# Response of Osteoblasts to Electric Field Line Patterns Emerging from Molecule Stripe Landscapes
## 3 Results and Discussion
### Cell Response to Molecular Landscapes  
MG-63 osteoblastic cells were seeded on BSA and NH\({}_{2}\)-terminated dendrimer printed glass samples and incubated for 24 h, allowing them to adhere and migrate to preferred locations. Subsequently, the cell culture medium was exchanged by HEPES to localize the molecular stripes by luminescence (GFP-filtercube). In areas where molecular stripe patterns were present, a significant amount of single cells ((89 \(\pm\) 3)% for BSA and (78 \(\pm\) 6)% for NH\({}_{2}\)-dendrimer-samples) adopted a distinct spindle shape, aligning parallel to the stripes (Figure 3). This observation remained true only when cells were separated from each other. In case of connected cells (e.g., via filopodia) the response to the molecular patterns seemed to be largely absent. Furthermore, the cells were often randomly oriented and did not necessarily maintain a spindle shape. Figure 3: Optical microscopy images of MG-63 cells after 24 h adhesion time on glass with molecule stripe landscapes. The scalebars in the bottom right corner show 100 \(\upmu\)m or 50 \(\upmu\)m for images (**a,b**) and (**c,d**), respectively. (**a**) Brightfield image of adhered cells. Most cells espcisally the ones in the lower half of the image orient diagonally and adopt a spindle shape. (**b**) Corresponding auto-fluorescence image, obtained by using a GFP-filtercube. The bright stripes (indicating NH\({}_{2}\)-terminated dendrimer layers) are oriented in the same direction as the cells. At spots where the stripe pattern is absent (top right) the cells orient randomly and do not necessarily take on a spindle shape. (**c**) Brightfield image of adhered MG-63 cells on BSA \(\upmu\)CP glass. The cell clusters (marked in blue) are oriented in the same way as the separated cells (marked in red). However, it is visible that the adhesion area is not so well defined for the cell-clusters. (**d**) Corresponding auto-fluorescence image. The separated cells orient along the bright BSA stripes, but adhere on the dark glass-stripes. In case of the cell clusters (marked in blue), orientation and confinement onto the glass site is largely absent. Taking only separated cells into account, we noticed a significant adhesion preference onto glass sites (see Figure 4). While for the BSA-samples (87 \(\pm\) 5)% of single cells residing on glass, the preference seemed to be sightly lower in case of PAMAM-samples with (70 \(\pm\) 5)%. We can exclude that this observation is due to different adhesion probabilities on glass or flat molecule layers. When we offered a flat unstructured layer of BSA or NH\({}_{2}\)-terminated dendrimer, the glass preference vanished. A similar amount of cells were then found on the glass and molecule site after 24 h incubation (see Figure 5). This may also serve to dispel doubts with respect to the fact that albumin is not fully innocent in terms of adhesion [15; 16; 17; 18]. The apparent glass preference when molecular layers are offered in the direct vicinity of cells is not explainable by means of \(\zeta\) potentials of the different species: the potential of glass is strongly negative (\(\approx\)\(-\)80 mV), BSA films on glass raise its potential by a few 10 mV. The amine groups of the dendrimer exposed to aqueous medium tend to bind a proton and become positively charged, resulting in a positive \(\zeta\) potential of 22 mV [42]. This would give rise to a preference to the molecular layers since \(\zeta\) potentials in the regime around 0 mV are favourable for MG-63 activity [7]. Especially on larger, covalently bound NH\({}_{2}\) terminated dendrimers (gen. 5) the osteoblasts exhibited tight cell attachment with well-organized actin stress fibers and well-spread morphology [42]. However, if a chemical gradient would be operative, the cells could sense and mistake/regard the amine for presence of cells or extracellular matrix. In terms of (pseudo-)tissue formation priority, i.e., swiftly covering a surface, it would make sense, that cells fill voids, i.e., the space inbetween the molecule stripes.