Neuron Viability and Maturation on Electroactive Materials and Effect of Electroactive Stimulation on Neural Cell Response

Electric cues are intimately related connected to physiological functions of cells and tissues. The ability to control and direct electric signals within tissue-engineered constructs is therefore increasingly important for proper tissue engineering strategies, as they influence cell behaviour and guide tissue development towards more functional and biomimetic outcomes. IN this context, smart materials, able to react in a controllable and reversible way to external stimuli by varying a specific physical or chemical quantity, show great potential for the development of advanced biomedical strategies, including biosensing, tissue regeneration and repair, immuno- and cancer therapy. Among the different types of smart materials, piezoelectric ones, that convert mechanical solicitations into electrical potential variations and vice versa, represent as suitable candidates to induce specific cell behaviors through electric cues, mainly by improving biomimicry of the cell microenvironment.<br/>The presence of piezoelectric properties in different tissues is known and the effect of surface charge and piezoelectric stimulation has been proven to be beneficial in a variety of cells, including mesenchymal stem cells, myoblast and osteoblasts, among others, has been evaluated,<br/>Since neurons were first cultured outside a living organism more than a century ago, a number of experimental techniques for their <i>in vitro</i> maintenance have been developed. These methods have been further adapted and refined to study specific neurobiological processes under controlled experimental conditions. Nevertheless, the impact of fundamental properties of the surfaces to which neurons adhere when cultured <i>in vitro</i> has not been sufficiently considered.<br/><br/>In the present work we have analyzed the response of primary neurons when cultured on piezoelectric poly(vinylidene fluoride), PVDF, surfaces with different surface electric charge and electric charge type, allowing to stablish the effect of the net charge on neuron behavior. It is shown that PVDF films promote the attachment, viability and maturation of primary neurons. More cells per unit area and longer neurites were already present in neurons cultured for one day on PVDF surfaces. Early neurite outgrowth was induced using poled PVDF as culture substrates, since by day 4 of culture neurons already show significantly increased neural process development, and by day 7, this development is even more obvious in neurons cultured on PVDF–. Negative surface charges particularly increased cell metabolism, being about 3 times higher than in control and around 1.7 times higher than in the absence of charge (PVDF NP). When adhering to PVDF–, neurons undergo maturation at a faster rate, as revealed by the higher expression levels of MAP2 and NeuN.<br/>Further, the effect of surface charged PVDF films (positive, negative, and non-poled (average neutral surface charge)) on adhesion, proliferation and differentiation of neural-derived cell line, under static and dynamic mechanoelectrical stimulation was assessed.<br/>Upon mechanoelectrical stimulation, PVDF with average positive surface charge allows to significantly improve cellular adhesion compared to PVDF with negative or without average surface charges. Independently of the PVDF surface charge type, the applied electrical stimulus throughout the assay improved neurite extension and differentiation.<br/>The obtained results highlight the importance of considering the electric charge of the culture surface for the in vitro maintenance of neurons, demonstrating the suitability of the use of 2D PVDF based surface-charged substrates. Further, these findings highlight the possibility of novel therapeutic strategies for the regeneration of neural tissues based on the dynamically surface charge variation that can be induced in the electroactive films.