Test procedure

The experimental process involved three steps. First, a count of n = 50.000 commercially available osteoblasts (PromoCell, Heidelberg, Germany) per millilitre of culture medium were cultured on the bottom of the cell-bearing surface (glass panel). Therefore, cells were seeded in a culture medium (cf. Appendix 2 for a detailed composition) at 37 °C. Prior to the test procedure, the cells were manually removed from the culture bottles’ bottom by gentle movements while adding 5 ml of Trypsin followed by 10 min of incubation. Finally, Trypsin residues were removed with, first, centrifugation (1600 rpm/5 min) of the cell fluid (culture medium with additives and loose osteoblast cells) and, second, by subsequently adding 10 ml of culture medium. After 24 h of incubation, cells showed adherent to the glass panel. A conventional petri dish was filled to 70% of its capacity with cell fluid (culture medium and additives (Appendix 2). The petri dishs' bottom formed the lower plate and the round glass panel the upper plate placed within the culture medium. Directly after, the circulation process for FSS induction was initiated. In brief, after 24 h of incubation at 37 °C and 5% CO₂ concentration, cells adhered to under side of the glass panel and the glass panel was incorporated into the device as described above. The circulation process (speed level = 200 rpm) started for 24 h under sterile incubation conditions (37 °C and 5% of CO₂). Via repeated computational simulations, a rotational speed level of 200 rpm was found as adequate to provide 10 dyn/cm² of shear force at the plates’ peripheral region. Lastly, light microscopic examination was conducted (Leica DC480^®, Leica Microsystems, Wetzlar, Germany) to verify the cell orientation (after 24 h with and without rotation), followed by phallacidin fluorescence staining according to the manufacturer’s protocol (Appendix 3) (BODYPY® FL Phallacidin, ThermoFisher Scientific, MA, USA) and fluorescence microscopy with Leica/Leitz DM RBE^® (Leica Microsystems, Wetzlar, Germany). The cell body and its longitudinal actin fibre orientation was put in relation to the total-force-vector (Fig. 5) (resulted from the flow velocity-vector and the centrifugal force-vector) which was calculated by formula 3. For differentiation into an oriented and non-oriented cell formation, an angle of 90° was set as threshold value.