*2.1. Cellular Activity*

In both cell types, no significant di fference in the metabolic activity between the control group and the group treated with 100–150 MPa was detectable (Figure 1). In contrast, HHPs ranging between 250–300 MPa and higher led to a significant decrease in the cell activity in comparison to the control group and the group treated with the lowest HHP range (osteoblasts: *p* = 0.0429 (control vs. 250–300 MPa); *p* = 0.0161 (control vs. 450–500 MPa); *p* = 0.0285 (100–150 MPa vs. 250–300 MPa); *p* = 0.0107 (100–150 MPa vs. 450–500 MPa); chondrocytes: *p* = 0.0155 (control vs. 250–300 MPa); *p* = 0.0150 (control vs. 450–500 MPa)). However, a significant difference in the metabolic activity of cells treated with 250–300 MPa and 450–500 MPa was not detectable.

**Figure 1.** Metabolic activity of (**a**) human osteoblasts (*n* = 4) and (**b**) human chondrocytes (*n* = 4) following treatment with different HHPs ranging from 100–150 MPa, 250–300 MPa, and 450–500 MPa. After HHP exposure, cells were incubated at 37 ◦C and 5% CO2 over a period of 24 h. Subsequently, metabolic activity was determined with water-soluble tetrazolium salt (WST-) 1 assay. Data are presented as boxplots. Significant differences between stimulation groups were determined via one-way ANOVA: \* *p* ≤ 0.05.

#### *2.2. Induction of Cell Death Following Exposure to HHP*

Cell death analysis after HHP treatment was carried out via Cell Death Detection ELISA. In human osteoblasts, HHP exposure at the lowest range (100–150 MPa) led to only a slight increase in necrosis and apoptosis without significant differences compared to untreated cells. In contrast, a significant increase in necrosis (Figure 2a; *p* = 0.0206) and apoptosis (Figure 2b; *p* = 0.0022) of human osteoblasts was shown after HHP treatment of 250–300 MPa. In addition, HHP treatment of 450–500 MPa also resulted in a significant increase in necrosis compared to the control group (*p* = 0.0233) while this HHP range apparently had no effect on osteoblasts regarding apoptosis (Figure 2a,b).

In Figure 2c,d the apoptosis and necrosis rates of human chondrocytes after HHP treatment are shown. A significant increase in necrosis took place in response to an HHP range of 250–300 MPa compared to untreated cells (*p* = 0.0184). In addition, the results of apoptosis (Figure 2d) revealed that the lowest HHP (100–150 MPa) induced mostly apoptosis while higher HHP ranges had no apoptotic effect. However, these data were not statistically significant.

#### *2.3. Di*ff*erentiation of Necrosis, Apoptosis, and Late Apoptosis Following HHP Treatment of Pelleted Cells*

Since Cell Death ELISA data revealed that HHP treatment of 250–300 MPa had the strongest effect on the tested cell types, a more detailed cell death analysis took place via flow cytometry. In comparison to this pressure, pelleted cells were also treated with a pressure of 100–150 MPa again for analysis via flow cytometry. The used gating strategy is shown in Figure 3 on the basis of untreated human osteoblasts. After gating the cells from the cell debris with the side scatter height (SSC-H) and forward scatter height (FSC-H) channels, fluorescence (FL)4-H on the y-axis for apoptosis and FL2-H on the x-axis for necrosis were set. Then, four quadrants (Q)1 to Q4 were adjusted, which represent annexin single positive cells (Q1; apoptosis), annexin/propidium iodide (PI) double positive cells (Q2; late apoptotic and necrotic cells), PI single positive cells (Q3; necrosis), and annexin/PI double negative cells (Q4; vital cells), respectively.

**Figure 2.** Analysis of cell death induced by HHP treatment of (**<sup>a</sup>**,**b**) human osteoblasts (*n* = 4), and (**<sup>c</sup>**,**d**) human chondrocytes (*n* = 4). Graphics (**<sup>a</sup>**,**<sup>c</sup>**) show necrosis and graphics (**b**,**d**) show apoptosis, both detected via Cell Death Detection ELISA. After HHP treatment, the supernatants were collected for necrosis detection. The residual cells were lysed for 30 min with 200 μL lysis buffer provided by the kit. After centrifugation at 118× *g* for 8 min, supernatants were collected for apoptosis detection. All data are presented as boxplots. Significant differences between groups were determined via one-way ANOVA. \* *p* ≤ 0.05; \*\* *p* ≤ 0.01.

**Figure 3.** Gating strategy for cell death detection with annexin and propidium iodide (PI) exemplified using untreated human osteoblasts. Analysis took place with FloJo. (**a**) Gating of cells from the debris using the side scatter height (SSC-H) and forward scatter height (FSC-H) channel. This gate was used to detect the different stainings in (**b**) where quadrant (Q)1 represents annexin single positive cells, Q2 annexin/PI double positive cells, Q3 PI single positive cells and Q4 annexin/PI double negative cells.

The majority of untreated groups of both cell types could be detected as double negative (alive, around 80%). The remaining parts of the cells were distributed between apoptotic, necrotic and late apoptotic/necrotic detection for both osteoblasts and chondrocytes. A similar distribution could be observed for cells treated with 100–150 MPa for 10 min (Figure 4a,b). Human osteoblasts (Figure 4a) were significantly late apoptotic/necrotic following HHP treatment of 250–300 MPa in comparison to the control group (*p* < 0.0001), while the live, apoptotic and necrotic cells were less than 5%. In contrast to this, human chondrocytes were detected as necrotic and late apoptotic/necrotic in a wide range after treatment with 250–300 MPa (Figure 4b). However, a significant increase in late apoptotic/necrotic cells after treatment with 250–300 MPa in comparison to the control group could be detected (*p* < 0.0001), and an increase in necrotic cells after HHP treatment of 250–300 MPa in comparison to the control group (*p* = 0.0041) could be observed.

**Figure 4.** Analysis of cell death detection after HHP treatment (100–150 MPa and 250–300 MPa, 10 min) of (**a**) human osteoblast cell pellets (*n* ≥ 4) and (**b**) human chondrocyte cell pellets (*n* ≥ 5) with annexin and propidium iodide (PI). For each cell type, cell pellets were prepared for HHP treatment. Afterwards, treated cells were washed with autoMACS® Running Buffer, stained with annexin and PI, and analyzed with the FACS Calibur. Data are shown as box plots. Significance between groups was detected via two-way ANOVA. \*\* *p* ≤ 0,01; \*\*\*\* *p* < 0.0001.

#### *2.4. Evaluation of the Cellular Damage of Osteoblasts after HHP Treatment Based on Field Emission Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM)*

Imaging techniques such as FESEM and TEM were used to visualize the HHP effects on cell structures. Therefore, osteoblasts were treated with HHP (100–150 MPa and 250–300 MPa for 10 min each) and subsequently prepared for FESEM and TEM analysis.

Untreated human osteoblasts could be detected via FESEM with an uneven cell surface with distinct elevations which appeared to be smooth (Figure 5a). In the sections, analyzed by TEM, cellular structures such as the nucleus, mitochondria, and the endoplasmic reticulum were observed (Figure 5b). Between the control group and the group treated with 100–150 MPa, only slight differences could be detected. Again, the cell surface appeared to be intact in FESEM, and TEM analyses revealed distinct structures as described before (Figure 5c,d). Only after treatment of human osteoblasts with 250–300 MPa, the cell surface appeared to be altered and became a sponge-like structure covered with small blebs (Figure 5e). While in the other two groups, the intracellular structures were clearly visible with TEM, these structures were destroyed following HHP treatment with 250–300 MPa (Figure 5f). Based on a comparison of the cell diameters of the differently treated cells, the untreated and 100–150 MPa treated groups had a diameter of approximately 20 μm while most of the cells treated with 250–300 MPa seemed to shrink approximately by one fifth.

*Int. J. Mol. Sci.* **2020**, *21*, 3836

**Figure 5.** Cell structure analysis of human osteoblasts following HHP treatment. Treated cells (**<sup>c</sup>**,**d** 150–200 MPa; **<sup>e</sup>**,**f**: 250–300 MPa) and untreated cells (**<sup>a</sup>**,**b**) were fixed to carry out field emission electron (**<sup>a</sup>**,**c**,**<sup>e</sup>** scale bar: 5 μm) and transmission electron (**b**,**d**,**f**; scale bar: 1 μm) microscopy. Abbreviations: Nc = nucleus (light green), Mc = mitochondrion (orange), ER: endoplasmic reticulum (white).
