*4.1. Cell Culture*

Human osteoblasts from femoral heads (*n* = 5) and human chondrocytes from hyaline knee cartilage (*n* = 5) were isolated from patients undergoing total joint replacements. Prior to isolation of human osteoblasts and chondrocytes, informed consent and ethical approval (A2010-10A (osteoblasts) and A2009-17 (chondrocytes) were obtained from the ethics committee of the University of Rostock, Germany) including IRB information. Harvesting and isolation of cells took place after patients signed agreemen<sup>t</sup> forms following the previously described protocols [16–18]. We have go<sup>t</sup> the written informed consent from all patients. The isolated osteoblasts were cultivated in a 25 cm<sup>2</sup> culture flask with a volume of 8 mL osteogenic medium (Dulbecco's modified Eagle's medium (DMEM), PAN-Biotech, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS, PAN-Biotech), 1% amphotericin B, 1% penicillin/streptomycin, 1% HEPES buffer, 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone (all: Sigma–Aldrich, Munich, Germany). Human chondrocytes were cultivated in 25 cm<sup>2</sup> culture flasks containing 8 mL DMEM medium (Gibco® Invitrogen, Paisly, UK) containing 10% FCS, 1% amphotericin B, and 1% penicillin/streptomycin. In addition, chondrocytes were supplemented with 50 μg/mL ascorbic acid. Incubation took place in a humidified atmosphere of 5% CO2 and at 37 ◦C. The respective medium was changed every second day so non-adherent cells could be aspirated. At a confluence of 100%, which was checked by light microscopy, the cells were transferred in a 75 cm<sup>2</sup> culture flask, and further culture took place under the same conditions as described above. Cells in the third passage were used for the experiments.

## *4.2. High Hydrostatic Pressure Treatment*

To treat cells with high hydrostatic pressure (HHP), the medium was discarded and adherent cells were detached with 2 mL trypsin per 75 cm<sup>2</sup> flask. After an incubation time of 3 min at 5% CO2 and 37 ◦C, the reaction was stopped with 6 mL DMEM. The cell suspension was collected and centrifuged at 118× *g* for 8 min. The medium was discarded, and the obtained cell sediment was resuspended in 1 mL DMEM. After cell counting, 50,000 cells were transferred as duplicates into 2 mL CryoTubes (ThermoFisher Scientific, Waltham, MA, USA), filled up with the corresponding medium, and centrifuged at 118× *g* for 8 min to generate freshly pelleted cells. These cell pellets were treated with di fferent HHPs ranging from 100–150 MPa, 250–300 MPa, and 450–500 MPa for 10 min using an HHP device (HDR-100, RECORD GmbH, Koenigsee, Germany). After HHP treatment, the tubes were centrifuged again at 118× *g* for 8 min; 200 μL of the supernatants were discarded, and the cells were incubated for 24 h under standard cell conditions. HHP-untreated cells served as controls; these cells were exposed to the same test conditions.
