Cellular Nanointerface of Vertical Nanostructures: Impact of Size‐Modulated Nanopillar Arrays on Neuronal Morphology, Maturation, and Synapse Formation

Investigating the cellular mechanisms facilitated by highly ordered nanostructures in vitro neuronal networks is crucial for advancing biomedical research. However, the intricate effects of these nanostructures on cellular behavior remain unclear. Herein, we explore how variations in nanopillar (NP) array dimensions, defined by the spacing‐to‐diameter ( S / D ) ratio, influence cortical neuronal responses—including cellular morphology, mechanosensing, neuronal maturation, and synapse formation. NP arrays with a low S / D ratio accelerate neurite protrusion and enhance neuronal maturation. These topography‐driven changes are attributed to the degree of cell confinement on NPs, which can be visualized using focused ion beam scanning microscopy. Furthermore, the synaptic density in cortical neurons declines as the S / D ratio decreases, with neurons preferring to form synapses along the NPs. By utilizing correlative light and electron microscopy, the spatial organization of these synapses is mapped relative to the NP geometries, revealing specialized synaptic regions with remarkable clarity. This approach to designing high‐aspect‐ratio nanostructures with various S / D ratios holds broad applications in materials engineering, offering insights into nervous system engineering and neural‐interfacing bioelectronics.