In the present study, we demonstrate a rapid and low-cost method, leveraging photolithographic and PDMS micromolding techniques, to design and prepare anisotropic, gradient micro-ridge/groove arrays with variable local pattern width. It was anticipated this study may demonstrate the ability of gradient micro-topographies to influence neuron adhesion, dendrite branching, and synapse formation, providing a comprehensive understanding of the cell-topography interaction for the design of neuroregenerative devices. The surface topography of biomaterials with specific spatial structure mimic the physical microenvironment of neurons and regulate their orientation, neurite outgrowth and synaptic elements. However, most topographical structures used in neuron culture are fabricated with single geometries. This necessitates bulk fabrication, which is both inefficient and laborious, while also increasing potential contamination. A single gradient micro-topographical structure with a range of patterns begins to ameliorate these issues, although few studies have examined the influence of longitudinal micro-ridge/groove structures. The physicochemical properties of the prepared gradient micro-ridge/groove arrays were characterized by analyzing the surface morphology and wettability. Cell experiments were subsequently carried out using primary rat hippocampal neurons. Following substrate fabrication, the patterning structure was confirmed to be intact and viable for cell contact. Additionally, cell adhesion was increased through surface treatment with poly-l-lysine and laminin, and verified through FTIR-ATR spectroscopy. Immunofluorescent images of neurons in culture for 14 days revealed smaller pattern widths regulate neuron growth and increase orientation along the linear direction. Neurons cultured on these substrates demonstrate a preference to attach in areas of smaller pattern width. Additionally, cell somas located within a 5μm groove demonstrate heightened aspect ratios. Dendrites of these neurons extended less when introduced to a linear topography, decreasing their coverage area with respect to decreasing geometry. Finally, the density of synapses formed significantly decreased in the presence of topography. Thus, the effect of gradient micro-topography on neuron behavior was achieved and systematically understood by a one-step screening on a single integrated chip, lending itself to potential advances in the design of neuroregenerative micro-devices.

Comprehensive screening of neuronal behavior on gradient micro-alignment topographies

256th National Meeting (2018)

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