## 2 Materials and Methods
### Electrical Stimulation of Human Primary Osteoblasts  
Human primary osteoblasts were isolated, cultivated, and electrically stimulated for the experiments, as previously described by [PERSON] et al. [30]. In brief, spongiosa was isolated from femoral heads allocated from patients undergoing total hip replacement (registration number: A 2010-0010, approval date: 27 January 2017). After digestion with collagenase a and dispase (both: Roche, Basel; Switzerland), the cell suspension was purified through filtration and centrifugation steps. The cells were cultivated in a cell culture flask with Dulbecco's Modified Eagle Medium without CaCl\({}_{2}\) to increase proliferation (DMEM, PAN-Biotech, Aidenbach, Germany), containing 10% fetal calf serum (FCS, PAN-Biotech, Aidenbach, Germany), 1% amphoteric B, 1% penicillin-streptomycinycin, and 1% HEPES buffer (all: Sigma-Aldrich, Munich, Germany). Ascorbic acid (final concentration: 50 \(\mu\)g/mL), \(\beta\)-glycerophosphate (final concentration: 10 mM), and dexamethasone (final concentration: 100 nM) (all: Sigma-Aldrich, Munich, Germany) were added to bring cells to a similar stage of differentiation. The cells were kept in cell culture under standard cell culture conditions (5% CO\({}_{2}\) and 37 \({}^{\circ}\)C) until the second passage and stored in liquid nitrogen until use.  
For the electrical stimulation experiment, osteoblasts from seven different human donors (four females (age: 77.25 \(\pm\) 2.21 years), three males (age: 73 \(\pm\) 9.54 years)) were thawed and cultured for another passage at 37\({}^{\circ}\)C and 5% CO\({}_{2}\). Rat tail collagen-coated coverslips (diameter: 15 mm, Neuvitro Corporation, Vancouver, WA, USA) were placed in a 6-well plate, and 30,000 cells were seeded on each coverslip. The cells adhered for 30 min, and 5 mL of cell culture medium (Dulbecco's Modified Eagle Medium (DMEM w/o calcium)) was added with 10% fetal calf serum (FCS, both: PAN-Biotech, Aidenbach, Germany), 1% amphotericin B, 1% penicillin-streptomycin, and 1% HEPES buffer (all: Sigma-Aldrich, Munich, Germany) containing the osteogenic additives ascorbic acid (final concentration: 50 \(\mu\)g/mL), \(\beta\)-glycerophosphate (final concentration: 10 mM), dexamethasone-sone (final concentration: 100 nM) (all: Sigma-Aldrich, Munich, Germany) and CaCl\({}_{2}\) (final concentration: 1.8 mmol/L) for mineralization. An in vitro stimulation electrode was used, which was designed similarly to the clinically used ASNIS III s-series stimulation screw system [31; 32]. The electrode consists of two 14 mm Ti6 Al4V cylindrical electrodes separated by a 5 mm long insulator made of polyetheretherketone. The detailed stimulation protocol can be found as described by [PERSON] et al. [30]. The stimulation electrodes were placed in each well. In addition to the electrically stimulated osteoblasts (ESO), unconnected electrodes served as a control (non-stimulated osteoblasts--NSO). A sinusoidal signal with 0.7 V\({}_{\text{rms}}\) and 20 Hz was applied 24 h after cell seeding using a Metrix GX 305 and GX310 function generator (Metrix Electronics, Bramley, Hampshire, UK). The distance between the electrode and the well bottom was 1 mm, which leads to an electric field of approximately 150 V/m in the cell medium and the well bottom under the center of the electrode [30]. The alternating current was applied 3 \(\times\) 45 min/d with 225 min breaks between stimulations and a longer 855 min break between the last and the first new stimulation. This was performed to keep comparability with clinical studies [31]. The supernatant was collected and refreshed after 7, 14, and 21 days. The supernatant was collected 20 h after the last stimulation interval had started. It was centrifuged, pooled, and frozen for indirect stimulation.