In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.

Heart failure (HF) is a leading cause of death worldwide. The most common conditions that lead to HF are coronary artery disease, myocardial infarction, valve disorders, high blood pressure, and cardiomyopathy. Due to the limited regenerative capacity of the heart, the only curative therapy currently available is heart transplantation. Therefore, there is a great need for the development of novel regenerative strategies to repair the injured myocardium, replace damaged valves, and treat occluded coronary arteries. Recent advances in manufacturing technologies have resulted in the precise fabrication of 3D fiber scaffolds with high architectural control that can support and guide new tissue growth, opening exciting new avenues for repair of the human heart. This review discusses the recent advancements in the novel research field of fiber patterning manufacturing technologies for cardiac tissue engineering (cTE) and to what extent these technologies could meet the requirements of the highly organized and structured cardiac tissues. Additionally, future directions of these novel fiber patterning technologies, designs, and applicability to advance cTE are presented.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the Western population, claiming 17000 deaths per year in the United States and affecting 25% of people older than 65 years of age. Contrary to traditional belief, CAVD is not a passive, degenerative disease but rather a dynamic disease, where initial cellular changes in the valve leaflets progress into fibrotic lesions that induce valve thickening and calcification. Advanced thickening and calcification impair valve function and lead to aortic stenosis (AS). Without intervention, progressive ventricular hypertrophy ensues, which ultimately results in heart failure and death. Currently, aortic valve replacement (AVR), surgical or transcatheter, is the only effective therapy to treat CAVD. However, these costly interventions are often delayed until the late stages of the disease. Nonetheless, 275000 are performed per year worldwide, and this is expected to triple by 2050. Given the current landscape, next-generation therapies for CAVD are needed to improve patient outcome and quality of life. Here, we first provide a background on the aortic valve (AV) and the pathobiology of CAVD as well as highlight current directions and future outlook on the development of functional 3D models of CAVD in vitro. We then consider an often-overlooked aspect contributing to CAVD: miRNA (mis)regulation. Therapeutics could potentially normalize miRNA levels in the early stages of the disease and may slow its progression or even reverse calcification. We close with a discussion of strategies that would enable the use of miRNA as a therapeutic for CAVD. This focuses on an overview of controlled delivery technologies for nucleic acid therapeutics to the valve or other target tissues.

A novel injectable hydrogel drug delivery platform introduces miRNA therapeutics coupled to gold nanoparticles to cells in a 3D bioprinted heart valve disease model.

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In vitro 3D model and miRNA drug delivery to target calcific aortic valve disease Chapter 2Engineering a 3D-bioprinted model of human heart valve disease using nano-indentation based biomechanics Chapter 3MicroRNA-214 attenuates formation of microcalcifications in a three-dimensional model of human calcific aortic valve disease Chapter 4Controlled delivery of gold nanoparticle-coupled miRNA therapeutics via an injectable self-healing hydrogel Chapter 5Non-coding RNAs in cardiac regeneration Chapter 6Summarizing discussion and future perspectives