The term plasma medicine was first used only two decades ago but research in this field has grown tremendously in recent years. Great advances have been made in the knowledge of the mechanisms involved in the interaction between cold atmospheric plasma and living cells and tissues . Numerous research groups across the globe, especially in the United States and Asia, are trying to gain a deeper understanding of these mechanisms. Results of numerous experiments in-vitro, ex-vivo and in-vivo have been published showing the excellent properties of this treatment and the arrival of this technology to our reference hospitals is getting closer.
By mid-1990s, the scientist Mounir Laroussi showed that cold atmospheric plasma could be used to inactivate bacteria . Based on these results, the Directorate of Physics and Electronics of the Air Force Office of Scientific Research of the United States (AFOSR) decided to finance a research program for the use of cold plasma in the treatment of wounded soldiers and for the sterilization and disinfection of surfaces. In the early years of the 2000s, numerous studies with eukaryotic cells were carried out and it was found that in small doses cold plasma treatment induces phagocytosis, accelerates fibroblast proliferation, produces mammal cell shedding without causing necrosis and, under certain conditions, leads to apoptosis [70, 71].
Efforts in this field multiplied recently and knowledge of plasma treatment applications has greatly grown [38, 72]. In 2008 the Food and Drug Administration (FDA)  approved the use of cold atmospheric plasma in dermatological applications and in 2010 the first clinical trial for wound treatment was carried out, resulting in the approval of the treatment in 2013. More recently, in mid-2019, the FDA has approved the first clinical study for cold atmospheric plasma cancer treatment in the United States .
Applications can be divided into five large areas that are, of course, closely linked:
Disinfection and sterilization
Plasma application is lethal to bacteria and other microorganisms. In addition, it does not generate resistance and it is capable of killing even antibiotic-resistant bacteria . This is of vital importance at present as the incidence of infections by resistant bacteria in the population is increasing. Cold atmospheric plasma provides an important contribution to the solution of this very serious global problem. According to a survey by the Centers for Disease Control and Prevention in the United States (CDC), every year more than 2,800,000 people suffer an infection with antibiotic-resistant bacteria and approximately 36,000 of them die as a result .
The wound healing process is frequently affected by the presence of bacteria and the substances they generate . The tissue reacts with what we know as infection if the amount of bacteria exceeds a certain limit . In addition, the presence of a high bacterial load not only causes infection but also inhibits the healing process. That is why the reduction of bacterial load favors healing . Several studies show that treatment with plasma inactivates bacteria very efficiently and reduces the bacterial load in chronic wounds. It also eliminates biofilm without producing side effects or allergic reactions.
The wound healing process is frequently affected by the presence of bacteria and the substances they generate . The tissue reacts with what we know as infection if the amount of bacteria exceeds a certain limit . In addition, the presence of a high bacterial load not only causes infection but also inhibits the healing process. That is why the reduction of bacterial load favors healing . Several studies show that treatment with plasma inactivates bacteria very efficiently and reduces the bacterial load in chronic wounds. It also eliminates biofilm without producing side effects or allergic reactions [80–86], even in complicated wounds such as venous leg ulcers .
It has been shown that cold atmospheric plasma treatment accelerates healing without necrosis, reduces bacterial load on wounds (even on those colonized by antibiotic-resistant bacteria ) and favors angiogenesis [89, 90]. The use of plasma in diabetic ulcers is especially convenient since these, in many cases, remain open for long periods of time. Recent studies published in the prestigious journal Nature show the excellent results obtained in experiments carried out in murine models [91, 92].
The application of cold plasma treatment in burns is also of great interest. These injuries are usually caused by accidents and have a higher incidence in children and elderly people. In second and higher degree burns the skin does not have the capacity to reepithelialize, this implies an aesthetic damage and a functional risk that can threaten the patient’s life if they are extensive . One of the main risks is infection; and the combination of disinfectant, regenerative and healing properties of cold plasma makes it an enormously useful tool in this field. Through in vitro models that reproduce different stages of angiogenesis, it has been shown that plasma induces nitric oxide production, promotes cell migration and epithelial cell attachment to form vessel-like structures, both indicators of the proliferation phase in wound healing .
Cold atmospheric plasma applications in dentistry include its use in cavity treatment, sterilization, biofilm removal, root canal disinfection, bleaching and improved adhesion at the junctions between dentin and composites [95-97]. Many oral conditions such as cavities and some periodontal or intraoral diseases are produced by bacteria; for this reason, the disinfectant capacity of cold plasma makes it an ideal tool as part of the treatment [98, 99]. The cold plasma allows to decontaminate cavities without drilling; this makes it possible to keep a larger part of the tooth and would be of special help to the professional in case of treating children [100, 101]. In the same way, the possibility of disinfecting the root canal through plasma treatment, without the need to use laser or mechanical methods that can cause destruction of healthy tissue, has attracted the attention of several research groups. Plasma treatment disinfects the root canal without pain feeling. The root canal has a complex structure with isthmus, deltas, ramifications and irregularities that facilitate bacteria places to stay but which the plasma can access [9, 102].
The World Health Organization estimated 14 million new cancer cases in the world in 2012 . The search for new complementary therapies, less invasive and with fewer side effects is rapidly growing. The cold atmospheric plasma induces the apoptosis of the tumor cells and, therefore, there is a growing interest on it as a tool for fighting against cancer. One of its main advantages compared to other therapies is its selectivity, since it attacks tumor cells without damaging the healthy ones [104-111].
From the mid-2000s, different experiments showed that cold atmospheric plasmas could destroy tumor cells in vitro. Subsequently, it was shown that these plasmas can also reduce the size of cancer tumors in animal models [105, 112-118]. In 2019, the first human clinical study was approved in the United States .
Implant treatment to increase the biocompatibility
Cold plasma treatment increases the wettability of the implant surface and modifies the oxide layer that interacts with the cells and proteins of the surrounding tissue. Consequently, its application can improve tissue adhesion [119-121].
Therapeutic action principles
The first question that arises when studying the applications and possibilities of cold atmospheric plasma treatment is:
How does it work?, what are the physico-chemical mechanisms involved?
Although this is a field in which much remains to be discovered, researchers agree to target reactive oxygen and nitrogen species (O, OH, H2O2, O3, NO, NO2) present in the plasma as responsible for the effects in eukaryotic and prokaryotic cells [122-127]. These reactive species appear naturally in cells as products of their biochemical processes and they have an important role in cell communication and homeostasis .
The second question that is natural to ask is:
How can cold plasma be a selective treatment? How can plasma kill bacteria and cancer cells without damaging healthy tissue?
It has been proven both by direct observation and by histological studies that sterilization is achieved with treatment doses one hundred times lower than those necessary to produce tissue damage . Bacteria are much smaller than mammalian cells (approximately two orders of magnitude), this makes their surface/volume ratio much lower and, therefore, the concentration in cytoplasm of reactive species is much greater than in eukaryotic cells for equal treatment times. Therefore, the concentration capable of producing cell death is achieved with much shorter treatment times in bacteria. In addition, there are biological differences between both organisms, for example, polysaccharides are easier to peroxidate than lipids.
In the case of cancer cells, it is precisely the alteration of cell cycle that they suffer what makes them sensitive to cold atmospheric plasma treatment. Cancer is essentially an uncontrolled cell division disease; because of this, the tumor cells are in a continuous reproductive process . During cell division, the nucleus disappears and the DNA is exposed in the cytoplasm making cells much more sensitive. In this state, the increase in concentration of reactive species inside the cell can produce the arrest of DNA replication, the disruption of double chains and the induction of apoptosis. Several scientific studies show that reactive species can interact with the cell membrane and even penetrate the cell, producing reactions that can induce cascades of cellular signals that ultimately result in the death of tumor cells [118, 123-126, 130-132].
Another of the key questions that arise around cold plasma treatment is whether the generated reactive species affect only the most superficial cell layer or if they can penetrate the tissue . Several experiments have shown that, in fact, reactive species also affect cells that are not directly on the surface and, therefore, have no direct contact with plasma [133-136]. The explanation for this phenomenon is still under study but one possibility is that there are chemical signals produced by the surface cells (in contact with the plasma) and sent to cells that are at deeper levels . These signals would cause reactions similar to those occurring in surface cells, including induction of apoptosis.