Integration of Graphene with Chitosan Membranes for Peripheral Nerve Regeneration

The employment of graphene in tissue engineering has been recently exploited for the repair and regeneration of nerve tissue. Among the possible applications, graphene-based materials display a great potential as peripheral neural interfaces, especially for their unique electrical, optical and physical properties^1–4.<br/>The integration of graphene with flexible biocompatible substrates represents a central point for the development of high-quality graphene-based nerve conduits. Many graphene-based nerve conduits use graphene flakes, however, the use of chemical vapor deposition (CVD) graphene would be promising due to its higher resistance to degradation, that avoids flakes release in the bloodstream⁵.<br/>In this work we present recent results on graphene (G) integration with flexible supports made of chitosan and glycerol-blended chitosan^6,7.The membranes were prepared in their isotropic flat form (FLAT) and with directional micro-topographies presenting different levels of symmetry [gratings (GR) and scalene triangles (SCA)]. We discuss the challenges encountered in CVD graphene integration with these supports. Different transfer techniques were tested to choose the best one in terms of yield of number of good samples, focusing mainly on graphene continuity. The micropattern and graphene integration was assessed using optical microscopy and Raman spectroscopy.<br/>Then we report preliminary results on the scaffolds’ interaction with a glial Schwann cell (SC) line <i>in vitro</i>. We performed adhesion and single cell migration experiments, observing that the presence of graphene reduced the guidance effect of the patterns, acting like a masking layer that impedes the cells from feeling the pattern. However, the interaction of SCs with scaffolds was highly boosted by graphene, inducing an increased adhesion in the graphene coated substrates.<br/><br/>Overall, our results aimed at understanding the possibility to integrate graphene with micro-structured polymeric membranes. Additional efforts have to be made to optimize the transfer process in order to obtain a repeatable transfer of high-quality large-scale CVD graphene. The substrates will be then tested to study their effects on the different players involved in nerve regeneration.<br/><br/>Keywords: Graphene, chitosan, scaffold, nerve regeneration, graphene-cell interaction<br/><br/>References<br/>1. Shin, S. R. <i>et al.</i> Graphene-based materials for tissue engineering. <i>Adv. Drug Deliv. Rev.</i> <b>105</b>, 255–274 (2016).<br/>2. Qian, Y. <i>et al.</i> Preclinical assessment on neuronal regeneration in the injury- related microenvironment of graphene-based scaffolds. <i>npj Regen. Med.</i> <b>6</b>, (2021).<br/>3. Convertino, D., Luin, S., Marchetti, L. & Coletti, C. Peripheral neuron survival and outgrowth on graphene. <i>Front. </i><i>Neurosci.</i> <b>12</b>, 1–8 (2018).<br/>4. Convertino, D., Trincavelli, M. L., Giacomelli, C., Marchetti, L. & Coletti, C. Graphene-based nanomaterials for peripheral nerve regeneration. <i>Front. Bioeng. Biotechnol.</i> <b>11</b>, 1–15 (2023).<br/>5. Huang, Q. <i>et al.</i> Aligned Graphene Mesh-Supported Double Network Natural Hydrogel Conduit Loaded with Netrin-1 for Peripheral Nerve Regeneration. <i>ACS Appl. Mater. Interfaces</i> <b>13</b>, 112–122 (2021).<br/>6. Scaccini, L. <i>et al.</i> Chitosan Micro-Grooved Membranes with Increased Asymmetry for the Improvement of the Schwann Cell Response in Nerve Regeneration. <i>Int. J. Mol. Sci.</i> <b>22</b>, (2021).<br/>7. Scaccini, L. <i>et al.</i> Glycerol-blended chitosan membranes with directional micro-grooves and reduced stiffness improve Schwann cell wound healing. <i>Biomed. Mater.</i> (2024).<br/><br/>We acknowledge: “Tuscany Health Ecosystem - THE” ECS00000017 – NextGenerationEU PNRR MUR - M4C2 –CUP J13C22000410001