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Graphene- and hexagonal boron-nitride-based in-vitro cell-culturing surfaces alter morphological properties of neonatal spiral ganglion neurons

A new generation of neural interfaces based on novel graphene-based materials is expected to address limitations of low spatial resolution and broad specificity of neuronal stimulation in current cochlear implant devices leading to poor pitch perception and significant variability in its efficacy. Graphene and other two-dimensional (2D) materials possess an array of properties, making them an attractive candidate for future cochlear implants. It was previously shown that graphene might support neural growth due to unique electric properties, biocompatibility with neuronal cells, and its capacity to interface with neuronal tissue effectively. Recent studies of hexagonal boron nitride (hBN) showed promising applications in neuroengineering. However, it is not known how graphene or hBN interacts with spiral ganglion neurons (SGN) morphology. In this study, three types of graphene-based substrates (single layer graphene - SLG, hydrogenated graphene - HG, and fluorinated graphene - FG) as well as single-layer hexagonal boron nitride - SLhBN, were used as a surface for in-vitro culturing of neonatal SGN extracted from rat pups. The cultures were immunocytochemically stained at eight days in vitro (8DIV), and the subsequent fluorescence images were analyzed with the custom-made machine learning- based image processing allowing automatic identification and segmentation of neurons. We compared morphological properties of neuronal cultures grown on graphene-based and single-layer h-BN surfaces with those grown on glass coverslips. We found that neonatal SGN neurons may grow on all four tested substrates with significantly altered morphological properties compared to standard glass coverslips. Analysis with the mixed effect model of different morphological parameters data revealed that HG, SLG, and SLhBN substrates represented a more favorable environment for spiral ganglion neurons growth compared with neurons grown on control coverslips. These findings reveal substantial interaction between material properties of graphene- and hBNbased substrates on in-vitro SGN cell cultures.

Graphene substrates, Spiral ganglion neurons, Morphological properties

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