## 4 Discussion  
While the treatment of osteosarcoma (OS) has undergone significant improvements over the course of recent years, the 5-year overall survival rate remains notably low at approximately 60% [7; 8; 9]. The present therapy for OS involves surgical intervention, chemotherapy and radiotherapy [8; 67]. While OS typically develops in the metaphysis of long bones, detecting tumors in other regions, such as the skull or spine, can raise difficulties due to the proximity of neurovascular structures [7]. According to [PERSON] et al., the existence of micro residual tumors, intraoperative contamination during radical surgery and residual microlesions in peritumal tissue that cannot be entirely excised are the primary factors responsible for local recurrence in patients with OS [8]. Moreover, chemotherapy can lead to drug resistance and unwanted side effects caused by its non-specific toxicity [9]. Therefore, it is imperative to explore novel treatment methods that can supplement or partially replace the existing therapies, thereby reducing recurrence rates and improving the survival rates of patients [10]. [PERSON] et al. presented CAP as a promising anti-tumor treatment due to its demonstrated efficacy and selectivity in treating more than 20 different cancer types [9]. Over the past few years, different plasma devices have exhibited the capability to induce cytotoxic effects in vitro on various OS cell lines, either through direct or indirect treatment, offering a new potential direction in treating OS. For OS treatment, plasma-activated solutions are a particularly promising option as they not only have similar antitumor effects as a direct treatment but also offer the potential to bypass the need for open surgical interventions to expose tumors to CAP [9]. Moreover, PAM provides a feasible approach for aiming tumors deeply embedded in the tissue or for treating cancer cells where the physical effects of CAP need to be avoided [68]. The objective of this study was to provide a brief characterization of the CAP generated by a custom-made plasma source, as well as to analyze the resulting PAM and its potential anti-tumoral effects. Furthermore, the study aimed to identify the optimal exposure parameters for inducing a cytotoxic effect on HOS cells. Figure 16: Stability of NO\({}_{2}^{-}\) from PAM-RPMI preserved for up to 3 months at \(-80\)\({}^{\circ}\)C. Values are means \(\pm\) SEM (\(n=3\)). Statistical significance was determined by two-way ANOVA followed by [PERSON]’s post hoc multiple comparison test. The use of indirect CAP therapy at atmospheric pressure is described in the specialized literature as having potential in the development of new antitumor future strategies. The most common way to provide this medication is with physiologically adapted plasma-activated solutions, including growth medium, phosphate-buffered saline, or Ringer's solution [69]. Although [PERSON] et al. observed that CAP-exposed PBS consistently had lower cytotoxic effects compared to CAP-exposed culture medium [70], [PERSON] et al. study comparing the effects of two solutions exposed to CAP, namely PBS and RPMI 1640, on A549 cells, demonstrated a more robust cytotoxic effect of CAP-exposed PBS [71]. Hence, in this study, we evaluated the anticumoral effects of two distinct solutions exposed to CAP generated by our custom-built source, Phosphate-Buffered Saline (PBS) and Roswell Park Memorial Institute 1640 medium (RPMI 1640), the aim was to determine which of these two CAP-exposed solutions had a superior cytotoxic effect on HOS cells. Our findings indicated that for exposure times of 30 and 60 s, the cytotoxic effects were relatively comparable between the two solutions, with a slightly stronger cytotoxic effect observed for PAM-RPMI. However, for an exposure time of 90 s, a significant difference was observed between the two solutions, with PAM-RPMI 90 s resulting in a reduction in HOS cell viability of approximately 78%.