Synthetic Hydrogels Reveal the Role of the Matrix Microenvironment on Aortic Valve Calcification

Abstract

Objective: Calcific aortic valve disease (CAVD) affects 2.5% of the US population over 75 and there remain no therapeutics, other than surgical replacement, to prevent or reverse disease progression. The aim of this study was to better understand the role of the matrix microenvironment in promoting or suppressing calcification, specifically with respect to the role that valvular interstitial cells (VICs) play in CAVD. Methods: We designed and characterized a hydrogel system for 3D VIC culture including proteolytically degradable crosslinkers and studied the evolution of VIC osteogenic markers and matrix mineralization in response to osteogenic stimuli. Further, type I collagen was introduced as a secondary interpenetrating network, a component known to be involved in mineralization processes. Results: VIC phenotype was characterized by measuring gene and protein expression for early and late osteogenic markers, RUNX2 and osteocalcin, respectively, as well as fibroblast marker vimentin. As a screening tool, increased optical density (600 nm) corresponded to increased matrix mineralization as early as 12 days. At selected time points, samples were evaluated histologically, and with immunofluorescent imaging for key proteins and colorimetric calcium assays. Finally, we screened for drug therapeutics that might prevent matrix mineralization in VIC-laden gels and identified zoledronic acid, a bisphosphonate, as effective in reducing mineralization by 86%. Conclusions: These results highlight the value of in vitro culture platforms to study VICs in an environment that more closely recapitulates the in vivo valve microenvironment and to identify potential pharmacological factors to prevent or reverse calcification that occurs in CAVD.