Plasma of Argon Treatment of the Implant Surface, Systematic Review of In Vitro Studies

This paper aims to review the evidence of the cellular activity on titanium samples exposed to Plasma of Argon (PoA) treatment. A systematic review was carried out based on the PRISMA statement by searching the Cochrane Library, PubMed, Web of Science, EMBASE and Scopus, up to October 2020. Papers were selected according to PICOS format that is: Population (P): osteoblasts, fibroblasts, gingival cells; Intervention (I): PoA disinfection treatment; Comparison (C): untreated controls; Outcome (O): cell culture; Setting (S): in vitro assays. The quality assessment was performed according to the CRIS Guidelines (Checklist for Reporting In vitro Studies). A total of 661 articles were found, of which 16 were included. The quality assessment revealed an overall poor quality of the studies analyzed. In vitro studies on the potential of PoA showed a potential effect in promoting higher cell adhesion and protein adsorption in the earliest times (hours). This outcome was not so evident when later stages of cell growth on the surfaces were tested and compared to the control groups. Only one study was conducted in vivo on a human sample regarding abutment cleaning. No meta-analysis was conducted because of the variety of experimental settings, mixed methods and different cell lines studied. PoA seems to be effective in promoting cell adhesion and protein adsorption. The duration of this effect remains unclear. Further evidence is required to demonstrate the long-term efficacy of the treatment and to support the use of PoA treatment in clinical practice.


Introduction
Implant rehabilitation is considered a successful treatment for most edentulous patients. Titanium is widely used as a gold standard implant material, which allows predictable clinical outcomes [1]. However, even if the long-term success of this rehabilitation is demonstrated, researchers are continuously focusing on how to improve, from biological to clinical relevance, it's potential. The long-term success rate of implant rehabilitation is determined by several factors [2][3][4]. Among others, the creation and maintenance of both osseointegration [5] and soft tissue sealing [6,7] around the implant are fundamental. In order to obtain these two key factors, correct and early cell adhesion is mandatory. Surface treatment [8][9][10] of the implant and its abutment have been investigated to obtain a more efficient and stable cell adhesion. Among all the properties that can influence cell response, such as surface topography and chemistry, hydrophilicity certainly covers an important role [11]. The benefits of high surface energy have been highlighted in terms of protein adsorption and early cell adhesion several times [12][13][14]. However, due to the time-dependent interaction with atmospheric hydrocarbon [15], the surface energy tends to decrease, possibly affecting the biological interaction [16,17]. In addition, since the customization process may be more prone to ambient contamination, carbon, aluminum, and siliceous particles, have been found on the titanium surface [18]. These particles are capable to hinder cell response [19] and their presence should be limited, otherwise implant surface may become more hydrophobic [20,21]. Different implant surface treatments, such as ultraviolet light [22][23][24][25][26] and plasma of Argon (PoA) [27], have been studied over the years in order to decontaminate the surface, increase wettability and enhance cell adhesion. In vitro, different researches have shown how PoA is able to activate the surfaces [28] thus increasing cell spreading and affecting the quantity of protein absorbed [29]. Moreover, these effects are achieved without changing the surface topography in order to predispose the best condition for optimal cell adhesion [30]. In vivo studies [31,32] also showed clinical benefits influencing hard and soft tissue levels and cell adhesion. In order to analyze all the results that have been described over the years, in the present study the authors aimed to perform a systematic review of the literature on PoA.

Protocol
This study was performed in accordance with PRISMA-P guidelines for systematic reviews and meta-analyses [33].

Information Sources and Search Strategy
The electronic search was conducted under the guidance of a librarian following the available guidelines [34,35]. A structured electronic search of the following electronic databases was performed on 6 October 2020 with specific search strategies (Table 1) for each database: Cochrane Library, PubMed, Web of Science, EMBASE and Scopus.  Indexes:  SCI-EXPANDED,  SSCI, A&HCI,  CPCI-S, CPCI-SSH,  BKCI-S, BKCI-SSH,  ESCI,  CCR-EXPANDED, IC.
('plasma argon' OR (('plasma'/exp OR plasma) AND ('argon'/exp OR argon))) AND ('dental'/exp OR dental) plasma argon dental Results  181  167  120  191  2 Hand search among references of included papers, related articles and grey literature was also performed but did not add any new paper or report. No publication date restriction was set and only papers in English were considered. Corrections specific to each database were performed to adapt the query in combination with database-specific filters, where available.

Study Selection and Data Collection Process
Papers have been selected according to inclusion criteria developed on the basis of the aforementioned PICOS checklist. Two conditions were set for inclusion: (I) in vitro hard and/or soft tissue periodontal cell culture and (II) comparison between PoA treatment and not-treated control.
The exclusion criteria were: review articles, case reports, retrospective studies, clinical studies, letters to the editor, personal opinions, guidelines, surveys, conferences and in vivo animal studies. All titles and summaries of studies retrieved in the initial search were evaluated and selected after removing duplicates according to eligibility criteria. The titles and summaries were independently selected by two reviewers (M.C. and Fr. M.), and the papers that met the inclusion criteria were identified and their inclusion was crosschecked by reading the full text to confirm their eligibility. Any disagreement between the two reviewers was solved through discussion. In case of further disagreement, a third reviewer (F.M.) who did not know the opinions of the other two reviewers was contacted and his opinion was determinant to decide whether the paper should be included. For data collection, relevant pieces of information were summarized into a spreadsheet of Microsoft Excel 2016: year of publication and authors, types of cell lines, cell culture methodologies and study outcomes.

Quality Assessment
To date, there is no standard Risk of Bias tool to assess the methodological quality of in vitro study [36]. The CRIS Guidelines [35] (Checklist for Reporting In vitro Studies) were developed around the necessity of standardized guidelines for improving quality and transparency in reporting and summarizing in vitro dental research. In this review, the CRIS checklist was applied to evaluate the inherent methodological reliability. It consists of five dichotomous questions, which evaluate the methodological aspects of each paper, that is sample size calculation, consideration of the meaning difference required to compute sample size, information on sample loss, clear description of allocation concealment, whether the outcome assessment is performed in blind.

Search Results
A total of 661 titles were found. After removing duplicates (326), title and abstract selection were carried out on 335 papers, of which 129 articles were selected for the evaluation of the full text. A total of 16 papers satisfied the inclusion criteria. The PRISMA flowchart reporting the selection process in detail is reported in Figure 1.  Titles of included studies, studied cell line and CRIS checklist are summarized in Table 2. The main characteristics of included studies are summarized in Table 3. As there was variation with regard to the methods between various papers as well as differences in the measurement of outcomes, it was impossible to perform any quantitative synthesis of the studies. Therefore, only qualitative descriptive analysis of the aforementioned papers is reported hereafter.

Results of the In Vitro Studies
In vitro studies on the potential of PoA showed a significant effect in promoting higher cell adhesion and protein adsorption in the earliest times (hours). This outcome was not so evident when later stages of cell growth on the surfaces were tested and compared to the control groups.
Even though no conclusive evidence could be drawn because of heterogeneity issues, it appears that all the studies taken into consideration conclude that PoA treatment is capable to increase the surface free energy of the treated surface. However, the duration of its effects appears to be limited in time.

Discussion
Implant osseointegration is a complex biological process in which cell spreading and attachment play a paramount role to ensure direct bone apposition to the fixture. In the last decades, several research strategies have focused on the enhancement of this process starting from the bone-implant interface features. Among the most promising technologies available to clinicians, one must take into consideration the chair side use of PoA before implant placement. The assumption of this technique is based simply upon that by increasing the surface energy of a given fixture it is possible to increase its wettability and consequently enhance cell adhesion, as a key preliminary phase leading the osseointegration. Hence, in the present literature review, we searched and identified articles assessing and comparing the biological response of cells seeded onto titanium samples either exposed or not to PoA. Although the heterogeneity of the experimental setting adopted in the papers examined (i.e., different cell types, titanium grades, time of exposure, PoA systems, measured outcomes) may preclude a whole metanalytic approach, a similar effect on cell activity was documented. All the authors reported faster cell adhesion on PoA-treated surfaces. This outcome was similar both in murine and human osteoblasts and fibroblasts, which highlighted the potential benefits of using PoA for the treatment of titanium intrabony fixtures and abutments.
Pistilli et al. [41] evaluated the effect of PoA treatment on three types of titanium surfaces (machined, double-etched Ti-AE and zirconium nitride) on the adhesion and proliferation of preosteoblastic cells and bacteria. Scanning electron microscopy (SEM) demonstrated a significant augmentation of colony forming units in machined and zirconium nitride surfaces. A sample size calculation was reported, but no detailed information regarding the source of the true difference between the experimental and the control mean was clearly provided.
As demonstrated by Canullo et al. [29], the increased cell adhesion elicited by PoA is time-dependent, and maximal soon after treatment, decreasing by the first 48 h. This seems not detrimental since the physiologic protein cascade reaches the highest level in the first 48 h [46], and the PoA treatment may be able to exert its action when most required, thus improving the hard and soft tissue healing process. This, however, might indicate also a major susceptibility to bacterial colonization even though it is unlikely to happen immediately after the insertion of the fixture unless gross clinical errors occur and has no influence on second-stage contamination since PoA has an effect only in the short term. No clear indication of the machine used nor the specific protocol used to perform PoA treatment was given in this study.
González-Blanco et al. [40] assessed the changes occurring in human osteoblasts on Grade IV and V titanium with a sand-blasted, acid-etched (SLA) surface treatment. They found out that cell viability was better in presence of surfaces treated with a vacuum cold PoA (Plasma Finish, PINK GmbH Thermosysteme, Wertheim, Germany) until 48 h when the measured cell viability no longer differed between groups treated with PoA and negative controls. Fluorescence microphotographs used for morphological analysis were performed only at the 24 h incubation period demonstrating a greater average cell area in comparison with surfaces not treated with PoA. The evaluation at the confocal microscope found that cells seeded on the titanium discs with a PoA treatment were enlarged compared to the ones on non-treated surfaces, this event is allegedly due to an improvement in surface hydrophilicity and increased surface energy that favors cell spreading. The different surfaces treated with PoA showed different mitochondrial activity: grade IV titanium treated with PoA showed higher values compared with controls, and grade V titanium showed instead the highest mitochondrial activity in non-treated samples. No clear explanation of this event is given by the authors in the discussion of their paper but stating as a limitation of the study to have a sample reduced in size. Therefore all the considerations taken from this study should be considered with caution. No sample size calculation was reported, thus limiting the internal and external validity of the conclusion of the study, nor was this substantial limitation discussed in the article. The analyzed sample is consistently reduced in size, which hinders the meaningfulness of inferential statistics reported.
Wang et al. [38] stated that the aim of their study was to investigate the capability of low-temperature PoA to maintain the activity of titanium dental implants and to restore the biological functions of implant materials after aging, thus accelerating the biological response of the peri-implant tissues to the implant.
Canullo et al. [37] aimed to assess whether PoA and ultraviolet light were able to have an impact on cell adhesion (fibroblast) on four different grade 5 titanium surfaces used for the manufacturing of healing abutments. The tested surfaces were machined surfaces and micro-grooved surfaces that had Ultrathin Threaded Microsurface; Anodized Ultrathin Threaded Microsurface or Thin Machined. However, no specification was given regarding the type of titanium surface treatment. These surfaces were divided into three groups: negative control, UV treatment and PoA (8 W and atmospheric pressure for 6 min, using a PoA reactor, Plasma R, Diener Electronic GmbH, Ebhausen, German). The sample size was reported and correctly performed. PoA had an effect on fibroblast morphology in all surfaces only during the first phases of adhesion (showing marked differences after 20 min, but a progressive reduction after 72 h, a time point in which the quantitative differences between treated and control discs were strongly reduced), UV did not produce any significant effect. With regards to cell adhesion, the differences statistically evident after 20 min were no longer present at the other evaluation time points, and no differences between surfaces were noted.
Henningsen et al. [42] performed a similar comparison between non-thermal plasma (NTP) and UV treatment. This study provided no sample size calculation nor a precise indication of the sample. In vitro cell culture studies are the first approach to test the safety of new methodologies before being approved for further pre-clinical and clinical assessments. The in vivo effects of PoA on titanium implants have been investigated in animal studies. Naujokat [47] analyzed the osseointegration of implants treated with cold PoA in pigs; these results showed slightly higher osseointegration when compared to nontreated implants. This is in an agreement with Canullo [48] and in contrast with Yi-Wen Hung [49] whose study investigated the effect of nonthermal PoA on sandblasted implants in beagle dogs, resulting in non-different implant stability and bone formation compared to the control group.
In vivo effects of PoA on titanium abutments have also been investigated in humans. Canullo et al. [31] led a randomized control trial over the span of two years to investigate the effect of PoA cleaning on implant abutments. This finding highlighted how the cleaning treatment may be favorable in terms of hard tissue level changes, confirming the results of the first 18 months of the study [17]. From a soft tissue standpoint, Garcia [30] investigated the influence of the PoA cleaning procedure on the soft tissue-abutment interaction in a randomized controlled study. The results indicated positive cell promotion and collagen fiber orientation, in accordance with a previous study by Canullo [32]. In accordance with a recent systematic review by Sanz Martin [50], all the in vivo studies obtained the results using one abutment-a one-time prosthetic approach.
In an in vitro study, Annunziata et al. [51] demonstrated how PoA is also capable of eliminating bacteria contaminations, suggesting its possible role in the treatment of peri-implantitis. This result was also confirmed in the in vivo study by Garcia et al. [30], which observed a more sterilized surface of the group cleaned with the PoA than the control group. This is also in agreement with Canullo et al. [13], who hypothesized the positive effects of PoA in patients with a history of peri-implantitis.

Limitations of the Study
The inner limitations of the present systematic review are related to the study design of the available studies on the subject, which are mainly in vitro studies and few in vivo studies. In fact, only one study was conducted in vivo on a human sample regarding abutment cleaning. The main question left unanswered by this paper is the net effect of PoA treatment on clinical practice. No meta-analysis was conducted because of the variety of experimental settings, mixed methods and different cell lines studied. Quality assessment of included studies revealed a low score, so a better adherence to shared guidelines in conducting in vivo and in vitro studies is advisable to be able to draw more solid evidence in the future. As a final note, even if sometimes not clearly stated, many authors declaring no conflict of interest appears to have several connections to the manufacturing companies of PoA devices (e.g., the companies producing the device sponsoring their private clinical courses on the same or on related subjects). While other forms of potential bias in biomedical research have been addressed through new processes and policies in the last decade, new approaches to identify and account for potential conflicts of interest are urged to ameliorate the objectivity of scientific literature [52].

Conclusions
Within the limitations of this systematic review, even though the quality of the most manuscript is in some cases low, most of them state that PoA seems to be a probable method to promote cell adhesion and protein adsorption, especially in the earliest time (hours).
The duration of this effect remains unclear and further research is needed to demonstrate the long time efficacy of the treatment. However, the results are to be interpreted with caution due to the low percentage of clinical human studies. In accordance, more in vivo studies are required to improve the scientific evidence on this topic and a better quality of in vitro study is advisable.  Data Availability Statement: Data are available upon request from the first author email address after reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.