## 4 Discussion  
Electrical impulses play a role in physiological processes in various tissues, such as bones [27]. Therefore, artificial electrical stimulation (ES) is a successful adjunct therapy in regenerative medicine [28; 29; 30; 31; 32], especially for bone regeneration [2; 33]. However, the mechanisms of ES on cells and the environment still need to be understood. A previous study showed that initial cell adhesion and signaling are improved under ES. We could exclude medium-mediated effects of ES, such as changes in temperature, pH, and O\({}_{2}\) content [12]. In the current study, we investigated the response of human MG-63 osteoblasts after 10 min of ES with the IonOptix multi-channel device under 1 and 5 V at a frequency of 20 Hz, respectively. The focus was on ES-mediated effects on cells' hyaluron coat, the zeta potential, the membrane potential, aquaporin expression, and on protein adsorption. We excluded an effect on cell viability after 24 h in earlier studies (Figure S1). We confirmed our adhesion results from previous experiments conducted by [PERSON] et al. [12]. The ES of suspended cells with 1 V/20 Hz enhanced their initial 10 min adhesion compared to the control (without ES). When calculated with a paired \(t\)-test, 5 V/20 Hz also yielded a significant result. The cell attachment depended on the serum content in the cell medium, because the cell adhesion was not increased without fetal bovine serum (FBS). The overall adhesion was higher in samples stimulated without FBS, but these values are not significant due to the high variability of cell behavior and resulting high standard deviation. For the tendency of higher cell adhesion, we could explain this with a study by [PERSON] et al. [34]. The group found that the initial adhesion of osteosarcoma cells was decreased on material surfaces that were coated with FBS, and only a few cells adhered after 5 or 30 min of incubation compared to coatings with poly-L-lysine, fibronectin, vitronectin, and collagen type I [34]. Also, hydroxyapatite sources coated with FBS induced less adherence and spreading in MG-63 cells than surfaces coated with fibronectin alone [35]. In FBS, both albumin and fibronectin are present. Still, albumin makes up the majority of proteins and, at 20-50 mg/mL, is 1000 times more abundant than fibronectin [36]. The positive effect of secreted fibronectin on cell adhesion and spreading was identified by [PERSON] et al. [37]. Fibronectin was strongly adsorbed in our experiments due to 10 min of ES. MG-63 cells present the adhesion receptors for fibronectin, the \(\alpha\)5\(\beta\)1 integrin subunit, which finds its prime target of fibronectin on the glass bottom [38; 39]. It was found that BSA adsorption was higher during electrical stimulation with 1 V/cm for 1 h on composite hydrogels [40]. This could be correlated with higher cell adhesion and spreading and mBMSC cell genotype and phenotype changes in the direction of differentiated osteoblasts [40]. The research group of [PERSON] found that the adsorption of complex (e.g., lysozyme) and soft proteins (e.g., BSA or IgG) on conductive substrates can be modulated by the application of an external electric potential at 800 mV [41]. The proteins of the FBS-containing medium formed multiple layers under ES, which could be why more cells adhered as a result of ES. The negatively charged pericellular matrix substance hyaluron was described as the first initiator of cell adhesion [21; 42]. Although electrically stimulated, osteoblasts did not adhere better if the hyaluron coat was enzymatically destroyed. The thick hyaluron coat [43] of osteoblasts could also be the reason for the unchanged surface charge of the osteoblasts. MG-63 osteoblasts exhibited a negatively charged cell surface of \(-13\) mV, independently of ES.