Features over a wide range of length scales affect the biological response to a surface. While the influence of micro-features has been extensively studied, the effect of nano-features has only rarely been systematically investigated. We have developed a simple method to produce nano-featured gradients by kinetically controlled adsorption of negatively charged silica nanoparticles onto positively charged, poly(ethylene imine) (PEI)-coated silicon wafers. Subsequent sintering of the particles allowed a tuning of the particle morphology and resulted in a firm anchoring of the particles to the surface. Particle-density gradients were characterized by atomic force microscopy (AFM). Cell experiments with rat calvarial osteoblasts (RCO) on nano-featured gradients exhibited a significant decrease in proliferation at locations with higher particle coverage. Seven days post seeding, the number of osteoblasts was eight times higher at positions without particles compared to positions with maximum particle coverage. While cells spread well and developed a well-organized actin network in the absence of particles, spreading and formation of a strong actin network was considerably hindered at locations with maximum particle density.