Layer-by-Layer biomimetic nanocoatings for 3D-printed scaffolds: a novel approach for cardiac disease models and cardiotoxicity screening platforms

The constant exposure to toxic substances in the environment may be correlated to the increase in cardiac pathological conditions, such as cardiac fibrosis or cardiovascular diseases (CVDs), that usually affect the elderly population. Nowadays there is a strong pre-clinical research’s dependence on animal models or 2D human in-vitro models, both of which present significant differences from human real-case scenarios. A new approach is represented by 3D in-vitro models that allow for the use of human cells, higher throughput, tunability of cell composition and tridimensional environment. In collaboration with H2020 Project ALTERNATIVE, the aim of the work is the development of a cardiotoxicity screening platform of industrial chemicals and pharmaceuticals, focusing most of all on the effects on the elderly population. The model of aged cardiac tissue has been obtained through a biomimetic nanostructured surface coating for 3D printed scaffolds. The coating aims to recreate the composition of cardiac extracellular matrix (ECM) and it is built via Layer-by-Layer (LbL) self-assembly of oppositely charged polyelectrolytes. Specifically, poly(𝜀-caprolactone) (PCL) is chosen as substrate, being a synthetic FDA-approved biocompatible, semi-crystalline and biodegradable polyester, with low degradation rate and tunable properties, while the polyelectrolytes are cardiac ECM components Collagen, in particular type I (Coll1), and Hyaluronic Acid (HA). Initial surface charge was achieved through both plasma polymerization of 3-aminopropyl-triethoxysilane (APTES) and aminolysis, allowing for the exposure of amino groups (-NH2) on the substrate. After pre-treatment optimization, a 6-bilayer LbL coating was deposited testing both dipping and spraying LbL techniques and a crosslinking strategy between layers using genipin (GP) was also investigated. The study of the layers deposition was carried out using Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). Both crosslinked and non-crosslinked coatings were extensively characterized in terms of wettability, chemical composition, topography, roughness and polyelectrolytes release. Results confirmed a slower and inconsistent release when GP is involved, so cross-linking was discarded to allow quick recruitment of cells and new ECM deposition. A second LbL configuration including cardiac ECM protein fibronectin (FN) in the last three Coll1 layers was also investigated. Both LbL coating configurations (Coll1/HA and Coll1_FN/HA) were finally biologically tested for cellular response, compared with uncoated PCL scaffolds, the coated ones showed better results in terms of cell viability, and cellular colonization. These results suggest that the LbL coatings enhance the scaffold’s biocompatibility and their support role for cellular functions, underlining their potential for improved cardiac tissue engineering applications.