Filamented Hydrogels as Tunable Conduits for Guiding Neurite Outgrowth

Anisotropic scaffolds with unidirectionally aligned fibers present an optimal solution for nerve tissue engineering and graft repair. This study investigates the application of filamented light (FLight) biofabrication to create hydrogel matrices featuring highly aligned microfilaments, facilitating neurite guidance and outgrowth from encapsulated chicken dorsal root ganglion (DRG) cells. FLight employs optical modulation instability (OMI) to rapidly and safely (<5 s) fabricate hydrogel constructs with precise microfilament alignment. The tunability of FLight matrices was demonstrated by adjusting four key parameters: stiffness, porosity, growth factor release, and incorporation of biological cues. Matrix stiffness was fine-tuned by varying the projection light dose, yielding matrices with stiffness ranging from 0.6 to 5.7 kPa. Optimal neurite outgrowth occurred at a stiffness of 0.6 kPa, achieving an outgrowth of 2.5 mm over 4 days. Matrix porosity was modified using diffraction gratings in the optical setup. While significant differences in neurite outgrowth and alignment were observed between bulk and FLight gels, further increases in porosity from 40 % to 70 % enhanced cell migration and axon bundling without significantly affecting maximal outgrowth. The incorporation of protein microcrystals containing nerve growth factor (NGF) into the photoresin enabled sustained neurite outgrowth without the need for additional NGF in the media. Finally, laminin was added to the resin to enhance the bioactivity of the biomaterial, resulting in a further increase in maximum neurite outgrowth to 3.5 mm after 4 days of culture in softer matrices. Overall, the varied matrix properties achieved through FLight significantly enhance neurite outgrowth, highlighting the importance of adaptable scaffold characteristics for guiding neurite development. This demonstrates the potential of FLight as a versatile platform for creating ideal matrices for clinical applications in nerve repair and tissue engineering.