Abstract
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.
Highlights
Calcific aortic valve disease (CAVD) is the most prevalent heart disease, affecting more than 1/4 of individuals over age 65 in the Western world [1]
The human AV is composed of three distinct layers with unique extracellular matrix (ECM) composition and valve interstitial cells (VICs) phenotypes [17]
To produce an in vitro model of human CAVD that was capable of recapitulating AV leaflet layer-specific mechanical properties, we employed a tunable hydrogel system that we had previously shown to be capable of maintaining porcine VIC quiescence under basal conditions in 3D [19]
Summary
Calcific aortic valve disease (CAVD) is the most prevalent heart disease, affecting more than 1/4 of individuals over age 65 in the Western world [1]. Despite this high prevalence and many risk factors shared with atherosclerosis, there are no pharmacological therapies for CAVD—aortic valve (AV) replacement currently is the sole treatment option. The onset and progression of CAVD is layer-specific: it preferentially initiates within the fibrosa and progresses to the spongiosa, while the ventricularis is largely unaffected until the final stages of disease [6,7]
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