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Biophysical parameters such as substrate topography and stiffness have been shown independently to elicit profound effects on neuronal differentiation and maturation from neural progenitor cells (NPCs) yet have not been investigated in combination. Here, the effects of various micrograting and stiffness combinations on neuronal differentiation and maturation were investigated using a polyacrylamide and N-acryloyl-6-aminocaproic acid copolymer (PAA-ACA) hydrogel with tunable stiffness. Whole laminin was conjugated onto the PAA-ACA surface indirectly or directly to facilitate long-term mouse and human NPC-derived neuron attachment. Three micrograting dimensions (2–10 µm) were patterned onto gels with varying stiffness (6.1–110.5 kPa) to evaluate the effects of topography, stiffness, and their interaction. The results demonstrate that the extracellular matrix (ECM)-modified PAA-ACA gels support mouse and human neuronal cell attachment throughout the differentiation and maturation stages (14 and 28 days, respectively). The interaction between topography and stiffness is shown to significantly increase the proportion of β-tubulin III (TUJ1) positive neurons and microtubule associated protein-2 (MAP2) positive neurite branching and length. Thus, the effects of topography and stiffness cannot be imparted. These results provide a novel platform for neural mechanobiology studies and emphasize the utility of optimizing numerous biophysical cues for improved neuronal yield in vitro.

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduit has been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfate polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 μm convex lenses, 2 μm gratings, and 10 μm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0 to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 μm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration.

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