**1. Introduction**

Musculoskeletal disorders are the world's leading cause of chronic pain and impaired physical function, which result in loss of life quality [1]. In particular, the treatment of bone-cartilage defects is challenging.

Reasons for a defective or unstable bone structure are on the one hand systemic bone loss due to osteoporosis and on the other hand local trauma or cancerous diseases [2,3]. Often, bone can regenerate itself, but in the case of larger bone defects, where whole segments are missing, this process fails [4]. The gold standard to heal bone defects is still the transplantation of autologous material, which has the advantage that rejections can be avoided [5]. However, the material is naturally limited, and the

transplantation of autologous tissue always requires a second surgical treatment which leads to the problem of donor site morbidity [5]. In contrast to autografts, the availability of allografts is less limited. However, the remodeling of allografts into the recipient tissue is critical. Numerous allografts are degraded by recipient's tissue instead of promoting bone remodeling [6].

Cartilage defects can lead to osteoarthritis when left untreated [7]. One of the main problems of cartilage regeneration is the avascularity of cartilage tissue and low cell turnover, which limit the ability of cartilage healing and self-regeneration [8]. Additionally, for the treatment of cartilage defects, no optimal healing method has been found since regeneration approaches resulted mostly in fibrous tissue instead of hyaline extracellular matrix [9]. The transplantation of sca ffolds loaded with mesenchymal stem cells seems to be the most promising approach so far. Nevertheless, an immunological reaction of the recipient in response to the transferred stem cells cannot be completely ruled out, and previously used sca ffolds can hardly imitate the complex physiology of osteochondral tissue [8,10]

A promising alternative for patients with musculoskeletal disorders could be the transplantation of allografts which morphologically correspond to the recipient tissue. Additionally, allogenic tissues have only a low potential of immunological response compared to xenogeneic grafts. For the preparation of allografts, strong chemical or physical methods are currently used, which are associated with a significant reduction in biomechanics and a change in the biological behavior regarding remodeling [11]. High hydrostatic pressure (HHP) technology which is widely used in the food industry for the decontamination of food with simultaneous retaining properties such as taste and vitamins, could be a way to devitalize tissues while maintaining their biomechanical properties [12]. If HHP is suitable for providing replacement materials, the kind of cell devitalization should be taken into account during tissue processing. Based on the level of the applied pressure, cells react in either an apoptotic or a necrotic manner [12]. Pressures around 200 MPa seem to induce apoptosis while pressures higher than 300 MPa are associated with necrotic-like pathways [12]. Induction of necrosis provokes a release of proinflammatory molecules and danger signals which can further trigger the immunological response [13]. With a guided initiation of apoptosis using HHP the immunological potential of allografts might be reduced without the need to use other tissue-destructive methods.

The aim of this study was the identification of necessary HHP ranges to initiate apoptosis in human osteoblasts and chondrocytes and establish cell-specific treatment protocols. To assess the potential of HHP treatment for allograft processing, the e ffects of di fferent HHP ranges on cell survival and cell death were analyzed. Hence, human osteoblasts and chondrocytes were treated with three di fferent HHPs, ranging from 100–150 MPa, 250–300 MPa, and 450–500 MPa, to determine cell viability and HHP-dependent cell death pathways. Additionally, di fferent methodological approaches were carried out to further characterize cell apoptosis and necrosis. To avoid a loss of treated cells due to the experimental setting, the HHP treatment was performed on freshly pelleted cells. Moreover, field emission scanning microscopy (FESEM) and transmission electron microscopy (TEM) were used to provide a detailed insight into cell structures after HHP treatment. Finally, cell specific characterization of HHP treatment served as an indication to transfer cell-specific protocols to tissues in a next step to generate treatment protocols for allogenic tissue such as en bloc bone grafts.
