Morphological risk assessment of cold atmospheric plasma-based therapy: bone marrow mesenchymal stem cells in treatment zone proximity

Cold atmospheric plasma (CAP), modern technology for medical purposes, has recently established its potential in cancer therapy both in vitro and in vivo, thanks to the plasma-induced formation of free radicals. Due to its growing demand and extensive use, comprehensive studies are necessary to ensure its safety, especially for healthy cells adjacent to the treatment area. To this end, in this paper and for the first time, use has been made of morphological analysis, including the cell/nucleus shape index (CSI/NSI) and fractal dimension (FD), to assess the risk of CAP therapy on normal bone marrow mesenchymal stem cells (BMMSCs). To find the best protocol of plasma treatment, BMMSCs were subjected to direct and indirect (adding plasma-activated-water (PAW) to the culture medium) treatments. Our bioassay and morphological assessments results showed that the PAW treatment was the better method for the optimal dosage equal to 80% of the culture media, provided that the maximum power was 30W and the exposure time was 90 s. Our research showed that high-dose PAW results in significant changes in FD, e.g. an equal proportion of PAW and culture medium increases the FD 1.38-fold, with respect to the control. Furthermore, while in the low-power direct treatment (<30W) NSI did not change significantly, in the high-power one (>30W) it approached 1, which is an indicator of apoptosis. It was also seen that high-dose PAW elongates the cells and therefore diminishes CSI. Any change in cell/nucleus shape could results in cancer initiation. Therefore, the observed changes in shape index and FD number might indicate cancer initiation. Our measurements of the BAX and BCL2 expression ratio were in favor of our results. Taken together, all of our results showed cell damage in high powers of plasma source and high-dose PAW. Moreover, it is concluded that the probability of cancer initiation is a severe issue that should be investigated in all CAP-based cancer therapies, even in the optimized indirect methods.