A novel approach was developed using PDMS-substrates with surface-aligned nanotopography gradients, varying unidirectional in amplitude and wavelength, for studying cell behavior with regard to adhesion and alignment. The gradients target more surface feature parameters simultaneously and provide more information with fewer experiments and are therefore vastly superior with respect to individual topography substrates. Cellular adhesion experiments on non-gradient aligned nanowrinkled surfaces displayed a linear relationship of osteoblast cell adhesion with respect to topography aspect ratio. Additionally, an aspect ratio of 0.25 was found to be most efficient for cell alignment. Modification of the surface preparation method allowed us to develop an approach for creating surface nanotopography gradients which innovatively provided a superior data collection with fewer experiments showing that 1) low amplitude with small wavenumber is best for osteoblast cell adhesion 2) indeed higher aspect ratios are favorable for alignment however only with features between 80–180 nm in amplitude and 450–750 nm in wavelength with a clear transition between adhesion and alignment efficiency and 3) disproved a linear relationship of cell adhesion towards aspect ratio as was found for single feature substrate analysis.
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