Calcific Aortic Valve Disease is the most common form of valve disease in the Western world and represents a major healthcare burden. The primary driver for valvular calcification is the differentiation of valvular interstitial cells (VICs) into a diseased phenotype, the osteoblastic-like cells.
Moreover, the disease induces significant changes in the organization, composition and mechanical properties of the extracellular matrix that it seems to contribute to the progression of the pathology altering cellular molecular signaling. Cells interact with ECM through the focal adhesion points (FAs). The FA number and distribution are modulated by the extracellular matrix rigidity and determine the intracellular tensions. FAs contain ECM receptors, called integrins, and cytoplasmatic protein, such as vinculin, connecting the actin stress fibers to the integrins. Vinculin plays an important role in mechano-transduction and an accumulation of this in FAs has a role in intracellular tensions regulation and ECM stiffness.
In recent years, the quest for biocompatible (nano)materials capable of mimicking the natural ECM for tissue regeneration has increased. Carbon nanotubes (CNTs) are optimal candidate in this context, showing dimensions comparable to fibril ECM constituents and both in-vitro and in-vivo cellular biocompatibility. In the present study, we show the mechanical, morphological and molecular properties of VICs grown on transparent, randomly oriented, CNTs. Using Atomic Force Microscopy and immunofluorescence experiments we assessed the biomechanics, morphology and the pathway involved in the mechanical stress in onset of pathology. We observedthat cell density does not vary between VICs grown on CNts or glass controls, indicating that CNTs are not toxic for VICs. Interestingly a morphological variation between VICs grown on glass and on CNTs was pointed out. In particular we observed that, on CNTs, the percentage of myofibroblast (Mfib) diminishes significatively with respect to glass. Mfib are correlated with VICs differentiation into osteoblastic-like cells and, therefore, with a diseased phenotype. A variation in the mechanical properties of fibroblast grown on CNTs was measured via AFM force-spectroscopy experiments, in particular Mfib grown on CNTs are a softer than those on controls. No difference has been observed, instead, for smooth muscle cell phenotype on both substrates. Our results identified CNTs as a promising material to engineer novel artificial scaffolds for aortic valve regeneration.