Plasma in wound healing

Plasmas, due to their electrical and chemical properties, release in the invironment a unique cocktail of charged species, energetic photons and active radicals. Plasmas have a miriad of applications. To mention just a few, plasmas are being used in the semiconductors industry, atomic layer deposition, plasma ion immersion implantation, eching, deposition of nano-structured layers, surface cleaning, gas cleaning, water cleaning, and other environmental technologies. When we think of biological surface processing, the largest added value was brought by the so called cold atmospheric plasmas. For biological applications, the temperature of the plasma should be maintained as low as possible so the biological structrures are not affected. The electrons in the discharge have a temperature of a few eV, but the gas can remain at room temperature or slightly above. Recent advances in cold atmospheric plasmas pushed the technology in such a way that it can be used for biomedical applications. Those plasmas are able to emit amounts of active species in such a way that they stimulate cell growth and wound healing, the emited radiation does not generate visible damage to the DNA, and they are operational at human body temperatures. Compared with low pressure plasmas, the design of such cold atmospheric plasma devices should enhance heat losses and reduce the applied power. A reduction in the heat losses can be achieved by using a reduction of plasma surface to volume ratio, while a low operating power can be achieved by reducing the operating voltage (lowest breakdown voltage for helium and argon). Plasmas operating in air require high breakdown voltages. In our experiments, we used two plasna devices: the plasma needle and the kINPen. The plasma needle is a device operating at 13.56 MHz. The device is using helium as feed gas with a ¿ow rate of 0.5-2 l/min. The power input ranges from 10 mW to several Watts. The plasma needle generates a micro-plasma (0.1-2 mm glow size). In the 10-300 mW regime the gas temperature is about the same as the body temperature, above 1 W it can increase to 1000C and more. The plasma needle has been used for experiments on living cells, bacterial inactivation and skin. The intended applications are: wound healing, and the stimulation of cell proliferation. The glow can be applied directly to the tissues without causing any damage or discomfort. The second plasma source that we used was the kINPen 09, produced by the INP Greifswald, Germany. The device consists of a hand-held unit for the generation of a plasma jet at atmospheric pressure, a power supply, and a gas supply unit. In the centre of a quartz capillary (inner diameter 1.6 mm) a pin-type electrode (1 mm diameter) is mounted. In the continuous working mode, a high frequency (HF) voltage (1.1MHz, 2-6 kVpp) is coupled to the pin-type electrode. The plasma is generated from the top of the centred electrode and expands to the surrounding air outside the nozzle. The kINPen can be operated in helium, argon and compressed air. Several cell lines have been exposed to the effects of cold atmospheric plasma. We used 3T3 cells, human dermal ¿broblasts (HDF), Cho K1, HeLa cells and keratynocytes. In the case of 3T3 cells, the plasma needle resulted in a higher proliferation rate (reduction of the doubling time with 40 %) and a faster wound healing. Best results were obtained when the duration of the treatment was 10 -15 seconds, when a complete wound model healed in 3 days compared with 7 days for the control. No visible DNA damage was observed. Application of the helium-operated kINPen resulted in a still higher proliferation rate compared with the control, but less signi¿cant compared with the plasma needle. The effect is partly due to the high electric ¿elds present in the plasma needle: the electrical ¿elds are lower in the kINPen. Keratinocytes did not show such a high growth rate enhancement. HeLa cancer cells proved to be very sensitive to plasma treatment: they die readily after even a brief exposure to the plasma medium. Treatment of burned skin with the Plasma Needle results in a faster outgrowth in terms of the number of nuclei per outgrown micrometer, but not in a correspondingly larger outgrowth length. The Plasma needle results in effects which are larger than for the kINPen, which in turn is more effective than the control (untreated). The enhancement of growth rates of ¿broblasts was also observed in static electrical ¿elds, without plasma. In 3T3 ¿broblast cell cultures, we have observed a threshold value of about 2.04 kV/m when measured in the air (around 1 V/cm in the liquid). Electrical ¿elds below this threshold do not modify the cell growth rate at all. Electrical ¿elds above this value do cause a remarkable increase in growth rate of about a factor 2. At the threshold value, longer exposures seem to result in inhibition of the growth. For electrical ¿eld values above 4.7 kV/m, this inhibition is not observed: the growth rate increase is maintained over many days. This remarkable effect has been observed in 3T3 cells only. CHO-K1 cells, keratinocytes and bacteria do not seem to react at all to electrical ¿eld exposure. In the case of bacteria, experiments had been performed on Pseudomonas Aeruginosa and Staphilococcus Aureus. When the plasma needle was used, with helium as feeding gas, no effects of the plasma treatment were observed compared with the control. A comparison between helium, argon and compressed air plasmas was performed using the kINPen. Helium plasmas are not very ef¿cient: even after 5 minutes of treatment a lot of CFU’s were still present on the plate. Promising results were obtained for argon, and the best results were obtained in compressed air plasmas. The basic difference between these three plasmas is the chemistry. Electrical ¿elds and ion bombardment are comparable for these conditions, but the chemical composition of the "cocktail" that arrives at the surface of the bacteria is very different. Compressed air plasmas produce a very reactive cocktail of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RON) like nitrogen monoxide, nitrogen dioxide, ozone, hydroxyl radicals, etc. In plasmas in helium or argon, these species are created in the air entrained in the plasma jet, which is, of course, less productive then a direct plasma in air. The results suggest that the composition and thickness of the outer cell membrane play an important role in the process of plasma based bacteria inactivation. Gram positive bacteria (S. Aureus) seem to be more sensitive to plasma treat-ment than gram negative bacteria (P. Aeruginosa). Different mechanisms have been proposed in the literature to explain different sensitivity of different bacterias against plasma treatment. The cocktail of reactive species produced by the plasma (NOx, H2O2, OH radicals, ozone, UV etc) triggers the breaching of the membrane. Apparently, the thick lipopolysacharide outer membrane of gram negative bac¬teria is a better shield to reactive plasma species than the thinner peptidoglycans membrane of gram positive bacteria. All these results clearly suggest that plasma treatment offers perspectives for wound healing: bacteria can be killed or inactivated, cancer cells die, but ¿broblasts are stimulated in their proliferation. Eventhough the results are promising, and clinical tests around the world indicate an enormous potential, the toxicity on longer terms still need to be addressed further before the technique can really be sent into the market