Development of Polycaprolactone/Polyaniline Conductive Nanofibrous Scaffolds for Enhancing Neural Tissue Growth and Maturation

There is an increasing prevalence of neuropathologies resulting from traumatic brain injury, as well as neurodegenerative diseases (ND) such as Alzheimer’s, Parkinson’s, Huntington’s, particularly among elderly patients. The nervous system regeneration necessitates the repair or replacement of damaged nerve cells which, however, exhibit limited renewal capacity, and the complex nature of NDs hinders the development of effective treatments. Limitations of standard 2D cell cultures, as well as in vivo animal models, can be overcome using 3D cultures and co-cultures such as cell spheroids, organoids and organs-on-chip, by enabling the study of cell-cell and cell–extracellular matrix (ECM) interaction and replication of brain functionality. Moreover, tissue-engineered models represent another promising approach for replicating cell structure and orientation allowing for the development of oriented co-cultures. Therefore, realizing novel in vitro models with greater physiological relevance may bridge the gap between current preclinical models and humans. In this project, an in vitro model replicating neural and glial cells behaviour was developed to study cell regeneration and interaction. This model consists of an electrospun nanofibrous scaffold made of a blend of polycaprolactone (PCL) and polyaniline (PANI), one of the main conductive polymers used in the biomedical field. The electrospun mats were treated with cold plasma, above which neural stem cells (NE4C) were seeded and treated with 10-6 M retinoic acid to enhance differentiation. Both random (RND) and aligned (ALG) nanofibers were electrospun for membrane characterization tests. The plasma-treated fibrous mats showed higher hydrophilicity than non-treated ones, thus favouring cell adhesion. Tensile stress tests revealed that ALG fibers stressed along the alignment direction exhibit higher stiffness than RND, while the ALG fibers’ stiffness decreases drastically if stressed perpendicularly; this validates our model's anisotropic properties, aiming at mimicking the native extracellular matrix. Moreover, PCL/PANI samples were found to have higher conductivity than PCL ones, corroborating the role of PANI as a conductive polymer. In vitro tests confirm the viability of NE4C cultured on the plasma-treated PCL/PANI scaffolds and show differentiation into neuronal phenotype (at day 7) and glial astrocytic phenotype (at day 14). ALG nanofibers allow both NE4C and dorsal root ganglion cells differentiation and neurite outgrowth along the same direction as fiber orientation, boosting the growth of astrocytes as well. The conductivity of the substrate helps neuronal regeneration by conveying the electrical impulse, which can be endogenous or provided externally. In this regard, preliminary dynamic culture was performed inside the IVTech bioreactor LiveBox1, able to provide an electrical stimulus to the scaffold. The dynamic culture showed positive cell viability, as well as differentiation into neural and astrocytic phenotypes. These preliminary results hold promise for developing platforms for neural regeneration and disease modeling with even more biomimetic properties, offering an important strategy to characterize disease mechanisms leading to the discovery of new therapies.