The physiological functions of specific areas of the nervous system, such as the spinal cord, are directional dependent, relying on the ordered arrangement of cells and extracellular matrix. Here, an automated electrospinning method is used to make fibers with different diameters by changing the concentrations of polycaprolactone/gelatine solutions. The impact of fiber diameter, orientation, and the inclusion of superparamagnetic iron oxide nanoparticles on neural stem cell (NSC) behavior and differentiation is investigated. Fibers with an average diameter of 1189 ± 274 nm greatly improve neuronal differentiation. The highest relative expression of the neuronal marker Tuj‐1 and the formation of interconnected neuronal networks with neurites aligned along the fiber axis demonstrate this. Incorporating SPIONS into the fibers do not damage the cells, and aligned fibers do much better than random fibers at cell proliferation, migration, and neurite alignment. Further, magnetoresponsive fiber‐based hydrogels are fabricated by embedding SPIONS‐loaded fibers within a collagen matrix, which enables remote alignment of the fibers via a magnetic field. Postcrosslinking maintains this alignment and induces significant neurite orientation within the hydrogels. The potential use of automated, magnetically responsive electrospun fibers as flexible supports for NSC differentiation and neural tissue engineering guidance is demonstrated.
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