Abstract
The development of novel three-dimensional (3D) nanomaterials combining high biocompatibility, precise mechanical characteristics, electrical conductivity, and controlled pore size to enable cell and nutrient permeation is highly sought after for cardiac tissue engineering applications including repair of damaged heart tissues following myocardial infarction and heart failure. Such unique characteristics can collectively be found in hybrid, highly porous tridimensional scaffolds based on chemically functionalized graphene oxide (GO). By exploiting the rich reactivity of the GO's basal epoxydic and edge carboxylate moieties when interacting, respectively, with NH2 and NH3+ groups of linear polyethylenimines (PEIs), 3D architectures with variable thickness and porosity can be manufactured, making use of the layer-by-layer technique through the subsequent dipping in GO and PEI aqueous solutions, thereby attaining enhanced compositional and structural control. The elasticity modulus of the hybrid material is found to depend on scaffold's thickness, with the lowest value of 13 GPa obtained in samples containing the highest number of alternating layers. Thanks to the amino-rich composition of the hybrid and the established biocompatibility of GO, the scaffolds do not exhibit cytotoxicity; they promote cardiac muscle HL-1 cell adhesion and growth without interfering with the cell morphology and increasing cardiac markers such as Connexin-43 and Nkx 2.5. Our novel strategy for scaffold preparation thus overcomes the drawbacks associated with the limited processability of pristine graphene and low GO conductivity, and it enables the production of biocompatible 3D GO scaffolds covalently functionalized with amino-based spacers, which is advantageous for cardiac tissue engineering applications. In particular, they displayed a significant increase in the number of gap junctions compared to HL-1 cultured on CTRL substrates, which render them key components for repairing damaged heart tissues as well as being used for 3D in vitro cardiac modeling investigations.