# Response of Osteoblasts to Electric Field Line Patterns Emerging from Molecule Stripe Landscapes
## 3 Results and Discussion
### Cell Response to Molecular Landscapes
The observation of mini-tissue, i.e., accumulation of two or more cells, that did not reside on the glass site supports this scenario. The UV-ozone pre-treatment of the glass slides as a reason for the cell response could be excluded since experiments without treatment yielded similar results. Contact guidance occurs beyond a threshold of \(\approx\)33 nm deep grooves [11; 12] for osteoblasts. For fibroblasts, the threshold for contact guidance was reported to be 35 nm with groove structures at 1:1 pitch ratio [11]. Therefore, we regard contact guidance as an  
Figure 4: Percentage of separated cells on glass samples with structured molecular landscapes after 24 h adhesion time. Cells either oriented along the structures (adhered on molecule layer or glass) or they did not respond to the landscape and remained unoriented. At least 4 independent experiments were performed, all graphs show significance (mean \(\pm\) sem) between oriented (glass and molecule layer) vs. unoriented cells and molecule layer vs. glass (Student’s \(t\)-test, \(p\) \(<\) 0.05). (a) Cell-distribution on BSA \(\upmu\)CP structured glass. (b) Cell-distribution on NH\({}_{2}\)-terminated dendrimer \(\upmu\)CP structured glass. unlikely process in our experiment since our structures are less than 4 nm in height. We suggest that the different \(\zeta\) potentials between glass and the respective molecular layer give rise to a horizontal electric field localized at the molecule-glass transition lines. Considering the low distance over which the electric potential drops (e.g., roughly assuming twice the molecule diameter), the electric field already amounts to the order of \(10^{6}\) V/m. Such high field strengths are usually not present in conventional electric chambers [8; 43]. In case of the BSA and NH\({}_{2}\)-terminated dendrimer landscapes, the electric field vectors would point in-plane, perpendicularly from the stripe edges towards the glass site, which is the retraction (or deformation) direction of osteoblastic cells in our experiments (see Figure 6). Often, mammalian cells appear to show cathodic taxis [30; 31], among them are keratinocytes [44], neuronal growth cones [45], meniscus cells [46], and SaOS-2 osteoblast-like cells [43; 47]. Some cell types show anodic taxis, e.g., osteoclasts [43] and dermal fibroblasts [44]. Furthermore primary, patient-derived osteoblasts [48] and calvariat osteosarcoma osteoblasts [47] show anodic taxis. A paucity of literature exists on the behavior of the osteosarcoma cell line MG-63 [8; 9]. This type of osteoblastic cell appears not to migrate to one or the other electrode, however, MG-63 adopts a crescent shape with the convex side towards the cathode [8], as has also been observed for migrating RCJ.1.20 osteoblastic cells [43]. This may possibly represent an attempt to minimize electric potential differences along the cell body. This is in agreement with our observation, since the cells adhered along the glass and thus avoided the horizontal electric field at the glass-molecule interface. To corroborate that the glass preference is due to electro-deformation, we assessed the cell response to COOH-terminated dendrimers, PDADMA and PSS landscapes. PSS shows a slightly lower \(\zeta\) potential (\(-88\) mV) than glass (if we assume \(-80\) mV for glass), which may allow us to alter the electric field vectors in comparison to BSA and NH\({}_{2}\)-terminated dendrimer printed glass samples [7]. COOH-terminated dendrimers and PDADMA were chosen to vary the electric field strength due to their \(\zeta\) potential (\(-47\) mV and \(51\) mV, respectively) [7; 42]. Through brightfield microscopy, we noticed an alignment and orientation of single cells, similar to previous observations with positively charged stripes. Unfortunately, the auto-fluorescence of the three molecule types was too faint to observe in liquid conditions, which is why we could only conduct spot checks to investigate the cell's preference with SICM.