Heterogeneous multi-laminar tissue constructs as a platform to evaluate aortic valve matrix-dependent pathogenicity

Designing and constructing controlled in vitro cell culture platforms is imperative toward pinpointing factors that contribute to the development of calcific aortic valve disease. A 3D, laminar, filter paper-based cell culture system that was previously established as a method of analyzing valvular interstitial cell migration and protein expression was adapted here for studying the impact of specific extracellular matrix proteins on cellular viability and calcification proclivity. Hydrogels incorporating hyaluronan and collagen I, two prevalent valvular extracellular matrix proteins with altered pathological production, were designed with similar mechanics to parse out effects of the individual proteins on cell behavior. Laminar constructs containing varying combinations of discrete layers of collagen and hyaluronan were assembled to mimic native and pathological valve compositions. Proteinaceous and genetic expression patterns pertaining to cell viability and calcific potential were quantified via fluorescent imaging. A significant dose-dependency was observed, with increased collagen content associated with decreased viability and increased calcific phenotype. These results suggest that extracellular composition is influential in calcific aortic valve disease progression and will be key toward development of future tissue-engineered or pharmaceutical calcific aortic valve treatments. STATEMENT OF SIGNIFICANCE: Calcific aortic valve disease (CAVD), a widespread heart valve disorder, is characterized by fibrotic leaflet thickening and calcific nodule formation. This pathological remodeling is an active process mediated by the valvular interstitial cells (VICs). Currently, the only treatment available is surgical replacement of the valve - a procedure associated with significant long-term risk and morbidity. Development of effective alternate therapies is hindered by our poor understanding of CAVD etiology. Previous work has implicated the composition and mechanics of the extracellular matrix in the progression of CAVD. These individual factors and their magnitude of influence have not been extensively explored - particularly in 3D systems. Here, we have bridged this gap in understanding through the employment of a heterogeneous 3D filter-paper culture system.