Myelination of Dorsal Root Ganglia Neurons Cocultured with Schwann Cells in a Three-Dimensional Tissue-Engineered Model

Investigating axonal myelination by Schwann cells (SCs) in three-dimensional (3D) tissue constructs is crucial to understand mechanisms underlying demyelination and remyelination, helping to gain insights into incurable disorders like neurodegenerative diseases. The existing 3D tissue models have shown a limited performance to permit visualization of direct between SCs and individual axons. In this study, a biocompatible gelatin-based hydrogel, gelatin methacrylate (GelMA), was refined and optimized to achieve the biocompatibility, porosity, mechanical stability, and degradability needed to provide high cell viability for dorsal root ganglia (DRG) neurons and SCs and to enable their long-term culture needed for myelination studies. The results of cell viability, neurite elongation, SC function and maturation, SC-axon interaction, and myelination were compared to the two other commonly used substrates, namely collagen and Poly-D Lysine (PDL). The tuned GelMA constructs enhanced single axon generation (unlike collagen) and promoted the interaction of DRG neurons and SCs (unlike PDL). Further adjusting the hydrogel properties to achieve two distinct ranges of relatively small and large pores supported SCs to extend their processes freely and enable physical contact with and wrapping around their corresponding axons. Staining the cells with myelin basic protein (MBA) and myelin-associated glycoprotein (MAG) revealed the myelination process on GelMA hydrogels. Moreover, the engineered porosity enhanced DRGs and SCs attachments and flexibility of movement across the substrate. Finally, various cellular mechanisms related to the superiority of GelMA properties for myelination studies were discussed. This engineered GelMA 3D structure can be further used to model demyelination in neurodegenerative diseases and study effect of various drug compounds on myelin regeneration.