized in Chapter 6, discussing the major findings and future directions.
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