Engineering a 3D-Bioprinted Model of Human Heart Valve Disease Using Nanoindentation-Based Biomechanics

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.

Impact of modified gelatin on valvular microtissues

“…Although many therapeutic targets have been identified, the mechanosensitive nature of VIC is a key limiting factor in current in vitro platforms for high throughput disease modeling or drug discovery, which are based on 2D cell culture tissue culture polystyrene with high nonphysiological stiffness (van der Valk et al, 2018). In vivo, the majority of VICs are in a quiescent state (qVIC) and have a fibroblast-like phenotype, low proliferation capacity, and minimal activity and express high vimentin and low alpha smooth muscle actin (α-SMA; Hinton et al, 2006;Rabkin-Aikawa, Farber, Aikawa, & Schoen, 2004;Rabkin et al, 2001).…”

“…In order to develop an in vitro platform that recapitulates key aspects of the valvular architecture and microenvironment and to reliably screen novel targets, microtissues need to be encapsulated in tunable hydrogels mimicking the layer-specific mechanical properties of heart valves (van der Valk et al, 2018) and allowing fusion of the microtissues into macrotissues.…”

“…In the past 10 years, several research groups have been evaluating polyethylene glycol, hyaluronic acid (HA), gelatin, and hybrid hydrogels as encapsulation material for VIC (Benton, DeForest, Vivekanandan, & Anseth, 2009;Chen, Srigunapalan, Wheeler, & Simmons, 2013;Kang et al, 2017;Masters, Shah, Leinwand, & Anseth, 2005). Encapsulated VICs in hybrid hydrogels are often the starting point for developing an organ on a chip to study physiological cardiovascular cell interactions Hjortnaes et al, 2015;van der Valk et al, 2018).…”

“…). These tunable hydrogel systems will be important for future 3D bioprinting of valvular tissues mimicking the layer-specific mechanical properties of heart valves(van der Valk et al, 2018). Above all, it is described in literature that soft hydrogels would lead to activation of qVIC into aVIC.…”

Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

“…Ultimately, our approach represents an efficient, cost-effective, and widely accessible strategy for studying the proteome and miRNAome of purified EVs isolated directly from fibro-calcific human, animal, or even engineered tissues. (8, 45)…”