3D Bioprinting of Graphene Oxide-Incorporated Hydrogels for Neural Tissue Regeneration

Hydrogels, especially natural hydrogels, have often been used for 3D bioprinting due to their numerous advantages, including excellent biocompatibility, biodegradability and similarity to natural extracellular matrix. However, natural hydrogels have shortcomings for 3D bioprinting, such as poor printability and very weak mechanical strength of printed structures. The incorporation of an additive (e.g., cellulose nanofibers, nanoclay particles, etc.) is an effective strategy to improve the performance (including mechanical performance) of natural hydrogels and may expand their potential applications in tissue engineering. The nervous system in human bodies is important as it regulates physiological functions and controls body activities. The native neural tissue is relatively soft and is sensitive to electrical stimulation. Therefore, materials used for neural tissue engineering should be soft and preferably have certain electrical conductivity. Owing to its good electron mobility and high surface-to-volume ratio, graphene oxidase (GO) is a promising material for creating electroactive hydrogels. Gelatin is a natural hydrogel obtained by denaturalization of collagen. It has intrinsic Arg-Gly-Asp (RGD) motifs and accessible active groups, which is desirable for tissue regeneration applications. However, gelatin alone is rarely used for 3D bioprinting because it is in the liquid state at 37 °C and hence cannot maintain the shape of a printed structure during its use in the body. Gelatin methacryloyl (GelMA) is a photocrosslinkable hydrogel that is derived from gelatin with the methacryloyl modification. After crosslinking, GelMA can maintain the shape of the printed structure. In this study, a natural hydrogel-based bioink (GO/Gelatin/GelMA) was developed by combining GO, gelatin, and GelMA, with the aim of obtaining a hydrogel with photocrosslinking ability, relatively soft nature, and improved electroactive properties. The performance of GO/Gelatin/GelMA composite hydrogels with different GO contents [0 ~ 0.1% (w/v)] was investigated in terms of microstructure, mechanical properties, degradation behavior, and water absorption properties. It was found that a low concentration of GO in the composite hydrogel had little effect on the microstructure of hydrogels, improved the mechanical strength slightly (but still at a kPa level), reduced the degradation rate, and increased water absorption. The electrical conductivity of GO/Gelatin/GelMA composite hydrogels was enhanced with the increasing GO contents. The printability of GO/Gelatin/GelMA composite hydrogels was studied by printing out multi-layered grid structures using a pneumatic extrusion-type 3D printer. It was observed that GO/Gelatin/GelMA hydrogels formed smooth filaments after extrusion and could be printed into 10-layer grids with good shape fidelity. After crosslinking, the printed grid structures could maintain their shapes and be held by hand without collapsing. Finally, neural cells (i.e., astrocytes) were added into GO/Gelatin/GelMA hydrogels to form bioinks for 3D bioprinting. It was found that astrocytes maintained high cell viability after bioprinting and grew well after culturing of the scaffolds for several days. The GO-incorporated gelatin/GelMA hydrogels have shown good biocompatibility, biodegradability, good printability, balanced softness and improved electroactivity, suggesting their high potential for 3D bioprinting, as well as applications in neural tissue regeneration.