## 4 Discussion
When using a real pig skin model, it is noteworthy that the overall absorbance decreases but the absorbance increases at 190 nm, 260 nm, and 280 nm. This measurement result can be considered the influence of nucleic acids having maximum absorption wavelengths at 260 nm and proteins with maximum absorption wavelengths at 190 nm and 280 nm. It is possible to consider that nucleic acids and proteins contained in skin diffuse together with active species during plasma treatment and are measured [49]. As a result, it is shown that the complexity of the actual skin can further reduce the permeation and diffusion tendency of reactive species. A recent publication by [PERSON] et al., stated that nitrated membranes appear to be more permeable to hydrophilic RONS than native and peroxid oxidized membranes [50]. They also found by applying computer simulations that hydrophilic RONS like HO2 and ONOOH are more prone to penetrate into nitrated membranes--which was not the case for hydrogen peroxide (H2O2) and hydroxyl radicals (OH). This study also reveals that hydrophobic RONS like molecular oxygen, nitrogen dioxide (NO2) and nitric oxide (NO) even have insignificant barriers for permeation. This further supports our findings. Based on these findings clinical trials should be performed in order to support the data with results from patient data. This might help to understand why plasma treatment is increasing skin microcirculation, and therefore supports wound healing in patients with chronic wounds. We were able to confirm the effect of agarose gel and SC model on RONS diffusion in real time using the UVAS method. This method has the advantage of being able to check the diffusion of RONS in real time and has the advantage that there is no interference with changes in pH. We tried to verify the UVAS method by chemical assays, but fluctuations according to the pH change had a very strong effect on the chemical measurement. There is a disadvantage also. RONS cannot be accurately distinguished when other salt componentsare included. The absorption spectrum overlaps with that of salts such as chloride. This method is thought to be helpful for kinetic studies that require real-time measurement. Applying agarose gels is a well-known way to investigate diffusion properties since the water content and density can be adjusted to mimic the properties of skin [22, 26], however, it does not completely mimic human skin, since the lipid layer or even the dead cells of the stratum corneum are missing. Therefore, we added a lipid layer isolated from a human skin cell line (HaCaT) in order to investigate the penetration and diffusion of different plasma components through such a more skin related barrier. This is in good accordance with the findings of [PERSON] et al.--they showed by computer simulations the effects in nitrated membranes, with similar tendencies. Adding a lipid layer to an agarose gel might be one further step to mimic real skin. However, the hydrophobicity was much lower than real SC. So, we tried mixing pure lipid components similarly to SC. However, it was difficult to physically manipulate the lipid film on the hydrophilic surface since it was not strongly combined. The actual SC has a very hydrophobic structure due to molecular crosslinking structures like bricks. Reports state that the properties of SC vary greatly according to the animal considered [51, 52]. A next step might be to cultivate human cells directly on agarose--e.g., Hacat cells in a multi-layer. However, such a cultivation is very complicated and time consuming--and ultimately would still not depict real skin, since it would lack fibroblasts, immune cells and melanocytes. Adding cells to such a complex model would also add more complex interactions--especially since those cells will introduce a variety of antioxidants and ROS-degrading enzymes--counteracting any attempt to investigate the penetration and diffusion of RONS generated by a NTAPP.