Interaction Of Graphene And WS₂ With Neural Cells, Neutrophils And Mesenchymal Stem Cells: Implications For Peripheral Nerve Regeneration

The employment of graphene and 2D materials in tissue engineering has been recently exploited for the repair and regeneration of nerve tissue. Among the possible applications, these relatively new materials display a great potential as peripheral neural interfaces, especially for their unique combination of electrical, optical and tribological properties^1-4.<br/><br/>In this work we present recent results on graphene (G) and tungsten disulfide (WS₂) interaction with some of the key players involved in nerve regeneration, namely peripheral neurons, neutrophils and mesenchymal stem cells (MSCs).<br/><br/>Firstly, we will discuss the effect of G on primary peripheral neurons, i.e. neonatal dorsal root ganglions (DRG) neurons. Graphene obtained via chemical vapor deposition (CVD), a promising candidate to realize functional nerve conduits, was used as a culture substrate. There, axons were found to be significantly longer (up to &gt;70%) with respect to controls during the early developmental phase (up to 2 days), confirming the trend previously reported by our group for embryonic DRG neurons on G on silicon carbide (SiC)⁵. Notably, with the same time-dependent fashion, we found a significative modification of the axonal transport of nerve growth factor (NGF), the neurotrophin involved in the development of peripheral neurons. The amount of retrogradely moving vesicles was strongly reduced in favor of a locally stalled population in the first two days of culture⁶. In addition, electrophysiological and ultrastructural analysis concurred to show the rearrangements that occur in axons developing on G⁶. Our results provide a broad structural and functional understanding of the impact of G on DRG neurons, key information toward the development of G-based devices for neural regeneration. Similarly, we tested WS₂, another 2D material with promising biomedical applications. CVD-grown WS₂, known for its optoelectronic properties, was used to culture neuronal-like cells (SH-SY5Y) displaying neurite length and viability comparable to the controls⁴.<br/><br/>However, the use of these innovative materials has raised questions about their interaction with the other players involved in nerve regeneration, including neutrophils, whose immune response affects the regenerative outcome⁷, and MSCs, a novel therapeutic avenue for peripheral nerve regeneration⁸.<br/><br/>Therefore, in the third part of the study we tested WS₂ on sapphire and different graphene, namely G on sapphire, G on SiC, both as-grown and H-intercalated, G-CVD transferred on glass, to assess the effect of the production method on neutrophils and MSCs⁹.<br/>We first characterized neutrophil activation and discussed how material properties influenced the NETs production and adhesion to the substrates. Furthermore, G resistance to NETs-induced degradation was carefully investigated and compared with the results reported for graphene oxide¹⁰. Ultimately, 2D materials cytocompatibility for MSCs are tested to estimate cell viability, morphology and mitochondrial health.<br/><br/>Overall, our results aimed at understanding the interface between 2D material and some of the key players involved in nerve injury, with special attention to the molecular mechanisms that govern axon outgrowth in neurons, a critical point for regenerative medicine.<br/><br/>References<br/>¹Shin et al., Adv. Drug Deliv. Rev. 105: 255–74 (2016)<br/>²Qian et al., NPJ Regen. Med. 6 (31) (2021)<br/>³Bramini et al., Front. Syst. Neurosci. 12 (Apr): 1–22 (2018)<br/>⁴Convertino et al., Front. Neurosci. 14 (Oct): 1–10 (2020)<br/>⁵Convertino et al., Front. Neurosci. 12 (Jan): 1–8 (2018)<br/>⁶Convertino et al., Nano Lett. 20: 3633–41 (2020)<br/>⁷Keshavan et al., Cell Death Dis. 10 (569) (2019)<br/>⁸Lavorato et al Int. J. Mol. Sci. 22 (572) (2021)<br/>⁹Convertino et al., In preparation<br/>¹⁰Huang et al., Adv. Healthc. Mater. 11 (2102439) (2022)<br/><br/>We acknowledge: the European Union's Horizon 2020 research and innovation programme_GA 881603; the Next Generation EU project ECS00000017 (THE); the University of Pisa under PRA 2020-2021 92.