Atmospheric-Pressure Plasma - Essay Example

? 31 January, “Atmospheric-pressure plasma” is essential not only for sterilization/disinfection, decomposition of hazardous substances and surface modification, but also for exploration and development of new composite fields that are based on multifaceted nanotechnology, biotechnology and medical sciences. In a recent research on modification and regeneration therapies that used pulsed plasma, plasma exposure was found to have healing effect on burns and cutaneous wounds caused by diabetic necrosis. The effect was thought to be caused by the nitrogen monoxide (NO) that was generated during the plasma exposure. However, the mechanism of regeneration of tissues through the short-time plasma exposure has not been explicitly revealed as yet in spite of its increased practical application. In order to improve the situation, it may be important to clarify the mechanism from multilateral standpoints including plasma science and engineering, molecular biology, and biochemistry. Therefore, we conducted a basic experiment on the direct irradiation of cells by using micro-spot atmospheric-pressure plasma source, which is hardly harmful to the living bodies both thermally and electromagnetically. In this experiment, murine fibroblast cell line (NIH3T3), which is usually used for cell experiments, was used and effect of plasma on the culture cells was considered. As a result of the experiment, it was revealed that cell multiplication is activated by plasma exposure. In response to the result, we considered factors related to the multiplication. Although there are many factors involved in the cell multiplication, we particularly focused on neoangiogenesis and NO production, and considered vascular endothelial growth factor (VEGF) and acidic fibroblast growth factor (bFGF). Schematic diagram of the experimental apparatus is shown in Fig 1. The apparatus comprised a coaxial structure with tungsten electrodes employed in a glass capillary (internal diameter of plasma genesis region: 8 mm, internal diameter of the tip: 1 mm) with cylindrical electrodes on the exterior. High voltage for plasma generation was generated by externally-controlled high-voltage power supply device. Plasma generation conditions were: applied voltage: 5-9 kV; frequency: 1-3 kHz; helium (He) gas flow rate: 1 L/min; and plasma exposure time: 1-100 sec. It was found in the earlier studies that NO gas is not generated by this apparatus. In the experiment, a culture containing 1 x 105 murine fibroblast cell lines (NIH3T3) was set in a 12-hole culture vessel. It was cultured for 24 hours in a CO2 incubator (culture conditions: temperature: 37 deg. C and CO2 gas concentration: 5%). The medium was replaced with serum-free medium. The unprocessed specimens (control), those processed with He gas flow and others exposed to plasma were compared. Processing time for each specimen group was 1, 10 and 100 sec, respectively. They were cultured for 24 hours in CO2 incubator after plasma exposure and then cell forms were observed with optical microscope. Afterwards, only live cells of NIH3T3 cell line attached to the bottom face of the culture vessel were peeled off by trypsin treatment and the number of cells under each condition was counted, followed by consideration of multiplication rate changes among the conditions. In addition to that, they were continuously cultured for 7 days in NIH3T3 cell line. Differences in the cell multiplication curves of specimens exposed to plasma for 90 sec per day and those processed with He gas flow were also considered. With this experiment, we considered the effects of plasma exposure on cell multiplication based on both the trends of exposure time dependent cell multiplication and the effects of plasma exposure on cell multiplication. Fig 2 shows cells exposed to plasma. Culture medium thickness was 6 mm. Plasma was radiated from capillary of 1 mm diameter to cells adhering to 6 cm2 base area at flow rate of 1 L/min. Irradiation distance was 5 mm from culture medium. Plasma was radiated onto a point where well center and capillary tip were matched. Expression analysis procedure for each growth factor was as follows. Total RNA was extracted from cells exposed to plasma, cells processed with He gas flow only and cells with no processing by using ISOGEN (Nippon Gene). Reverse transcription reaction of the total RNA 1mg was then caused by using Super Script III RTase (Invitrogen) to create cDNA. PCRs of b-actin (internal standard), vascular endothelial growth factor (VEGF) and acidic fibroblast growth factor (bFGF) were amplified with the created cDNA used as a template (primer used for each PCR was designed as indicated in Table 1). After the PCR reaction, PCR production was confirmed by electrocataphoresis using 2% agarose gel containing ethidium bromide. As a result of the plasma exposure experiment on NIH3T3 cell line, cells to which He gas flow was applied were peeled off from base area of culture dish by stirring the culture medium with the gas. Therefore, cell multiplication was inhibited in proportion to processing time. However, it was found that this inhibition effect is suppressed by plasma irradiation and cells are rather on an upward trend. In addition to that, hematoxylin and eosin (HE) stain for cells cultured for 24 hours after plasma irradiation for 90 sec, and He gas flow processing caused obvious difference in cell density at the center of the dish. These results indicate that plasma irradiation promotes multiplication of cultured cell line. Consequently, we created cell multiplication curves by performing He gas flow and plasma irradiation processing on NIH3T3 cell line for 90 sec per day and plotting increase in the number of cells of each group for 1 weak. In comparison with the multiplication curve with the He gas flow group, the curve of the plasma irradiation group evidently shifted to the left. This indicates that NIH3T3 cell line causes a change in cell group, which is cell multiplication promotion, caused by plasma irradiation. Doubling time calculated from the multiplication curves was 25.8 hrs for the flow group and 18.8 hrs for plasma irradiation group. As doubling time of NIH3T3 cells normally cultured in this facility is approximately 23 hours, it was revealed that plasma exposure significantly accelerates cell doubling. As this experiment’s result was not enough to identify the significance of this phenomenon in terms of cell biology, we assumed that an interaction caused by collision of ion radical to plasma electrical field or culture medium surface influences multiplication and growth factors in the cells, and conducted expression analysis in the subsequent experiment with a focus on factors related to neoangiogenesis. As a result, it was found that the transient VEGF expression is observed in 24 hours from the plasma irradiation processing on NIH3T3 cell line. As this expression was not observed in the controlled group and He gas flow processing group, this strongly implies that VEGF is associated with cell multiplication promotion due to plasma exposure. This research revealed several facts about the effects of atmospheric-pressure plasma on cultured cells. First of all, irradiation of atmospheric-pressure plasma significantly activates multiplication of cultured cell line in comparison to the unexposed group. Secondly, since no NO gas is generated from the apparatus used in this experiment, it is likely that this multiplication promotion effect is associated with endogenous NO produced by cells, and not with NO generated by plasma. Further, as we had observed acceleration of wound healing in our animal experiment, we assumed that neoangiogenesis affects the influence of plasma on living bodies and considered expressions of VEGF and bFGF, which are typical factors inducing neoangiogenesis. As a result, we found significant difference between the plasma-exposed group and unexposed group. These factors stimulate endothelial precursor cells or enthodelial cells forming neoangiogenesis and activate intracellular signal transduction pathway primarily controlled by protein phosphorylation reaction. Past researches show that these factors cause activation of PI3K-Akt path, subsequent eNOS activation increase and NO production increase, and induce neoangiogenesis. Although detailed mechanism of the way NO produced through eNOS activation controls neoangiogenesis has not yet been revealed, various effects of podokinesis and cell death suppression to maintain “healthy” endothelial cells have been reported. It is, therefore, safe to say that production of fairly low-concentrated NO positively influences living bodies. In this research, it was concluded that some stimulus from atmospheric-pressure plasma to cells promotes NO production through factors such as VEGF and bFGF and activates cell multiplication. The “element” of plasma that causes the stimulus producing these effects is unknown. However, it is possible for the collision of ions and radicals generated from plasma to cells and tissue surface to produce stimulus that would promote various growth factors within cells and production of cytokine. We assume that this can formulate highly attractive healing approach that “enhances self healing ability,” which is clearly distinguished from healing approaches that “fill a gap” with medicines. Finally, we would like to express our sincere appreciation to Prof. Kanako Sawada and Prof. Kazuko Sato of Biomedical Engineering Department of Tokyo City University and Prof. Shigeru Inomata, Prof. Yuko Sugihara, and Prof. Yuichiro Suzuki of Department of Electrical and Electronic Engineering of Tokyo City University for their cooperation. This research was conducted as part of a research based on grant-in-aid for scientific research (B) from Ministry of Education, Culture, Sports, Science and Technology, “Activation of Nerve Cell Tissue by Less-invasive Micro-spot Atmospheric-pressure Plasma Source” and an assistance project for strategic research basis formulation of private universities by the ministry, “Development of Nano Carbon/Quantum Device Composite Biotip for Biological Interface.” Read More