Abstract 19321: Novel 3D Microengineered Heart Valve Construct Mimics Early Calcific Aortic Valve Disease

Abstract

Objective: In calcific aortic valve disease (CAVD), evidence suggests that pro-inflammatory cues stimulate valvular interstitial cells (VIC) to undergo phenotypic modulation to osteoblast-like cells, leading to the formation of calcific nodules. However, the exact mechanism is unknown. We aim to engineer a novel 3D tissue model of CAVD to study the pro-inflammatory mechanisms of CAVD. This model uses a microengineered construct of natural ECM polymers of the native valve that mimics the extracellular matrix (ECM) environment of native tissue. Methods and Results: Porcine aortic VICs were encapsulated in photocrosslinkable hydrogels comprised of hyaluronic acid and gelatin methacrylate. These cell-laden 3D constructs were cultured in control (NM) (n=3) and osteogenic media (OM) (n=3), with (n=3) and without pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNFα) (10 ng/mL) for up to 21 days. VIC viability was more than 90% after 21 days in culture. In healthy valves VICs are characterized by minimally expressed α-Smooth Muscle Actin (α-SMA). Under pathological conditions, VICs differentiate into activated myofibroblasts with abundant expression of α-SMA. We showed that, when our heart valve constructs were cultured in OM, α-SMA expression gradually decreased while an increase of Runx2, a transcription factor upregulated in osteoblast-like cells, was observed. Constructs exposed to OM revealed a time-dependent induction of calcified noduli formation, where alkaline phosphatase (ALP) activity showed a peak at day 14 (d1: 0.45±0.11 vs d14: 1.96±0.11 vs d21: 0.88±0.07 U/mL (p <0.05)) and calcium deposition follows ALP activity and reaches a plateau at day 16 to day 21 (d1: 0.4 μ g/mL vs. d16 11.9 μ g/mL (p<00.5) vs d21: 10.6 μ g/mL (p >0.05)). Stimulation of heart valve constructs grown in OM with TNFα showed an increase in the amount of calcified noduli compared to without TNFα (d21: 69±7 vs 47±4 noduli (p<0.05)) and promoted Runx2 expression, suggesting a direct effect of pro-inflammatory cues on valvular calcification. Conclusions: Our novel in vitro 3D heart valve model simulates events that occur during early CAVD in vivo, in which pro-inflammatory mechanisms play an important role. This model provides a new platform to study mechanisms of CAVD.