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Published in final edited form as: Biol Chem. 2018 Dec 19;400(1):77–86. doi: 10.1515/hsz-2018-0235

Cold atmospheric plasma is a viable solution for treating orthopedic infection: a review

Ly Nguyen 1, Peng Lu 2, Daniela Boehm 3, Paula Bourke 4, Brendan F Gilmore 5, Noreen J Hickok 6, Theresa A Freeman 7,*
PMCID: PMC12105529  NIHMSID: NIHMS1935160  PMID: 30138104

Abstract

Bacterial infection and antibiotic resistance are major threats to human health and very few solutions are available to combat this eventuality. A growing number of studies indicate that cold (non-thermal) plasma treatment can be used to prevent or eliminate infection from bacteria, bacterial biofilms, fungi and viruses. Mechanistically, a cold plasma discharge is composed of high-energy electrons that generate short-lived reactive oxygen and nitrogen species which further react to form more stable compounds (NO2, H2O2, NH2Cl and others) depending on the gas mixture and plasma parameters. Cold plasma devices are being developed for medical applications including infection, cancer, plastic surgery applications and more. Thus, in this review we explore the potential utility of cold plasma as a non-antibiotic approach for treating post-surgical orthopedic infections.

Keywords: bacteria, biofilm, cold plasma, orthopedic infection, Staphylococcus aureus, titanium

Introduction

Orthopedic procedures often require the implantation of medical devices and hardware composed of metals; these metals are rapidly coated with serum proteins and thus present a preferred site for biofilm formation. Due to the presence of biofilm, infections associated with orthopedic implants are notoriously difficult to cure and when the specter of antibiotic-resistant organisms is included, it becomes clear that new therapies have to be defined. Although infections are rare, the presence of the bacterial biofilm on titanium or other implant materials, create complications that not only threaten the surgical outcome and the patient’s health, but also incur significant health care expense. In this review, we explore the potential of cold atmospheric plasma treatment to combat post-surgical infection of orthopedic implants, hardware and surgical site tissues. Application of this technology has the potential to provide a low cost, efficient and effective treatment which can be seamlessly integrated into current post-surgical infection treatment practices. Additionally, the use of cold plasma to eradicate orthopedic infection does not preclude treatment with antibiotics to synergistically alleviate the infection, and the associated pain, disability and suffering.

Post-surgical orthopedic infection

Surgical site infection accounts for a majority of complications in hospitalized patients, falling only slightly behind adverse drug events (Bakkum-Gamez and Dowdy, 2017). These infections cause approximately 88 000 deaths and cost 4.5 billion dollars per year. Adding to this is the global crisis of antibiotic resistance (Banin et al., 2017). The US Center for Disease Control and Prevention has declared antibiotic resistance to be among the world’s most pressing public health concerns. Tens of thousands of Americans are estimated to die from infections caused by antibiotic-resistant bacteria each year, and these number of infections are predicted to rise to 10 million people per year by 2050 (Banin et al., 2017). Infections associated with orthopedic implants are notoriously difficult to cure and when the specter of antibiotic-resistant organisms is included, it becomes clear that new therapies have to be defined.

Post-surgical infection of an implant, while relatively rare, can have devastating consequences, and when a prosthetic joint implant is involved (Bozic and Ries, 2005; Kurtz et al., 2012a,b); or the causative organism is antimicrobial resistant (Engemann et al., 2003), the cost of treatment can exceed $90 000/infection. In severe cases, implanted devices must be removed, which compromises patient mobility, results in long hospital stays, pain and disability, and at times even death (Hedequist et al., 2008). The number of orthopedic reconstructions and arthroplasties has steadily increased for the past five decades; with the majority being hip and knee replacements (Kurtz et al., 2012a,b; Tande et al., 2014). Taken together, this means that even if the rate of infection stays constant, the number of these types of difficult cases will continue to increase. Orthopedic hardware is also used in many other procedures, including another frequent procedure, spinal surgery for intractable, chronic back pain, degenerative lumbar stenosis or traumatic fractures. Even when an actual implant is not required, these operations frequently require other implanted materials or hardware, such as rods, plates and screws to permit reconstruction and to stabilize the spine and skeletal geometry while healing (Lalli et al., 2015). Like joint implants, these sites become infected in up to 14% of cases (Kurtz et al., 2012a,b), with even fewer options than exist for the joint replacements.

Biofilm formation and consequences in orthopedic infection

The most common pathogens associated with orthopedic infections are the staphylococci, with Staphylococcus aureus as the most common strain (Shirtliff et al., 2002); coagulase-negative staphylococci and other Gram-positive and Gram-negative bacteria account for the remaining infections (Gbejuade et al., 2015). Of further concern is the presence of methicillin-resistant S. aureus (MRSA) (Schimmel et al., 2010) which significantly lowers the chance of successfully eradicating joint infection. MRSA are found in 17.8% of deep spinal fusion infection cases and in these cases even the use of antibiotics is rarely successful (Pull Ter Gunne et al., 2013). While the most prevalent implant- and hardware-associated infections are due to staphylococci (Trampuz and Widmer, 2006; Corvec et al., 2012; Tande et al., 2014), other Gram-positive cocci are also involved at lower frequency, including streptococci (1%–10%) and enterococci (3%–7%). Gram-negative bacilli causing infection include Pseudomonas aeruginosa and Enterobacteriaceae 6%–17%, (Trampuz and Widmer, 2006; Corvec et al., 2012; Tande et al., 2014) and in rare cases the anaerobes (Propionibacteria and peptostreptococci) (Trampuz and Widmer, 2006; Corvec et al., 2012; Tande et al., 2014). In shoulder implants, Propionibacterium acnes have been more prevalent, at up to 38% (Achermann et al., 2013).

While the identity of the pathogen determines treatment, to some extent, all bacteria behave similarly when in the presence of an implant. Specifically, upon insertion of an implant into a physiological environment, these materials (commonly alloys such as Ti6Al4V, CoCrMo, and stainless steel as well as polymers/ceramics) are rapidly coated with serum proteins. Because of this coating, pathogens use their normal physicochemical interactions, as well as, extracellular matrix-specific binding proteins to rapidly adhere to the implanted hardware, ultimately forming biofilms. In addition, in the physiological environment, contaminating bacteria can aggregate with each other to form floating biofilms that, like the implant-associated biofilms, exhibit an altered phenotype with decreased metabolism, altered virulence factor production and decreased antibiotic sensitivity (Dastgheyb and Otto, 2015; Dastgheyb et al., 2015). These physiological biofilms are minimally comprised of bacteria, fibronectin, fibrin, polysaccharide intercellular adhesion (PIA) and extracellular DNA (134, 146). Together this clinically-relevant biofilm appears to be a hybrid of bacteria adhered to cross-linked proteins which are all encased in biofilm slime. In addition to the bacteria being encased in the protective extracellular polysaccharide layer, metabolic heterogeneity exists within the biofilm matrix, leading to elevated tolerance to antimicrobial challenge and an accumulation of antibiotic-degrading enzymes, or expression of specific antibiotic-resistance genes (de la Fuente-Nunez et al., 2013). Thus, the central challenge in treating orthopedic infection is to eradicate bacteria within the biofilm structure which sequesters them from the immune system and antibiotic eradication.

Current treatment of post-surgical infection

Early detection of infection can be difficult as symptoms may take up to a week to manifest. The surgical site may be painful, warm, swollen or red, but not be infected (Atkins and Bowler, 1998; Bauer et al., 2006). Classical radiographic, microbiologic and clinical signs can be diagnostic, ambiguous or not present (Parvizi et al., 2014). Prevention of the infection is also difficult, as no clear advantage has been demonstrated by a prolonged course of antibiotic therapy, and effectiveness is not observed past 24 h post-surgery, most likely due to the antibiotic recalcitrance of adherent and biofilm bacteria (Antoci et al., 2007; Lewis, 2008). In general, the orthopedic infection is treated by removal of any infected tissue/implants, disinfection and prolonged, aggressive antibiotic treatment. Specifically, the surgical approach used to combat an orthopedic infection can include extensive tissue debridement with prosthesis retention (Tande et al., 2014), or a one-stage or two-stage arthroplasty exchange, which includes debridement of the infected tissues, removal of the implant and insertion of a new implant. While it would be preferred that essential instrumentation remains in place to prevent instability and loss of corrective purposes, when possible, removal of instrumentation for optimal disinfection may give the best outcomes. Because of the requirements for surgical stability, removal of instrumentation becomes a special problem for infections associated with spinal hardware. In these cases, disruption using irrigation solutions (e.g. dilute betadine, acetic acid, hydrogen peroxide, chlorhexidine, etc.) along with physical disruption (e.g. pulse lavage, VersaJet, etc.) are used; but these techniques possess their own problems, such as toxicities to normal cells and tissues and causing the bacteria to be spread and lodged in other tissues. Superficial extra-fascial infections are often controlled with antibiotics, in combination with surgical incision and drainage (Tande et al., 2014). Deep sub-fascial wounds necessitate debridement and elimination of all affected necrotic tissues and it is becoming increasingly more common to place supratherapeutic levels (1–2 g) of vancomycin (VAN) directly into the surgical site before closure (Hegde, 2013). VAN is used because of its effectiveness against Gram-positive methicillin sensitive and methicillin-resistant S. aureus (MSSA and MRSA). However, this treatment may indirectly facilitate the establishment of Gram-negative bacterial infections (Ghobrial et al., 2014). Ideally, the development of cold atmospheric plasma as a safe, effective treatment that could decontaminate the implant surface and the surgical site in situ would prevent the risks associated with spinal instability and reduce patient pain, immobility and the incidence of pseudarthrosis.

Cold atmospheric plasma: composition, mode of action and bacterial response

Cold atmospheric plasma is an ionized gas created by high-energy electrons colliding with atmospheric molecules at tissue tolerable temperatures. The species generated within the ionized gas can be tuned for specific biological applications by varying plasma parameters and the gas composition in which the plasma is generated. We and others have shown that cold plasma can kill bacteria, degrade biofilm matrices, modify extracellular matrix properties and direct cell signaling to promote specific cellular behaviors (Ermolaeva et al., 2011; Flynn et al., 2015, 2016; Ziuzina et al., 2015; Eisenhauer et al., 2016). Other applications include elimination of cancer stem cells, induction of basal dermal keratinocytes proliferation and promotion of wound healing through the inhibition of the gap-junction proteins (Hasse et al., 2016; Schmidt et al., 2017a,b).

The composition of cold plasma allows for various modes of action depending on the plasma parameters and target. The specific reactive species (whose nature and relative prevalence depends on the type of inducer gas used) generated by cold plasma can cause oxidative stress, resulting in rupture of bacterial cell membrane and intracellular damage (Alkawareek et al., 2014). Physical stimuli can include local and global electric fields and biofilm permeabilizing shock waves produced by pulsed dielectric barrier discharged plasmas (Beebe et al., 2004; Babaeva and Kushner, 2010). However, it is the short-lived reactive oxygen and nitrogen species reacting with local molecules that appear to be the critical effectors of antimicrobial effects where the reactive species include potent and stable antimicrobial compounds such as nitric oxide (NO2), peroxide (H2O2), hypochlorous acid (HOCl), ammonium chloride (NH2Cl) and other species. These oxidizing agents, whose concentrations increase proportionally with increases in applied voltage, target bacterial DNA, RNA, proteins and lipids (Naitali et al., 2010).

The sensitivity to cold plasma inactivation differs between bacterial type and with biofilm formation capacity. The cell wall composition differs between Gram-positive and -negative bacteria. Gram-positive bacteria are characterized by the presence of the peptidoglycan lipoteichoic acid which decorates the cell wall. Gram-negative bacteria possess an outer cell membrane with abundant lipopolysaccharide (LPS); these differences in membranes/walls are critical for activity for some classes of antibiotics. Cold plasma generated reactive oxygen species induce the breakdown of both lipopolysaccharides and peptidoglycans but, the mechanism by which the bacterium dies appears to differ (Figure 1) (Han et al., 2015). In the Gram-negative Escherichia coli, cellular leakage was observed during- and post-cold plasma treatment. No such leakage was measured in the Gram-positive S. aureus. Thus, it was proposed that peroxidation of the E. coli cell membrane, specifically of the lipid content in the lipopolysaccharide layers, may lead to destabilization of the cell envelope and death though membrane leakage. Similar results would be expected in other Gram-negative bacteria, such as P. aeruginosa. Gram-positive species (L. monocytogenes and S. aureus), on the other hand, showed evidence of cell envelope shrinkage, and significantly higher intracellular ROS concentrations associated with DNA damage, leading to death (Han et al., 2015). A recent study shows cell wall thickness correlated with cold plasma inactivation times, but cell membranes and biofilm matrix also affected the time required to inactivate bacteria (Alkawareek et al., 2014; de Mesy Bentley et al., 2017). Bacterial inactivation efficacy by direct application of cold plasma is determined by the power, time of exposure, and composition of carrier gas (Adeli and Parvizi, 2012). For more detailed reviews readers are referred to the articles by Bourke et al. (2017) and Gilmore et al. (2018).

Figure 1:

Figure 1:

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Differences in the effect of cold plasma treatment on Gram-positive vs. Gram-negative bacteria (adapted from Han et al., 2015).

Plasma activated liquids

In addition to direct application of cold plasma, an indirect method has also been employed using plasma activation of liquids (PAL); these liquids include; water, saline and cell growth media. Indirect treatment of microbes by PAL also results in significant inactivation (Naitali et al., 2010; Shen et al., 2016; Lu et al., 2017). Water that has been activated by plasma discharges consistently showed powerful antimicrobial activity, where this activity was thought to be due to the presence of stabilized reactive nitrogen and oxygen species (Naitali et al., 2010). Specifically, OH radical, ozone, atomic oxygen and hydrogen peroxide are the main agents responsible. Reactive nitrogen species meanwhile include nitric oxide and products of its reaction with water, such as nitrites, nitrates and peroxynitrites (Shen et al., 2016). This antimicrobial activity appears to be due to the combined action of, acidified nitrates and hydrogen peroxide added to nitrites, rather than oxidation caused by the former two alone (Naitali et al., 2010). The advantages of using PAL for bacterial inactivation include less emission of toxic gases and elimination of the transportation/storage of hazardous chemicals, especially chlorine-based products that contain potentially carcinogenic chlorinated organic compounds (Shen et al., 2016). Extensive publications detail the use of both direct and indirect treatments to kill spores, Salmonella typhimurium, L. monocytogenes and E. coli biofilms and bacteria internalized in produce, as well as destruction of P. aeruginosa biofilm architecture and viability (Mody et al., 2009; Ziuzina et al., 2014; Dastgheyb et al., 2015; Mai-Prochnow et al., 2016).

It is interesting to note that the synergistic effects of these oxidizing agents and an acidic pH seem to determine the final antimicrobial effects of PAL in that a low pH creates favorable conditions for penetration of the cell membrane by ROS. Chemical reactions can also be accelerated by acidic conditions, especially those created by hydroperoxyl radicals that have strong oxidizing powers and cause peroxidation of membrane fatty acids. Because of the similar chemical makeup, plasma-activated liquids impose damage in the same manner as direct cold plasma (lipid peroxidation and subsequently cross-link reaction of the fatty acid side chain), with leakage of intracellular DNA and proteins being the direct cause of cell death (Han et al., 2015). Remarkably, attack by O and ·OH radicals formed transient pores in the membrane, which eventually lead to depolarization and permeabilization.

To employ PAL as an antibacterial therapy, however, its efficacy has to be standardized by controlling the reactive species contained with the liquid. Using different plasma working gases has been the main approach to regulate the reactive species composition. One plasma source that has been used extensively for PALs generation is based on plasma jet configuration. By changing the composition of plasma working gas and plasma jet surrounding gas, the reactive species composition can be controlled (Stoffels et al., 2008; Wende et al., 2015; Shen et al., 2016). Gas bubbles discharge in liquids with different working gases is another approach for selective generation of reactive species in PALs (Shen et al., 2016). A recent advance in this area is that without using any additional or noble working gases, an open-air discharge with different discharge modes (spark discharge and glow discharge) above liquids will produce reactive species specificity in plasma activated water (PAW) (Lu et al., 2017). Furthermore, using hydrogen peroxide and nitrite as principal reactive species’ indicators it has been shown that the cytotoxicity of PAW can be removed and/or enhanced by formulating their concentrations and composition through adjusting the discharge mode and time, again without the addition of working gas or chemical scavengers (Lu et al., 2017). These publications provide insights into how plasma treated liquids may be harnessed to create safe and effective antimicrobial liquids that can be applied during surgery to provide a specific and controllable treatment with increased efficacy.

Immunogenic enhancement by plasma modification of bacterial antigens

In addition to eradicating biofilms and bacteria, cold plasma treatment could also stimulate a more robust immune response against the bacteria that may survive the initial treatment. This is highly desirable considering the challenging treatment environment associated with a surgical site. Specifically, in the process of killing bacteria, cold plasma treatment of biological tissues and/or bodily fluids could generate additional reaction products during the course of treatment including: Cl2−, ClO− and hypochlorous acid (HOCl) as a result of peroxide generation by the plasma (Wende et al., 2015). Oxidation of bacterial macromolecules by HOCl can increase the uptake and processing of bacterial neoantigens by antigen-presenting cells (APCs) (Chiang et al., 2006, 2008; Biedron et al., 2015). This mechanism is similar to that employed by neutrophils to generate antimicrobial effects and antigen immunogenicity through myeloperoxidase activity (Winterbourn et al., 2016). Interestingly, in a clinical trial, patient-derived dendritic cells (DCs) were activated by tumor lysates oxidized by HOCl and successfully used for vaccination (Chiang et al., 2013). Our work shows that direct in situ nanosecond pulsed plasma treatment of tumor nodules resulted in a 60% eradication of the primary tumor, and our preliminary data and other published studies strongly suggested in vivo promotion of immune recognition (Chernets et al., 2015; Mizuno et al., 2017). When rechallenged after primary tumor eradication, the growth of a second tumor was either unsuccessful or dramatically slowed, indicating immune recognition and memory of tumor antigens. Thus, we propose the exciting possibility that cold plasma treatment stimulates an extensive platform of mechanisms that, as explored in the tumor, can kill bacteria, eradicate biofilms and produce bacterial neoantigens to strongly promote an immune response against any remaining bacteria.

Extracellular matrix modification by cold plasma

Another potential effect of cold plasma treatment of surgical site tissues could be extracellular matrix modification. Several studies have shown electric fields, ultrasound, mechanical strain and other biophysical stimuli enhance fracture repair and endochondral ossification through modification of cell extracellular matrix (ECM) interactions (Bassett et al., 1982; Brighton et al., 1985; Goodship and Kenwright, 1985; Kenwright and Goodship, 1989; Pilla et al., 1990; Joyce and Bolander, 1992; Heckman, 1994; Fredericks et al., 2000). Investigation of direct biophysical stimuli and reactive species effects on ECM found ‘matricryptic’ sites on ECM proteins and carbohydrate groups that are exposed by structural or conformational alterations (Davis et al., 2000). Thus, biologically active ECM peptide fragments, termed ‘matricryptins’ are generated after exposure to biophysical stimuli and injury, creating favorable conditions for tissue remodeling, promoting cell proliferation and migration (Beattie et al., 2008; Reing et al., 2008; Tottey et al., 2011). Our study comparing bone formation after cold plasma treatment with either microsecond or nanosecond pulsed dielectric barrier discharge plasmas (DBD) suggests that modification of amino acid side chains may occur (Eisenhauer et al., 2016). Our analysis of both Matrigel and type IV collagen by Fourier-transform infrared (FTIR) spectroscopy and Western blot showed no detectable structural changes or cleavage of the molecules occurred. Production of H2O2, NO and ONOO− by cold plasma can modify amino acid side chains and thus alter the basic nature of collagen. Specifically, carbonyl groups in proline/hydroxyproline (prevalent in collagen) can be convert by ONOO− to a nitroso will produce the more basic n-nitrosopyrrolidine amino acid (Ahmad and Ahsan, 2011). Differences in the production of ONOO− can produce different modifications of the amino acid side chains to permit positive or negative cell-matrix interactions.

Tissue tolerance

Despite its promise of effectively eliminating bacteria and destroying biofilms, the strong oxidizing nature of cold plasma-generated reactive species can also pose a significant cytotoxicity problem for mammalian tissues. The efficacy of bacterial inactivation is determined by power, mode of exposure, time of exposure and composition of carrier gas. Similarly cytotoxic effects of cold plasma on eukaryotic cells, such as human keratinocytes and fibroblasts (Brun et al., 2012), also appear to be mediated predominantly through H2O2 generated by the plasma discharge (Bekeschus et al., 2014; Winter et al., 2014). However, cold plasma-treatment induced damage to tissues such as the stratum corneum of skin tissues are usually confined to the upper cell layers, suggesting the extracellular matrix and superficial nature of cold plasma may protect tissues from plasma-induced injury (Fluhr et al., 2012). Thus, the main objective would be to determine if cold plasma can be tuned using specific technical parameters (power, time of treatment, gas flow rate) to eradicate biofilm bacteria but maintain a ‘tolerable’ cytotoxicity to the tissues involved in the post-surgical orthopedic field. The extensive extracellular matrix surrounding bone and cartilage should have a protective effect against cold plasma to render them less vulnerable. Unfortunately, most studies to date have been confined to in vitro cell culture and surface tissues such as skin. In vivo studies of mouse skin models showed neither localized nor systemic adverse effects to repeated cold plasma treatments after follow-up periods of up to 1 year (Schmidt et al., 2017a,b; van der Linde et al., 2017) and indicated improved wound healing (Hung et al., 2016). Clinical data available from human trials to-date indicate an absence of adverse effects such as inflammation and no increased risk of a formation of pre-cancerous skin alterations (Isbary et al., 2012; Heinlin et al., 2013; Metelmann et al., 2013). However, it is imperative to test cold plasma-induced cytotoxicity in relevant tissues to permit assessment of all factors within the heterogeneous tissues, including growth factors, cells, vasculature, extracellular matrix composition, etc., to determine appropriate human in vivo conditions. Currently, clinically approved cold plasma generators, such as the Bovie Medical J-Plasma® device and the kINPen® MED, permit changing between multiple settings or levels during the course of a treatment thus permitting the use of different power levels, gas flow or pulsed modes to eradicate biofilms adhering to titanium in acellular areas and then adjust to a tissue tolerable level for the surrounding tissues (Parsa, 2015; Hilker et al., 2017; Gentile, 2018).

A few studies have been performed to show the feasibility of this. Disinfection of ocular cells and tissues using a helium-based portable plasma device to generate low power cold plasma showed low cytotoxicity (Brun et al., 2012). Fibroblasts and keratinocytes treated with cold plasma showed negligible decreases in number an hour after treatment, while bacterial viability using CFU counts, were reduced by 90% (Ermolaeva et al., 2011). The same treatment time increased to 5 min significantly reduced human cell viability. Short treatment times of 2 min using an argon plasma jet were also shown to effectively reduce high bacterial numbers of P. aeruginosa by up to 6 log without affecting the viability of dermal fibroblasts or keratinocytes in vitro (Boekema et al., 2013). Increased intracellular ROS is observed in both microbial and mammalian cells, however, the antioxidant response is greater in some cell types which serve to protect their viability (Wende et al., 2010; Pai Kedar et al., 2015) and promote proliferation to restore the numbers over time. Therefore, there is evidence supporting that minimal tissue damage can be achieved at specific settings and the possibility of increased cell proliferation (Ermolaeva et al., 2011; Brun et al., 2012; Fluhr et al., 2012; Hasse et al., 2016; Suzuki and Yoshino, 2016; Choi et al., 2017) and migration (Brun et al., 2014) of mammalian tissue after cold plasma treatment to restore tissues after exposure.

Justification of cold plasma as a viable treatment option for post-surgical orthopedic infection

Cold atmospheric conditions have consistently been shown to have antimicrobial effects and has real promise as a new non-antibiotic candidate for treating infections. It is already being used to inhibit Listeria sp. on vegetables, processed milk and dairy products in place of thermal pasteurization, and inactivates P. acnes bio-films that infect post-surgical artificial joints, heart valves, shunts and catheter implants. It has been shown that repeated application of cold plasma does not induce resistance in S. aureus biofilms and neither primary nor acquired resistance occurs in MRSA strains exposed to cold plasma treatment (Zimmermann et al., 2012; Matthes et al., 2014), reinforcing the novelty of the approach in treating infection. Importantly, FDA and EU approved (or pending approvals) for several cold plasma devices exist under different commercial device names (J-Plasma®, kINPen® MED, Canady Hybrid Plasma® Scalpel). Thus, it is plausible that development or adaptation of a cold plasma device to treat infected orthopedic surgical sites could be quickly adapted for use within the constraints of the current medical protocols that guide treatment of surgical site infection. Additionally, plasma treated saline or water could be used instead of sterile saline, or in addition to other disinfecting solutions commonly used to rinse the site. A diagram illustrating the use is shown in Figure 2. After debridement of the necrotic tissue the hardware or implant could be removed or remain and be directly treated with cold plasma at higher powers to destroy biofilm and kill bacteria when removal is unnecessary or undesirable. Low power plasma or plasma activated saline could also be used directly on the tissue. It is plausible that while some surface tissues and cells may be damaged, underlying tissues would be stimulated by the diffusion of reactive species within the plasma, which stimulate tissue growth and wound healing. Cold plasma is currently being tested on facial skin to remove wrinkles. It is most likely that the mechanism by which this works is through micro-wounding, which stimulates wound healing and tissue remodeling. Application of a cold plasma treatment would take only a few minutes, and thus will not prolong the normal surgical procedures. Importantly, through the combined effects of direct cold plasma treatment and persistence of any remaining plasma-activated liquids, long-term efficacy of the treatments would be enhanced.

Figure 2:

Figure 2:

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Diagrammatic representation depicting treatment of spinal hardware site with direct cold atmospheric plasma (dCAP) and plasma activated liquid (PAL) to eradicate biofilm contamination.

Conclusion

In conclusion, multiple studies need to be performed to (1) determine the technical parameters of cold plasma, alone and in combination with PAL, that have biofilm-eradicative properties but also maintain tissue viability, (2) determine the tolerable level of cold plasma-induced cytotoxicity and measure healing response and immune cell activation, and (3) determine long-term effects of cellular damage and recovery of tissue viability.

Funding:

National Institutes of Health (Funder Id: 10.13039/100000002, Grant No: RO1 AR069119). National Institutes of Health’s funded under Award Numbers RO1 AR069119 and RO1 AR072513 were also used to support this work and for Dr. Bourke the Science Foundation Ireland under the Grant Number SFI/16/BBSRC/3391 and the BBSRC under Grant Reference BB/P008496/1.

Contributor Information

Ly Nguyen, Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107-5099, USA.

Peng Lu, College of Science and Health, Dublin Institute of Technology, Dublin, Ireland.

Daniela Boehm, College of Science and Health, Dublin Institute of Technology, Dublin, Ireland.

Paula Bourke, College of Science and Health, Dublin Institute of Technology, Dublin, Ireland.

Brendan F. Gilmore, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK

Noreen J. Hickok, Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107-5099, USA

Theresa A. Freeman, Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107-5099, USA.

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