Orthogonal nanometer-micrometer roughness gradients probe morphological influences on cell behavior

Surface gradients facilitate rapid, high-throughput, systematic investigations in cell biology, materials science, and other fields. An important surface parameter is the surface roughness on both the micrometer and nanometer scales in the lateral direction. Two approaches have been combined to create two-dimensional roughness gradients by adding a nanoparticle density gradient onto a gradient of micro-featured roughness. All fabricated gradients were extensively characterized by SEM, AFM and optical profilometry to ensure their quality and to determine the roughness parameter Ra along the gradient. Additionally, a Fourier-transform approach was applied that allows a wavelength-dependent analysis of the surface topography. Since cell-culture assays require replicate experiments, a replica technique was used to create copies of the master gradient. Creating a negative replica in an elastomeric material served as a mold for a subsequent ceramic-casting process. A positive replica was then formed from epoxy resin, which was subsequently coated with titanium and used for cell studies. Finally, these gradients were used in cell-culture assays to determine cellular response to surface roughness. The results clearly demonstrate the influence of surface roughness on the production by osteoblasts of markers for osteogenesis. It was shown that high roughness in the micrometer range, combined with an intermediate nanofeature density (30-40 features/μm2), leads to the highest degree of osteopontin production after 14 days.