# Ion Beam Nanopatterning of Biomaterial Surfaces
## 3 Ion Beam-Nanopatterned Surfaces in Contact with Biomolecules and Cells
### Cell Response
#### 3.2.1 Mammalian Tissue Cells
Whereas the cell densities on untreated and 75\({}^{\circ}\)-irradiated surfaces were comparable, cell density on the 30\({}^{\circ}\)-irradiated surface was reduced by about 40%. Finally, [PERSON] et al. irradiated Ti6 Al4V surfaces with 300 eV He\({}^{+}\) ions under normal incidence by direct extraction from a plasma [129]. This resulted in surface morphologies composed of networks of pores uniformly distributed over the complete sample surfaces. Pore formation was attributed not to material removal by sputtering but rather to the formation of embedded He bubbles. By varying the applied ion current, the pore diameter could be adjusted in the range from a few hundred nanometers to several microns in diameter, with the smaller pores occurring in higher density than the larger ones. The influence of these porous surfaces on cellular response was again studied using human fetal osteoblasts. No remarkable differences in cell proliferation, cell density, and cell spacing were observed for irradiated and non-irradiated substrates. Cell viability assays, on the other hand, indicated that the irradiated surfaces displayed decreased initial cell adhesion. Once cells were established on the irradiated surfaces, however, cellular activity was increased again. Nanopatterned Ti6 Al4V surfaces were also produced by [PERSON] et al. using 1 keV Ar\({}^{+}\) irradiation at various incident angles [93]. At normal and oblique, non-grazing incidence, surface topographies similar to those described by [PERSON] et al. [126] were obtained, i.e., sub-100 nm ripple patterns with different orientations on different grains. At grazing incidence (80\({}^{\circ}\) with respect to the surface normal), however, the surface displayed a homogeneous pattern of ripples with about 50 nm periodicity oriented parallel to the ion beam direction that crossed different grains without a change in direction. Such patterns are frequently observed for grazing incidence irradiation of polycrystalline metal surfaces [127]. Human aortic smooth muscle cells (HASMCs) were cultured on these surfaces to evaluate the effect of the different surface topographies on cell viability, adhesion, and morphology. While no differences between the ion beam-irradiated and the untreated control surfaces with regard to cell viability could be established, larger cell spreading was observed for the nanopatterned surfaces. Most intriguingly, the authors found an increased filopodia density for HASMCs cultured on the surface irradiated under 30\({}^{\circ}\) incidence with respect to the surface normal (see Figure 7). All these findings indicate that the nanoscale surface to popgraphic features promote HASMC-surface interactions with cell filopodia preferentially attaching to nanorippled grains. Apart from titanium-based alloys, other materials have been nanopatterned as well to evaluate the effects on cell response. Alumina, for instance, is another material frequently used in orthopedic and dental implants [130]. To evaluate the effect of nanoscale surface topography on cellular response to alumina surfaces, [PERSON] et al. patterned the surfaces of Al\({}_{2}\)O\({}_{3}\)(0001) single crystals using 500 eV Ar\({}^{+}\) and 35 keV Xe\({}^{+}\) irradiation at 50\({}^{\circ}\) incidence with respect to the surface normal [89]. This resulted in ripple patterns with periodicities of 24 and 179 nm, respectively. Furthermore, these patterns had a very low aspect ratio with corresponding peak-to-peak amplitudes of only 0.7 and 11.5 nm, respectively. Cell viability and morphology in response to these nanopatterns as well as flat control substrates were evaluated using the human osteosarcoma cell line MG-63. For the larger ripple pattern with 179 nm periodicity, significantly enhanced proliferation of the MG-63 cells was observed. While this pattern did not induce any cell alignment, the SEM images shown in Figure 8 revealed preferential alignment of pseudopodia along the ripples.