Developing a Three-Dimensional Type-1 Collagen-based Construct Model of Valve Calcification

Abstract

Introduction: Calcific aortic valve disease (CAVD) is a condition characterized by mineralization and fibrosis of the aortic valve leaflets, causing cardiac insufficiency if the diseased valve is not replaced. Current research models of CAVD include two-dimensional cell culture models of calcification. However, two-dimensional models lack the biological, mechanical, and biochemical properties of the native tissue. In this study, we developed a three-dimensional model of valvular calcification using valvular interstitial cells (VIC) seeded in collagen type I constructs. We treated the VIC constructs with osteochondrogenic conditions. We hypothesize that treating VIC collagen constructs with osteogenic stimuli promotes osteogenic differentiation and mineral deposition, better modeling disease conditions, as measured by calcium deposition and gene expression changes of VICs. Methods: Non-human primate VICs (BVIC) were seeded in collagen constructs and treated with DMEM (4.5 g/L D-glucose) in control [1 mM inorganic phosphate (Pi)] or osteogenic [2.8 mM Pi] media. Collagen constructs were lyophilized and rehydrated in 0.6 M HCl to extract calcium or emulsified using a tissue homogenizer to extract the RNA from samples. Gene expression was quantified through qPCR analysis after RNA collection from two gels combined. Three seeding densities of human valvular endothelial cells (hVECs) were tested and validated using live/dead staining to determine the optimal seeding density for a co-culture model of valvular calcification. BVICs and hVECs were treated with varying BVIC and hVEC media compositions. Results: There was little significant difference between the calcification of 125,000 and 250,000 cell densities of BVIC-seeded gels treated in osteogenic conditions for 7 days. Osteogenic treatment of BVIC-seeded collagen constructs for 7 days showed no significant difference in SOX9 and αSMA expression compared to control media only. The hVEC seeding density of 380,000 cells had the greatest cell adhesion and viability 24 hours after seeding the hVEC monolayer on collagen constructs. hVECs and BVICs treated in 50% BVIC and 50% hVEC media had the greatest cell viability after 7 days of treatment. Conclusion: Overall, we found that significant calcification of BVIC-seeded collagen constructs with 125,000 cells is measured at 7 days of osteogenic treatment, but additional studies are needed to determine the treatment time point for optimal calcification. Additionally, 50% BVIC and 50% hVEC media would best support both cell types in a future co-culture collagen construct model of valvular calcification.