J. Biophotonics 9, No. 6, 637–644 (2016) / DOI 10.1002/jbio.201500189

FULL ARTICLE

Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants: An in-vitro-study Saskia Preissner**, 1, Henrik C. Wirtz**, 1, Anne-Kristin Tietz2, Shady Abu-Sirhan1, Sascha R. Herbst1, Stefan Hartwig3, Philipp Pierdzioch1, Andrea Maria Schmidt-Westhausen4, Henrik Dommisch2, and Moritz Hertel*, 4 1

Department of Operative and Preventive Dentistry, Charité Universitätsmedizin Berlin, Aßmannshauser Str. 4–6, 14197 Berlin, Germany 2 Department of Periodontology and Synoptic Dentistry, Charité Universitätsmedizin Berlin, Aßmannshauser Str. 4–6, 14197 Berlin, Germany 3 Department of Oral and Maxillofacial Surgery/Clinical Navigation, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany 4 Department of Oral Medicine, Dental Radiology and Oral Surgery, Charité Universitätsmedizin Berlin, Aßmannshauser Str. 4–6, 14197 Berlin, Germany Received 1 July 2015, revised 27 July 2015, accepted 27 July 2015 Published online 9 September 2015

Key words: tissue tolerable plasma, cold atmospheric plasma, dental implants, peri-implantitis

Surface decontamination remains challenging in peri-implant infection therapy. To investigate the bactericidal efficacy of tissue tolerable plasma, S. mitis biofilms were created in vitro on 32 microrough titanium dental implants. Biofilm imaging was performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The implants were either rinsed with 1% NaCl as negative control (C) or irradiated with a diode laser (DL) for 60 sec as positive control or plasma (TTP60, TTP120) for 60 or 120 sec. Subsequently, colony forming units (CFU) were counted. Post-treatment, implants were further examined using fluorescence microscopy (FM). Median CFU counts differed significantly between TTP60, TTP120 and C (2.19 and 2.2 vs. 3.29 log CFU/ml; p = 0.012 and 0.024). No significant difference was found between TTP60 and TTP120 (p = 0.958). Logarithmic reduction factors were (TTP60) 2.21, (TTP120) 1.93 and (DL) 0.59. Prior to treatment, CLSM and SEM detected adhering bacteria. Post-treatment FM recorded that the number of dead cells was higher using TTP compared to DL and C. In view of TTP’s effectiveness, regardless of resistance patterns and absence of surface alteration, its use in peri-implant infection therapy is promising. The results encourage conducting clinical studies to investigate its impact on relevant parameters.

Experimental Design

* Corresponding author: e-mail: [email protected], Phone: +49 450 562 692, Fax: +49 450 562 905 ** These authors contributed equally to this work. © 2015 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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1. Introduction Microrough titanium dental implants provide domains with a scale of tens of micrometers to a few nanometers enhancing adsorption of proteins and cells to improve healing and tissue remodeling after implant placement to accelerate osseointegration and to increase the amount of bone-to-implant contact. Unfortunately, this specific surface is also inevitably susceptible to bacterial adhesion and subsequent biofilm formation. An antimicrobial treatment, including biofilm destruction, is crucial for the therapy for peri-implant infections (peri-implant mucositis and peri-implantitis) [1], which is crucially complicated not only because of the implant’s microroughness but also by its threads, its convexity, and shape. Therefore, antibacterial efficacy in therapy of periimplant infections remains a major challenge. Bactericidal agents, such as diode laser irradiation, have widely been studied in titanium specimens providing microrough surfaces [2–7]. Yet impairments caused by the implant’s macrostructure have not been thoroughly examined but for a few exceptions [8]. Laser irradiation has been suggested for decontamination of titanium implant surfaces, whereby there is no consensus neither on the laser type, on setting, nor irradiation parameters in peri-implant infection therapy due to limited evidence [9]. Amongst others, diode (gallium-aluminum-arsenide (GaAlAs)) lasers are eligible for this purpose. Their bactericidal efficacy is known to be dose dependent, similar to erbium-doped, yttrium-aluminum-garnet (Er-YAG) and carbon dioxide (CO2) lasers, ranging, for example, between 45% at 0.5 W and 99.9% at 2.5 W [3]. An increase in energy, determined by dose and time of radiation applied on a specimen, raises the likelihood of complete decontamination and for surface alteration likewise. Different titanium surfaces, moreover, show different decontamination results [10]. The clinical impact of laser use, however, remains uncertain. A study using an Er-YAG laser showed no better clinical long-term outcomes than curettes, cotton pellets, and sterile saline application in progressed peri-implantitis [11]. Plasma is known as the fourth physical state, next to solid, liquid, and gaseous. It is a partially ionized gas, which can be generated via pulsed direct current in plasma jet sources. Plasma induces a mixture of ultraviolet photons, charged particles, reactive atoms and molecules like reactive oxygen (ROS) and nitrogen species (RNS). Recent studies have shown antimicrobial effects of such in chemical reactions with organic matter [12–14]. The effects of tissue tolerable plasma (TTP, syn. cold atmospheric plasma, i.e., CAP) have been investigated in vitro [15–23] and described in case reports as well as clinical studies in animals and humans, the latter in dermatology and otorhinolaryngology [24–28]. No harm to viable host

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tissue or its function has been reported [29, 30]. An in-vitro-study using titanium discs detected no microstructure altering and complete ex-vivo-biofilm removal if combined with aerosol spray [31]. Therefore, TTP is a promising approach in peri-implant infection therapy due to its bactericidal efficacy, irrespective of microbial resistance [32, 33]. Etiology of peri-implant infection includes the presence of biofilm on the implant’s surface [34, 35]. Streptococcus mitis (S. mitis) is a Gram positive and facultative anaerobe bacterium. It is not motile and forms short chains. Physiologically, it is a frequent part of the oral flora and adheres to dental hard tissues, mucous membranes, as well as dental implants [36]. In terms of bacterial colonization, S. mitis acts as a pioneer organism. Regarding initiation of biofilm formation, it promotes co-adherence of late colonizers which modify, diversify, and mature oral biofilms. Despite its commensalism character, S. mitis opportunistically acts as a pathogen [37]. The aim of the present study was to investigate the bactericidal efficacy of TTP on microrough titanium dental implants compared to diode laser in vitro. It was hypothesized that TTP would greatly reduce the CFU number, compared to the diode laser. It was furthermore assumed that extended plasma application time would result in even lower CFU counts, as increased irradiation intensity and a second treatment cycle are known to enhance biofilm removal in vitro unto totality on titanium discs [31].

2. Material and methods 2.1 Bacteria cultivation S. mitis (DSM strain 12643) cultures were obtained by incubation for 24 hours at 37 °C on Columbia agar (Sifin, Berlin, Germany) plates under anaerobic conditions. Brain-heart-infusion (BHI) broth (Sifin, Berlin, Germany) was supplemented with 1 g/L l-cysteine (Sigma Aldrich, St. Louis, MO, USA) and 5 g/L yeast extract (Yeast Extract Servabacter, Serva Electrophoresis, Heidelberg, Germany). After autoclaving, 100 mg/L vitamin K and 100 mg/L hemin were added. BHI was subsequently inoculated with bacteria to an optical density of 1.0 at 600 nm using a photometer (Novaspec II Visible Spectrophotometer, GE Healthcare, Solingen, Germany). Bacteria broth was stored with gas-generating sachets (Oxoid AnaeroGen, Thermo Fisher Scientific, Waltham, MA, USA) in anaerobic jars at 37 °C. Indicator stripes (Oxoid Anaerobic Indicator, Thermo Fisher Scientific, Waltham, MA, USA) were used to confirm anaerobic conditions at all times.

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2.2 Sample processing Thirty-two, two-piece titanium dental implants with a sandblasted and acid-etched hydrophilic microrough surface, 0.5 mm machined collar, and an external hexagon (Tiny Implant, 2.5 × 13 mm, BTI Biotechnology Institute, Miñano, Spain, REF: IRT2513) were incubated in 1 ml of S. mitis culture at 37 °C for 84 hours under anaerobic conditions to allow biofilm formation. Implants were divided into four groups, each containing 8 implants. Each implant was subsequently coupled to an insertion tool (connection post long, BTI Biotechnology Institute, Miñano, Spain, REF: CPI22) and placed into a handpiece (Sirona Implant, Sirona, Wals bei Salzburg, Austria) mounted on a adjustable bench, allowing constant feed of the specimens. The implants were treated under constant rotation and feed according to the following parameters: Negative control (C): control-rinsing with 1% sodium chloride (NaCl) (5 ml) time: 60 sec rotation: 60 rpm feed: 0.22 mm/sec

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ma jet were 0 mm and 8 mm, respectively. Laser beam and plasma jet were applied perpendicular to the implant’s surface (Figures 1 and 2). After treatment, the interior of all implants was cleaned with absorbent paper points (Antaeos ISO 70, VDW, München, Germany) and 70% ethanol for 10 sec, dried for 5 sec and sealed with light curing resin (Tetric Flow A1, Ivoclar Vivadent, Schaan, Liechtenstein). Prior to polymerization, a stainless steel canula (0.92 × 23 mm yellow, Transcodent, Kiel, Germany) was placed into the implant’s interior to enable connection to an ultrasonic tip for direct sonication. As planktonization of S. mitis, according to procedures applied in titanium specimens or prosthetic joints [38], did not enable detachment of fertile cells in several preliminary investigations, each implant was processed successively as follows: 1. Incubation in 1 ml BHI followed by vortexing for 30 sec, direct sonication (ds) at 28 kHz (VDW Ultra, VDW, München, Germany) at 20% power/irrigation activation mode for 60 sec and repeated vortexing for 30 sec.

Positive Control: diode laser (DL): treatment with GaAlAs diode laser (λ = 980 nm) (Lina-10D, Intros Medical Laser, Heilbad Heiligenstadt, Germany) in direct contact mode (300 μm optical fiber) at 2.0 W (continuous wave) time: 60 sec rotation: 60 rpm feed: 0.22 mm/sec Tissue tolerable plasma (TTP60): treatment with plasma jet (kinpen MED, Neoplas Tools, Greifswald, Germany) at 4.3 bar/argon gas flow 4.3 slm time: 60 sec rotation: 60 rpm feed: 0.22 mm/sec

Figure 1 Tissue tolerable plasma irradiation of the implant’s surface.

Tissue tolerable plasma (TTP120): treatment with plasma jet (kinpen MED, Neoplas Tools, Greifswald, Germany) at 4.3 bar/argon gas flow 4.3 slm time: 120 sec rotation: 60 rpm feed: 0.11 mm/sec Laser beam and plasma jet were applied under constant irrigation with 1% NaCl (5 ml) to avoid surface exsiccation. Time, rotation, and feed were determined based on the diode laser and plasma jet manufacturer’s instructions. The distance between the implant surface and the tip of the fiberglass and plas-

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Figure 2 Diode laser irradiation of the implant’s surface.

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S. Preissner et al.: Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants

2. Brushing the implants’ surface using a cell sampler (cs) (Orcellex Brush, Rovers Medical Devices, KV Oss, Netherlands). To standardize brushing, the implant was again coupled to an insertion tool, rotated at 60 rpm and brought into contact with the cell sampler, which was rotated simultaneously. After 90 sec of clockwise rotation, the direction was switched to counterclockwise for another 90 sec. The head of the brush was removed and incubated in 2 ml BHI for sonication. 3. The interior of the implant was sealed again with resin and placed into 1 ml BHI. 4. The cell sampling brush and the implant were separately vortexed for 30 sec followed by mild indirect sonication (is) for 60 sec using a 35 kHz ultrasonic bath (BactoSonic, Bandelin Electronic, Berlin, Germany) at 40% power and repeated vortexing for 30 sec. Subsequently, each obtained specimen was serially diluted and 100 μl of each dilution was dispersed onto Columbia agar plates. After 24 hours of cultivation at 37 °C under anaerobic conditions, the CFU were counted, and fertile bacteria cell number was calculated. The experiments were carried out under sterile conditions.

Saw EXAKT 300 CL, EXAKT Advanced Technologies, Norderstedt, Germany). After fixation to an object holder (Plexiglas, Patho-service, Oststeinbek, Germany), incubation was carried out as described above. Five minutes after addition of 50 μl of fluorescein-ethidium bromide stain, examination was performed by applying dual fluorescence mode at up to 50× magnification.

2.4 Fluorescence microscopy Four implants were incubated as described and treated according assignment to Groups 1–4. A fifth implant remained sterile and served as control. After fixation to an object holder, 100 μl of live/dead stain (fluorescein-ethidium bromide) was added to each implant. After five minutes, fluorescence microscopy (Olympus Vanox-T, Olympus, Hamburg, Germany) was carried out at 40× magnification on the apex, middle, and neck of the implant. The examination was performed by two experienced investigators (S.P. and M.H.). Statistical analysis was performed using MannWhitney U test (IBM SPSS® 21.0, IBM Corp., Armonk, IL, USA). P-values ≤0.05 were considered as significant.

2.3 Bacteria identification Gram staining and a biochemical identification system (API Rapid ID32 Strep, bioMérieux, Nürtingen, Germany) were used to confirm the identity of S. mitis after incubation on Columbia agar plates.

2.3.1 Scanning electron microscopy and confocal laser scanning microscopy A scanning electron microscope (SEM) (CamScan Maxim 2040S, CamScan Electron Optics, Cambridgeshire, UK) and a dual fluorescence and confocal microscope (LSM 700; Zeiss, Jena, Germany) (CLSM) using the ZEN software (ZEN blue edition; Zeiss, Jena, Germany) were exemplary utilized to examine the implant’s surface after cultivation in S. mitis broth to visualize adherent bacteria. One sterile implant and one implant incubated as described (4.25 × 15 mm, BTI Biotechnology Institute, Miñano, Spain, REF: IIPU4215) above were comparatively examined using SEM recording secondary and backscatter electron images (SEI/BEI) at a magnification of up to 3000× (acceleration voltage = 10–15 kV). For CLSM, a flat 3.5 × 1 mm specimen was sawed off from one implant’s apex using a band saw (Band

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3. Results 3.1 CFU count Total logarithmic CFU/ml median values were (C) = 3.29 (mean: 4.59), (DL) = 3.61 (mean: 4.0), (TTP60) = 2.19 (mean: 2.38), and (TTP120) = 2.2 (mean: 2.66). Implants treated with tissue tolerable plasma showed significantly lower CFU counts compared to control implants (TTP60 vs. C: p = 0.012; TTP120 vs. C: p = 0.024). CFU number reduction within TTP groups was irrespective of irradiation time (TTP60 vs. TTP120: p = 0.958). The difference in CFU numbers found between implants treated with diode laser compared to control implants was not significant (DL vs. C: p = 0.674). Mean logarithmic reduction factor values were (DL) = 0.59, (TTP60) = 2.21 and (TTP120) = 1.93, respectively (Figure 3). Total bacteria mortification could not be observed in any group. The identity of S. mitis was confirmed by incubation on agar plates and subsequent testing after the experiments. Different cell detachment efficacies were observed for the three described processing methods applied after treatment. Brushing, using a cell sampler, obtained the highest mean CFU numbers, even though it was only secondly applied, fol-

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Figure 3 CFU count mic reduction factor log CFU/ml (mean grey = log reduction value); * = p ≤ 0.05).

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and logarith(dark grey = value); light factor (mean

Table 1 CFU count retrieved from subsequent direct sonication (ds), cell sampling (cs) and indirect sonication (is). C

average CFU

DL

TTP60

TTP120

ds

cs

is

ds

cs

is

ds

cs

is

252.13

1131.75

2512.88

2.63

997.25

11.13

3.63

18.75 1.88

ds

cs

is

2.0

38.75

5.25

C = control, DL = diode laser, TTP = tissue tolerable plasma

lowing direct sonication. CFU numbers retrieved from direct sonication were significantly higher than from indirect sonication, with the exception of TTP60 (Table 1).

3.2 SEM/CLSM SEM examination of incubated implants revealed disseminated round and oval shaped structures of approximately 0.5 μm, corresponding to coccoid bacteria covering large parts of the surface. Their presence was more evident on the machined part due to

Figure 4 SEM image (SEI, 10 kV, 960×) of machined implant collar after incubation.

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the rough part’s microstructure ranging within the size of bacteria cells (Figures 4 and 5). In contrast, no bacterial colonization was detectable on the sterile control implant’s surface (Figure 6). CLSM confirmed the results retrieved from SEM. Green fluorescence indicated viability of bacteria. Signs of cell cleavage were visible (Figure 7).

3.3 Fluorescence microscopy Intense, mostly green fluorescence was observed on all examined regions of the incubated control im-

Figure 5 SEM image (SEI, 10 kV, 3000×) of microrough implant surface after incubation.

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S. Preissner et al.: Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants

Figure 6 SEM image (SEI, 15 kV, 2000×) of sterile microrough implant surface.

Figure 8 Fluorescence microscope imaging of implant apex (a), middle (m), and neck (n) (fluorescein-ethidium bromide stain, 40×).

4. Discussion

Figure 7 CLSM imaging of S. mitis layer on the implant’s surface (fluorescein-ethidium bromide stain, 50×; * = cleavage of bacteria cells).

plant, whereas variable amounts of red fluorescence were observed on implants treated with DL or TTP. The amount of red fluorescence emitted from the surface of the implants treated with TTP exceeded that of DL; TTP60 and TTP120 did not differ notably. All irradiated specimens showed inhomogeneous emission within and between the examined regions, especially in the DL group (Figure 8). No fluorescence was found on the sterile implant.

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The aim of the present study was to investigate the bactericidal efficacy of TTP on titanium dental implants. SEM and CLSM showed viable bacterial biofilms covering the implant’s surface after incubation prior to treatment. Planktonization of adhering bacteria cells after treatment was feasible, at least through application of several techniques applied consecutively. Among these methods, standardized brushing of the implant’s surface using a cell sampler was most effective. Nevertheless, its efficacy without prior direct sonication was not found in several pretrials. It may be assumed that approaches described for other devices [38] are not suitable for dental implants due to their small dimension, resulting in a low total amount of adhering bacteria compared to, e.g., joint prostheses. Their microstructure might cause further impairments in terms of greater biofilm adherence, impeding its removal. The latter highlights problems related to biofilm elimination from microrough dental implants. Bactericidal efficacy of TTP significantly exceeded DL compared to control implants. Irradiation of up to 60 sec. per cm2 is recommended by the manufacturer, at least if applied on skin or wounds. Doubling TTP treatment time to imply the area increase through surface roughening did not provide any efficacy improvement, at least not in terms of further CFU number reduction. Assumedly, TTP’s CFU count reduction potential was exhausted within 60 seconds. The in-vivo-efficacy remains uncertain as the access to the implant surface might be compromised; so 60 seconds of treatment might not carry out the uppermost bactericidal effect. Diode laser irradiation, applied as positive control, reduced CFU numbers by log (0.59) related to negative control im-

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plants, but no significance was found. The observed, incomplete bactericidal results of diode laser irradiation confirmed findings from earlier studies [3, 5]. However, other literature reported bacteria killing rates of 100%, depending on the applied irradiation parameters [4, 6]. Tosun et al. reported 100% CFU number reduction of Staphylococcus aureus at 1.0 W [6] on sandblasted and acid-etched titanium discs. Findings in the present study, however, indicate a relevant impact of dental implants’ macrostructure, diminishing the efficacy of laser irradiation. A recent review article revealed that in-vitro-susceptibility of bacteria to laser irradiation depends on the investigated species [9]. In contrast, antimicrobial efficacy of cold plasma has widely been shown [13, 39–46] and is known to be independent from resistance patterns [32, 33]. As plasma spreads out on surfaces and even penetrates porous structures its antimicrobial efficacy exceeding diode laser is assumedly due to an extended radius of effect and good penetrance. Thus, a complex three-dimensional surface, as found on microrough titanium, can be efficiently disinfected. In contrast, the effectiveness of laser irradiation is limited to areas directly exposed to the laser beam or at least affected by its thermal effects including vaporization of water. Hence, shadowing caused by pits, cavities, threads and the implants’ convexity results in an impairment of efficacy. Even though it could be improved by increasing the deployed energy, the risk of surface alteration is augmented. Plasma alone reduces viability of oral biofilms as well as protein amounts and provides thinning. Mechanical biofilm removal exceeding these effects requires aerosol spray administration [31]. These findings indicate that plasma should be used in addition to mechanical surface debridement, whereby techniques without surface alteration, such as aerosol spraying, may be preferred. Plasma application on microstructured titanium does not alter its surface, which has been confirmed by SEM imaging [31]. The suggestion of combining TTP with other therapeutic approaches is further supported by the recorded log reduction factor of 2.21 for TTP60 and 1.93 for TTP120, respectively. Disinfection quality demands log reduction of at least 5, which was not obtained in any investigated group. Future investigations should therefore assess the hypothesized potential regarding efficacy improvement. Hydrophilization of surfaces can be additionally provided by plasma [47] which is favorable to cell attachment regarding potential re-osseointegration. Hydrophilic surface properties have not been investigated in the present study, but their potential impact should be considered in further examinations. Within the limitations of the present study, it can be concluded that tissue tolerable plasma is a promising approach in peri-implant infection treatment,

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whereas its’ bactericidal efficacy alone will not sufficiently overcome the therapeutic problems. Clinical studies are needed to investigate the impact of TTP on relevant outcome parameters, such as bleeding on probing and probing depth. Acknowledgements The authors thank Ms. Verena Karnitz (Charité Universitätsmedizin Berlin, Department of Periodontology and Synoptic Dentistry, Berlin, Germany), Dr. Herbert Renz (Charité Universitätsmedizin Berlin, Department of Craniofacial Developmental Biology, Berlin, Germany) and Prof. Dr. Sebastian Paris (Charité Universitätsmedizin Berlin, Department of Operative and Preventive Dentistry, Berlin, Germany) for their most professional and valuable assistance. Conflict of interest The implants examined in the study were kindly provided by BTI Biotechnology Institute, Vitoria, Spain. The plasma jet system used in the study was kindly provided by NeoPlas Tools, Greifswald, Germany. Neither of these companies nor any other company or institution provided financial support for the study. Author biographies online.

Please see Supporting Information

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Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants: An in-vitro-study.

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