*4.3. Metabolic Activity Analysis*

After an incubation time of 24 h, HHP-treated and untreated cells were centrifuged at 118× *g* for 8 min. The supernatants were discarded, and the metabolic activity cells was analyzed by incubating the cells for 1.5 h under standard culture conditions with 250 μL of a 1:10 dilution of WST-1 reagen<sup>t</sup> (Takara, Bio, Saint-Germain-en-Laye, France) and the respective cell culture medium. The amount of formed formazan dye, which correlates directly with the number of metabolic active cells, was determined by an extinction measurement using a Tecan-Reader Infinite ® 200 Pro (Tecan, Maennedorf, Switzerland); (absorption: 450 nm, reference: 600 nm).

## *4.4. Analysis of Cell Death*

For cell death detection, supernatants of exposed and unexposed cells were collected and stored at −20 ◦C for further necrosis detection by Cell Death Detection ELISA (Roche, Penzberg, Germany). For apoptosis detection, the residual cells were incubated with 200 μL lysis bu ffer provided by the kit for 30 min at room temperature. The cell lysates were centrifuged at 200× *g* for 10 min, and the supernatants without the cell debris were stored at −20 ◦C for further use.

To analyze the kind of cell death, all supernatants were thawed and transferred as duplicates into a streptavidin-coated microtiter plate with the related positive, negative, and background controls. In each well, 80 μL immunoreagent, containing incubation bu ffer, anti-histone-biotin, and anti-DNA-POD, all made available by the kit, were added to each well. The microtiter plate was covered with the cover foil and incubated for 2 h at room temperature on the shaker at 300 rpm. The supernatants were then discarded, and the plate was washed three times with 250 μL incubation bu ffer. Afterwards, the bu ffer was aspirated thoroughly, and 100 μL ABTS Solution was added. After an incubation period of 10 min at room temperature on the shaker at 250 rpm, a color reaction was visible and it was stopped by adding 100 μL ABTS Stop Solution. The quantification took place in a Tecan-Reader Infinite ® 200 Pro at a wavelength of 405 nm and a reference wave length of 490 nm.

For precise analysis of cell death after HHP treatment, human osteoblasts and chondrocytes were analyzed by flow cytometry with the APC Annexin-V Apoptotis Detection Kit with propidium iodide (PI) (Biolegend, San Diego, CA, USA). Cells were cultured as described above (all cell types *n* = 5) and a cell number of 2 × 10<sup>5</sup> cells were treated with an HHP of 100–150 MPa and 250–300 MPa for 10 min, whereas cell pellets were treated and analyzed. Afterwards, cells were centrifuged at 118× *g* for 8 min. The supernatants were discarded and the cells were washed with 1 mL autoMACS ® Running Bu ffer (Myltenyi Biotec, Bergisch Gladbach, Germany) at 1400 rpm for five minutes. A mastermix of 5 μL Annexin V FITC and 10 μL PI per sample was added, and the cells were incubated for 15 min at room temperature in darkness. Afterwards, 100 μL Annexin Binding bu ffer was added, and analysis took place with the FACS Calibur (BD, Franklin Lakes, NJ, USA). For the evaluation of the results, FloJo (BD, Franklin Lakes, NJ, USA) was used, and the percentage of positive cells was assessed.

#### *4.5. Analysis of Cellular Damage by Field Emission Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM)*

Human osteoblasts and human chondrocytes (100,000 cells each) were transferred into 2 mL CryoTubes (ThermoFisher), filled up with the corresponding medium, and centrifuged at 118× *g* for 8 min. These cell pellets were treated with di fferent pressure ranges (w/o HHP, 100–150 MPa and 250–300 MPa). After treatment, tubes were again centrifuged at 118× *g* for eight minutes and the supernatant was discarded. The cells where then fixed with fixation bu ffer (1% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium phosphate bu ffer, pH 7.3) and stored at 4 ◦C. Afterwards, cells were washed with sodium phosphate bu ffer, adhered to glass coverslips previously coated with poly-L-lysine (Co. Sigma) for one hour and then dehydrated with an acetone series, followed by critical point drying using CO2 (Emitech K850/Quorum Technologies Ltd., East Sussex, UK). The glass coverslips were mounted on aluminum sample holders and coated with gold under vacuum (SCD 004, Baltec, Balzers, Liechtenstein). With a field emission scanning electron microscope (MERLIN VP Compact, Carl Zeiss, Oberkochen Germany), images were taken from the selected regions (applied detector: HE-SE2; accelerating voltage: 5.0 kV; working distance: 5.2 mm).

For TEM preparation, fixed specimens were washed in 0.1 M sodium phosphate bu ffer (pH 7.3), and cell pellets were enclosed in 3% low melting agarose (Fluka, Munich, Germany) in water at 40 ◦C by centrifugation in Eppendorf tubes. After staining with 1% osmiumtetroxide (Roth GmbH, Karlsruhe, Germany) for 2 h, the specimens were dehydrated through an ascending series of acetone prior to embedding in Epon resin (Serva, Heidelberg, Germany). Resin infiltration started with a 1:1 mixture of acetone and resin overnight, followed by pure resin for 4 h. After transfer to rubber casting moulds, specimens were cured in an oven at 60 ◦C for at least 48 h. Resin blocks were trimmed using a Leica EM Trim 2 (Leica Microsystems, Wetzlar, Germany). Ultrathin sections (approx. 70–90 nm) were cut with a Leica UC7 ultramicrotome using a diamond knife (Diatome, Nidau, Switzerland). Ultrathin sections were mounted on formvar-coated copper grids and were contrasted with uranyl acetate and lead citrate. Ultrastructure was inspected with a Zeiss EM902 electron microscope operated at 80 kV (Carl Zeiss, Oberkochen, Germany). Digital images were acquired with a side-mounted 1x2k FT-CCD Camera (Proscan, Scheuring, Germany) using iTem Camera control imaging software (Olympus Soft Imaging Solutions, Muenster, Germany).
