The Emerging Role of Cold Atmospheric Plasma in Implantology: A Review of the Literature

In recent years, cold atmospheric plasma (CAP) technologies have received increasing attention in the field of biomedical applications. The aim of this article is to review the currently available literature to provide an overview of the scientific principles of CAP application, its features, functions, and its applications in systemic and oral diseases, with a specific focus on its potential in implantology. In this narrative review, PubMed, Medline, and Scopus databases were searched using key words like “cold atmospheric plasma”, “argon plasma”, “helium plasma”, “air plasma”, “dental implants”, “implantology”, “peri-implantitis”, “decontamination”. In vitro studies demonstrated CAP’s potential to enhance surface colonization and osteoblast activity and to accelerate mineralization, as well as to determine a clean surface with cell growth comparable to the sterile control on both titanium and zirconia surfaces. The effect of CAP on biofilm removal was revealed in comparative studies to the currently available decontamination modalities (laser, air abrasion, and chlorhexidine). The combination of mechanical treatments and CAP resulted in synergistic antimicrobial effects and surface improvement, indicating that it may play a central role in surface “rejuvenation” and offer a novel approach for the treatment of peri-implantitis. It is noteworthy that the CAP conditioning of implant surfaces leads to an improvement in osseointegration in in vivo animal studies. To the best of our knowledge, this is the first review of the literature providing a summary of the current state of the art of this emerging field in implantology and it could represent a point of reference for basic researchers and clinicians interested in approaching and testing new technologies.


Introduction
In recent years, plasma-enabled biomedical technologies have emerged as a promising approach for non-chemical, low-temperature decontamination in the biomedical, food manufacturing, and food service industries. Their use in medicine extends into synergistic and personalized plasma-enabled therapeutics for tissue regeneration [1,2], oncotherapy [3,4], and dermatology [5]. A rapidly growing body of evidence documents their use in disinfecting living and abiotic targets, promoting cell differentiation and migration, and enhancing tissue regeneration and wound healing. Among the necessarily related to surface damage or to the enhancement of surface roughness [17], although this aspect is still under debate.

Plasma's Potential Biomedical Applications
CAP has showed encouraging results in decontamination, blood clotting, skin disease treatment, cancer therapy, and oral medicine [12].

Systemic Applications
In medicine, CAP treatment may be successfully applied in dermatology [5], blood coagulation [31], surgical instrument and consumable decontamination [32], and the hydrophilic property enhancement of the surfaces of biomaterials [27]. Due to its antimicrobial effects, CAP has also been proposed for water disinfection since its application determines a series of exposure and postexposure channel reactions, which result in water purification [33].
The potential use of CAP in clinical oncology has been recently analyzed and it is obtaining a growing interest in the scientific community [11]. As currently reported by Semmler et al. [4], plasma application would induce tumor cell death (i.e., necrosis, apoptosis, senescence, and autophagy) in a dose-dependent manner, as well as decrease their adhesion, migration, and invasion, reducing cancer cell diffusion and metastasis forming ability [4]. Specifically, the same authors reported promising results in the treatment of head and neck squamous cell carcinomas in terms of lesion regression as well as pain reduction. However, the underlying mechanism determining the tumor cell arrest and the relative immune response have not been elucidated yet and need further evaluation.
Another promising field for CAP application is dermatology [5], as demonstrated by the absence of both damage of the skin barrier and a reduction in skin hydration following plasma usage [13]. Moreover, when applied in vivo, plasma treatment may speed up tissue granulation and enhance wound healing [14,34,35]; Daeschlein et al. [36] showed the ability of CAP to restrain the microbial colonization of chronic wounds. Although promising, the dermatological application of CAP should be studied in depth to render the treatments more effective and stable over time.

Oral Applications
In the oral medicine field, CAP has been applied to the treatment of dental caries, periodontal disease, implantology, teeth whitening, endodontic infection, tooth remineralization, an increase in the bonding efficacy of composite resin, and the disinfection of dental instruments [11,12,17,37]. The idea of using CAP for innovative dental procedures was first proposed by Goree et al. [38], who demonstrated the ability of a plasma needle to reproducibly kill the most cariogenic bacterium, Streptococcus mutans, thus attracting the interests of researchers from the dental field.
Since then, the largest area of investigation into CAP has dealt with endodontics. Indeed, the elimination of bacteria in infected root canals, especially with persistent periapical lesions, still remains an unsolved issue, as conventional chemical irrigants fail to achieve the eradication of bacteria in the root canals [39]. For these reasons, CAP can be seen not only as an alternative but also as an adjunct to investigate synergistic treatments. An interesting study comparing the antimicrobial efficiency of plasma jets with chemical irrigation solutions, such as chlorhexidine (CHX) and Sodium hypochlorite (NaOCl) against the principal organism responsible for endodontic treatment failures, Enterococcus faecalis, was conducted in a standardized simulated root canal model [39]. The results of this in vitro study showed that the plasma treatment achieved a significantly higher microbial reduction than chemical 0.1% CHX irrigation (p < 0.001) and a comparable one with 0.6% NaOCl. However, the conditions used in this study were far from a "real life" situation. A step forward in that direction was done by Simoncelli et al. [40], who investigated two different procedures for the inactivation of bacteria in realistic tooth models, resembling procedures conventionally adopted in endodontic practice, and using wet and dry canal models. They suggested the possibility of combining direct and indirect treatments in an innovative multi-phase endodontic plasma-based procedure with increased overall antibacterial efficacy. Nevertheless, technical difficulties related to the length of penetration of the plasma plume are hampering its clinical application. Schaudinn et al. [41] used disinfected root canals of extracted teeth to study the effect of non-thermal plasma on ex vivo biofilm and they found an efficacy of biofilm removal lower than that achieved by the traditional treatment of 6% NaOCl, probably due to CAP's inability to act on bacteria over a longer distance. Therefore, the authors advocated for progress in the development of devices equipped with fine, flexible needles that will ease the disinfection of root canals along their whole length in clinical practice, and future studies aimed at assessing plasma's effect on the integrity of the treated dental tissue are needed for CAP to reach the dental chair.
The antimicrobial and surface modification plasma potential demonstrated in in vitro and in vivo models would suggest CAP as a promising option in the treatment of peri-implantitis [30,56], although further evidence is necessary to draw final conclusions. CAP may enhance the elimination of bacterial plaque from implant surfaces, in inaccessible pockets or during open-flap debridement, and should stimulate the process of the re-osseointegration of affected dental implants [57] by enhancing their wettability. Indeed, the potential to determine a super-hydrophilic surface may stabilize the blot clot and promote the early wound healing immediately after implant insertion [17,23]. To better analyze the encouraging application of CAP in implantology, its effects were divided by biocompatibility property, surface improvement, and antimicrobial activity.

Biocompatibility
The influence of the CAP treatment of titanium and zirconium discs on cell activities has been investigated in a few in vitro studies [7,12,27,28,48,52,54], and they generally agree on its supportive role in cell adhesion, spreading, and proliferation. Specifically, when the treatment was conducted on titanium surfaces, Duske et al. [7] reported that the size of osteoblastic cells grown on argon-oxygen plasma-treated titanium discs was significantly larger than on non-treated surfaces irrespective of surface topography (machined, sandblasted and acid-etched SLA, SLActive, subjected to airflow or diamond bur application). Accordingly, higher osteoblastic cell adhesion and positive cell morphology were reported on plasma-conditioned titanium surfaces than untreated surfaces [43,44]. Moreover, the combination of CAP treatment with the mechanical brushing of titanium samples seemed to determine a clean surface with cell growth comparable to the sterile control [48]. Enhanced osteoblastic cell proliferation and viability have also been shown on zirconia surfaces treated with oxygen CAP, providing significantly better results than in the same cells cultured on nontreated, UV-treated, and argon plasma-treated specimens [54]. In the same way, CAP appeared to improve fibroblast cell colonization and adhesion both on titanium and zirconia surfaces [28,42], mainly in the early phase of culture.
Besides, Tominami et al. [58] observed the effect of CAP irradiation on culture media containing plated pre-osteoblastic MC3T3-E1 cells, concluding that an accelerative effect on cell mineralization due to alkaline phosphate (ALP) activity enhancement and osteoblastic differentiation improvement could be suggested.

Surface Improvement
The potential of CAP to change the physico-chemical properties of the titanium surfaces, without affecting their microstructure [32], may play a central role in surface "rejuvenation" that, in turn, promotes the re-osseointegration of previously affected dental implants [59]. The enhancement of wettability could be considered as a promising tool in the treatment of peri-implantitis [48], inducing not only an improvement in osteoblast as well fibroblast cells spreading, but also increasing the immune cells essential to eliminating residual bacteria [48,60].
CAP application has demonstrated its efficacy to reduce the in vitro water contact angle (WCA) of treated titanium surfaces [7,28,42,52], resulting in an improvement of hydrophilic surface features. Yang et al. [12] even demonstrated an improvement in surface roughness after plasma treatment, that may contribute to the enhancement of subsequent cell adhesion. As demonstrated by a recent in vivo study [22], CAP conditioning of sand-blasted and acid-etched titanium dental implants prior to implant placement resulted in the absence of morphological surface alterations, as well as an improvement in osseointegration parameters (expressed as bone-to-implant contact-BIC) after 8 weeks of healing. Histological analysis provided by the same study [22] showed the homogenous mineralization of newly formed bone, suggesting a promising use of CAP therapy before dental implant positioning.
Zirconia has been demonstrated to positively respond to CAP treatment, showing an absence of structure alterations and surface oxidation and an increase in wettability following oxygen plasma or argon plasma applications for 12 min [54]. In addition, helium CAP treatment on zirconia discs only demonstrated a change in surface chemistry but not in surface topography, suggesting a promising role in the decontamination of zirconia abutment, as well as an improvement in soft peri-implant tissues, which may prevent peri-implant lesions over time [21].

Antimicrobial Activity
The effect of CAP in surface decontamination, as well as antimicrobial efficacy, may indicate plasma as a suitable device in the treatment of peri-implantitis. The presence of plaque as an etiological factor of peri-implant lesions [61] stresses the need for a therapy with a reliable cleaning efficacy, even in an anatomically disadvantaged situation. In this regard, Pei et al. [62] assessed the CAP depth of penetration using a mobile (wireless) handheld plasma device to inactivate an Enterococcus faecalis biofilm of 25.5 µm in thickness, which is essentially 17 layers of cells. The authors demonstrated effective penetration of the plasma-generated reactive oxygen species to the very bottom layer after 5 min of treatment at a 5 mm distance.
Argon plasma has demonstrated, in cell culture, a significant reduction of Streptococcus sanguinis biofilms [63] and the ability to disinfect titanium discs contaminated with Aggregatibacter actinomycetemcomitans [57]. Accordingly, helium plasma showed a bactericidal effect against Porphyromonas gingivalis biofilms grown on sandblasted, large grit, acid-etched (SLA) discs, mainly when applied for more than 3 min [37], as well as a bacterial growth inhibition and a decrease in biofilms of Streptococcus mutans and Porphyromonas gingivalis on zirconia specimens [21].
When compared to different titanium implant decontamination methods, such as laser radiation with diode devices, air abrasion, and chlorhexidine (CHX), the exposure of titanium machined discs to CAP significantly reduced the viability and quantity of oral biofilms compared with the other tested treatments, although a complete biofilm removal was not obtained [18]. Accordingly, three different plasma devices were more effective in removing multispecies human saliva biofilms grown on titanium discs, when compared to CHX application [64]. Preissner et al. [53] compared the in vitro effect of CAP, for both 60 and 120 s, to that of diode laser irradiation for 60 s on Streptococcus mitis biofilms cultivated on microrough titanium dental implants. Both type of CAP treatments resulted in a greater reduction of adhering bacteria than laser application [53]. Similar results were reported by Ulu et al. [55], who compared contact and non-contact Er:YAG (erbium-doped yttrium aluminium garnet) lasers with CAP used on SLA discs contaminated by Staphylococcus aureus biofilms. CAP not only performed better than the evaluated lasers, but may also be used in a safer way since the potential thermal damage to the bone and surrounding tissue was mainly caused by the contact laser that showed a focal temperature increase of up to 58 • C [55].
The treatment of peri-implant lesions was demonstrated to be more effective when different surgical or non-surgical approaches were combined [65][66][67]. In this view, Shi et al. [68] showed that the combination of the conventional techniques (e.g., the elevated flaps, curetted plaque, calculus, and granulation tissue, irrigated with 0.2% CHX digluconate and sterile saline solution) and CAP could lead to a higher bone level, a significantly decreased detection of bacteria (Porphyromonas gingivalis and Tannerella forsythia), and to a significant improvement in the clinical examination. Precisely, the association of mechanical treatments (such as mechanical brushing or air polishing) and CAP [23,40,44] have already provided promising results for dental implant decontamination, highlighting the synergistic antimicrobial effect and surface improvement that may represent an encouraging method in the treatment of implants affected by peri-implantitis.

Results from In Vivo Studies
To the best of our knowledge, only five studies [22,[45][46][47]49] compared the differences in the osseointegration of untreated as well as CAP-conditioned rough titanium [22,46,47] and calcium phosphate-coated (CaP) [47,49] or zirconium implants [45] placed in vivo in animal models ( Table 2). The assessment of osseointegration has been conducted on bone biopsies retrieved at different time points (1, 3, 6, and 8 weeks, and 1 or 2 months) and histomorphometrically analyzed. All studies agreed that CAP is a promising option to hasten osseointegration; indeed, significantly higher bone formation was found in Ar CAP treated rough and CaP-coated implants at 3 [45,49] and 8 weeks [22] and in zirconia (ZirTi) at 2 months [38], whilst less evident differences were detected using CAP with the same air composition as the regular atmospheric composition (16% oxygen, 1% hydrogen, and 78% nitrogen). None of the studies above investigated the mechanism underneath the improvement of the quantity and quality of bone healing. Only Naujokat et al. [22] tried to gain information about the chronological sequence of bone formation by labeling bone metabolism, but they did not find a relevant discrepancy of fluorescence between untreated and CAP-treated implants. Therefore, it can be concluded that CAP's beneficial effect on osseointegration and "reosseointegration" is worthy of further investigation in prospective clinical trials.

Future Trends in Oral Surgery and Implantology
In addition to the highly efficient removal of biological residuals from implant surfaces [21,37,53,55,57,[62][63][64], plasma treatment can also be an effective tool for lasting and highly controlled surface modification [69], including, but not limited to, chemical functionalization, deposition of antibacterial thin films and coatings [70,71], and surface structuring to create antifouling surfaces. Plasma can also be used for the deposition of highly complex, ordered surface nanostructures from a range of material sources [72], which can exhibit a higher level of control over the attachment behavior of cells and micro-organisms, providing a more selective control tool. Furthermore, plasma deposition has not been limited to dental implants only but may have significant potential in 3D porous scaffolds as well. It has been successfully shown to impart chemical gradients inside porous structures to enhance cell viability in comparison to untreated materials [73,74].
The capacity of activating liquids as carriers of antibacterial reactive agents via plasma use may provide a significant advantage in overcoming the limitations related to a lack of direct access to certain areas of the implant surfaces. In fact, with the application of plasma in dentistry (plasma stomatology), the saliva may have a role in the decontamination process. Although, to the best of our knowledge, there is still no research published on the plasma-saliva interactions, the studies on the plasma-liquid interactions, which are among the emerging research lines in the field of plasma science and technology, would promote further specific studies about the abovementioned interaction, which is of special interest for clinical practical applications in the field of plasma stomatology.