# 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  
The first study to investigate cellular response to ion beam-nanopatterned surfaces employed high-aspect-ratio ripple patterns fabricated in a two-step process [124]. Here, periodic ripple patterns with periodicities between 60 and 140 nm were produced on Si surfaces by 1-8 keV nitrogen ion irradiation. This, however, resulted also in the formation of a thickness-modulated Si\({}_{\text{x}}\)N\({}_{\text{y}}\) top layer [74]. The thickness of this top layer was largest at the ripple slope facing the ion beam. Therefore, it could be used as a hard etch mask in a subsequent reactive ion etching (RIE) step to increase the depth of the ripple valleys to about 200 nm (see Figure 6a,b). Cellular response to the so-fabricated high-aspect-ratio nanopatterns was investigated using human corneal epithelial cells (HCEC). In standard epithelial growth medium, the authors observed HCEC contact guidance parallel to the ripples for periodicities of 80 and 140 nm but not for 60 nm. In serum-free EpiLife(r) medium, however, significant contact guidance occurred also at 60 nm periodicity. In addition, further analysis also revealed a stronger degree of HCEC alignment at the larger periodicities. It should also be noted that in this particular medium, the cells were aligned perpendicular to the ripples, which underscores the importance of soluble factors (see Figure 6c,d). Finally, it was demonstrated that HCEC contact guidance by lithographically produced micropatterns with 4 um pitch can be strongly enhanced by overlay with a 70 nm ripple pattern [124]. Figure 6: **(a,b)** SEM images of rippled substrates with 90 nm periodicity. **(a)** Top view. **(b)** Cross-sectional view. **(c,d)** Fluorescence images of HCEC showing **(c)** parallel alignment in epithelial growth medium and **(d)** perpendicular alignment in EpiLife® medium on 140 nm ripple patterns. Reprinted from Ref. [124] with permission from Elsevier. A subsequent set of studies by [PERSON] et al. investigated the potential of ion beam nanopatterning for controlling cellular response toward biomedical Ti6 Al4V ELI, one of the most widely employed orthopedic implant materials [125]. For this alloy, a hierarchical surface topography was obtained after normal incidence irradiation with 700 and 1100 eV Ar\({}^{+}\)[126], which can be attributed to its polycrystalline microstructure and comparably large surface roughness [127]. In particular, randomly distributed plateaus and valleys were observed on a micrometer scale, while some of the grains additionally displayed ripple patterns with about 20 nm periodicity. Using rat mesenchymal stem cells (MSC), the authors observed an increase in early cell spreading and mobility in the first seven days of culture on the ion-irradiated surfaces. Effects on long-term cellular response, however, were less conclusive [126]. To obtain more regular topographic features on Ti6 Al4V surfaces, [PERSON] et al. also employed oblique ion beam irradiation, which resulted in the formation of tilted, needle-like structures with tips about 200 nm in size [128]. Depending on the incident angle, two different surface morphologies were obtained after 1.2 keV Ar\({}^{+}\) irradiation. At an incident angle of 30\({}^{\circ}\) with respect to surface normal, the needles were around 4 \(\upmu\)m in height. At 75\({}^{\circ}\) incidence, however, their height was reduced to less than 2 \(\upmu\)m. Cellular response to these surfaces was investigated using human fetal osteoblasts. Within the first seven days of culture, pronounced differences in cell morphology were observed for the two irradiated surfaces and an untreated control. Compared to the flat control, the cells on the surface irradiated at 30\({}^{\circ}\) incidence showed rounded morphologies and reduced spreading. For 75\({}^{\circ}\) irradiation, however, preferential cell elongation and alignment along the needles was observed. The differences in surface topography also influenced cell density.