Nanomaterials 2021, 11, 2724. https://doi.org/10.3390/nano11102724 www.mdpi.com/journal/nanomaterials Article Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells Luisa Severino Ulloa 1 , Fabio Perissinotto 1 , Ilaria Rago 1 , Andrea Goldoni 2 , Rosaria Santoro 3 , Maurizio Pesce 3 , Loredana Casalis 2, * and Denis Scaini 4,5, * 1 Dipartimento di Fisica, Università di Trieste, Piazzale Europa 1, 34127 Trieste, Italy; luisa.ulloaseverino@unityhealth.to (L.S.U.); fabio.perissinotto@inserm.fr (F.P.); ilaria.rago@uniroma1.it (I.R.) 2 Elettra-Sincrotrone Trieste S.C.p.A., Basovizza, 34149 Trieste, Italy; andrea.goldoni@elettra.eu 3 Unità di Ingegneria Tissutale Cardiovascolare, Centro Cardiologico Monzino, IRCCS, 20138 Milan, Italy; rosaria.santoro@cardiologicomonzino.it (R.S.); Maurizio.Pesce@cardiologicomonzino.it (M.P.) 4 Scuola Internazionale Superiore di Studi Avanzati, via Bonomea 265, 34136 Trieste, Italy 5 Faculty of Medicine, Imperial College London, London W12 0NN, UK * Correspondence: loredana.casalis@elettra.eu (L.C.); d.scaini@imperial.ac.uk (D.S.) Abstract: The quest for surfaces able to interface cells and modulate their functionality has raised, in recent years, the development of biomaterials endowed with nanocues capable of mimicking the natural extracellular matrix (ECM), especially for tissue regeneration purposes. In this context, car- bon nanotubes (CNTs) are optimal candidates, showing dimensions and a morphology comparable to fibril ECM constituents. Moreover, when immobilized onto surfaces, they demonstrated out- standing cytocompatibility and ease of chemical modification with ad hoc functionalities. In this study, we interface porcine aortic valve interstitial cells (pVICs) to multi-walled carbon nanotube (MWNT) carpets, investigating the impact of surface nano-morphology on cell properties. The re- sults obtained indicate that CNTs significantly affect cell behavior in terms of cell morphology, cy- toskeleton organization, and mechanical properties. We discovered that CNT carpets appear to maintain interfaced pVICs in a sort of “quiescent state”, hampering cell activation into a myofibro- blasts-like phenotype morphology, a cellular evolution prodromal to Calcific Aortic Valve Disease (CAVD) and characterized by valve interstitial tissue stiffening. We found that this phenomenon is linked to CNTs’ ability to alter cell tensional homeostasis, interacting with cell plasma membranes, stabilizing focal adhesions and enabling a better strain distribution within cells. Our discovery con- tributes to shedding new light on the ECM contribution in modulating cell behavior and will open the door to new criteria for designing nanostructured scaffolds to drive cell functionality for tissue engineering applications. Keywords: carbon nanotubes; chemical vapor deposition; nanomorphology; valve interstitial cells; cell membrane 1. Introduction Among all aortic valve diseases widely diffuse in the Western world, Calcific Aortic Valve Disease (CAVD) is the main cause of aortic stenosis and represents a major healthcare burden [1]. In adults, the aortic valve (AOV) is predominately composed of two types of cells: valve endothelial cells (VECs) and valve interstitial cells (VICs). CAVD is a sclerotic process that goes together with a phenotypic modification of VICs. In healthy aortic valves, VICs are a heterogeneous population constituted mostly of smooth muscle cells and fibroblasts, with a small percentage of myofibroblasts (about 5%) [2–4]. It has been shown that in the CAVD pathological condition, the fraction of myofibroblasts rises within the overall cellular population (up to 30%) [4–6]. The disease is also associated with changes in the composition, organization, and mechanical properties of the extracellular Citation: Ulloa, L.S.; Perissinotto, F.; Rago, I.; Goldoni, A.; Santoro, R.; Pesce, M.; Casalis, L.; Scaini, D. Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells. Nanomaterials 2021, 11, 2724. https://doi.org/10.3390/ nano11102724 Academic Editor: Joseph Kost Received: 27 August 2021 Accepted: 12 October 2021 Published: 15 October 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (http://crea- tivecommons.org/licenses/by/4.0/).