**3. Discussion**

The need for tissue replacement materials, e.g., in the head and neck area, has increased in the last years, particularly with regard to the increasing amount of cancerous diseases [14]. The gold standard

of using autologous material has the disadvantage of being limited, and patients have to undergo two operations. Allografts, which are also common, have to be treated with strong chemicals to reduce the immunogenic potential. At the same time, the matrix integrity is lost [15].

Synthetic materials, which are not limited in availability, often cannot withstand the biomechanical strain and are resorbed quickly, which makes them unsuitable for use in major bone defects [5]. The treatment of allogenic material with high hydrostatic pressure (HHP) could be a gentle and effective alternative to already existing methods for providing decellularized tissue replacement materials without negative e ffects on the tissue matrix [12]. The aim of this study was to evaluate an optimal HHP protocol for di fferent cell types (osteoblasts and chondrocytes) regarding devitalization and induction of apoptosis or necrosis. Rivalain et al. already described the e ffect of di fferent HHPs on mammalian cells and observed, that after treatment of around 200 MPa, cells underwent apoptosis whereas a treatment higher than 300 MPa resulted in necrosis [12].

We were able to characterize the influence of high hydrostatic pressure on cell survival and cell death in human osteoblasts and chondrocytes. We further aimed to distinguish di ffering HHP-e ffects when cells were exposed as cell pellets. The first observation was that the lowest HHP range of 100–150 MPa had no significant e ffect on the metabolic activity of all tested cell types. In contrast, treatment of cells with HHP ranging from either 250–300 MPa or 450–500 MPa had the same e ffect regarding the significant reduction of metabolic activity. This observation was a first hint that HHP treatment of 250 MPa and higher destroyed cells and resulted in devitalization. The previous work of Hiemer et al. [8] showed that an HHP of 480 MPa led to successful devitalization of hyaline cartilage cells. However, the limitation of the work was that this devitalizing e ffect with respect to apoptosis and necrosis was not investigated. As mentioned before, this point is of high relevance because cell death is decisive for the induction of immunological processes. The requirement for tissue replacement materials is low to negligible immunological potential for the acceptor. Therefore, the devitalization process should not have a necrotic e ffect because this would lead to inflammatory responses regarding the release of, e.g., damage-associated molecular patterns (DAMPS) which further stimulate the pro-inflammatory response [13]. To analyze the type of cell death induced by HHP, necrosis and apoptosis were first detected by ELISA. The collected data showed that the untreated control groups also underwent apoptosis and necrosis partly, which can be attributed to the fact that the controls were exposed to non-standard conditions (e.g., room temperature) during this time that the treated groups underwent the HHP treatment. Aside from this finding, HHD treatment of 100–150 MPa had no significant e ffect in comparison to the control group. A balanced apoptosis- and necrosis-inducing effect on osteoblasts was observed at 250–300 MPa, whereas an HHP treatment of 450–500 MPa induced primarily necrosis. In contrast, an HHP range of 100–150 MPa induced apoptosis in chondrocytes derived from hyaline cartilage whereby necrosis was already induced by a range from 250–300 MPa. The most e ffective HHP treatment should first and foremost lead to metabolically inactive cells that have, ideally, not ye<sup>t</sup> undergone necrosis. Thus, taking the results of both, the WST-1 assay and ELISA-derived apoptosis and necrosis analysis into consideration, these findings sugges<sup>t</sup> that an HHP of 200–300 MPa may be the most e ffective for gentle and noninflammatory cell devitalization. Analysis via flow cytometry, providing a more precise separation of vital, necrotic, apoptotic, and late apoptotic/necrotic cells, revealed that most osteoblasts were vital after HHP treatment of 100–150 MPa, while exposure to 250–300 MPa led to necrosis and apoptosis only in a small number of cells. Moreover, most of these cells were detected as being in a late apoptotic/necrotic condition. Identical observations were made for chondrocytes. We assumed that cells treated with the higher HHP range were apoptotic at an earlier time point and underwent necrosis. This result can be explained, among other things, by the fact that there was a time delay between the HHP treatment and the subsequent FACS analysis due to the availability of equipment at di fferent locations. Therefore, it cannot be excluded that necrosis was induced in the cells after a short time. Additionally, the absence of any phagocytic cells in the experimental setting led to the absence of cell debris removal. Consequently, the membrane of the treated cells became permeable over time, and intracellular contents could be released, which might further stimulate the immunological response [13]. Additionally, permeable and destroyed cell membranes as well as cellular components were found in TEM analysis of osteoblasts treated with 250–300 MPa. In summary, the results support the idea that in the early phase after HHP, apoptosis was induced following a pressure of 250–300 MPa, but the initiation of intracellular material packaging was incomplete or aborted. As confirmed by FACS analyses, the cells were in a late apoptotic/necrotic state, which led to an uncontrolled destruction of cell components in the intracellular cavity, which was additionally supported by TEM. Interestingly, these results were not observed for the lower HHP range of 150–200 MPa. Therefore, we assumed that either a reduction of the HHP level or a shortening of the HHP exposure time could initiate apoptotic devitalization. This aspect has to be confirmed in future studies.

The aim of this study was to examine the cell-typical reactions to various HHP treatments to assess the tissue-specific reactions to HHP in the next step. Furthermore, our results on devitalization efficiency and cell death detection will help to better evaluate the results of a previous animal study on devitalized autografts [10]. Applied pressure of 480 MPa to osteochondral cylinders led to an extensive tissue remodeling process following tissue re-transplantation. However, excellent integration and revitalization were detected [10]. Possible reasons for these findings might be the autograft transplantation in the experimental setting and the animal-specific immune system, which plays an important role in tissue integration. For better understanding, it is even more important to perform targeted analysis of HHP-mediated cell death which is of high relevance for the subsequent translation of specific tissue types into the clinical use of allografts. It is also important to study additional tissue-specific cells to prove whether the respective high pressure successfully devitalizes all resident cells in the tissue through apoptosis. Taking late apoptosis/necrosis into account, rapid purification of the HHP-processed tissue is still mandatory so that cell residues can be removed gently and in sufficient time. These aspects will be analyzed in more detail in further studies.

## **4. Materials and Methods**
