Decontamination of Dental Implant Surfaces by the Er:YAG Laser Beam: A Comparative in Vitro Study of Various Protocols

Oral rehabilitation with dental implants has revolutionized the field of dentistry and has been proven to be an effective procedure. However, the incidence of peri-implantitis has become an emerging concern. The efficacy of the decontamination of the implant surface, by means of lasers, is still controversial. Previous studies have revealed a reduction in osteoblast adhesion to carbon-contaminated implant surfaces. This in-vitro study aimed to evaluate the decontamination of failed implants by assessing the carbon proportion, after irradiation by low-energy erbium yttrium-aluminum-garnet laser (Er:YAG) (Fotona; 2940 nm, Ljubljana, Slovenia) for a single and for multiple passages, until getting a surface, free of organic matters; to find the appropriate procedure for dental-implant surface-decontamination. Ninety implants were used. Thirty sterile implants were kept as a negative control. Thirty failed implants were irradiated by the Er:YAG laser, for a single passage, and the other thirty, for multiple passages. The parameters used in our experiments were an irradiation energy of 50 mJ, frequency of 30 Hz, and an energy density of 3.76 J/cm2. A sapphire tip, with a length of 8 mm, was used with concomitant water spray irrigation, under air 6 and water spray 4. Super short pulse mode (SSP) was of 50 μs; irradiation speed being 2 mm/s. We used energy-dispersive X-ray spectroscopy (EDX) to evaluate the carbon proportion on the surfaces of the sterile implants, the contaminated, and the lased implants, with one (LX1) and with three passages (LX3). Statistical analysis was performed by ANOVA. Results showed mean difference between the three groups (contaminated, LX1, and LX3) with p < 0.0001, as between LX1 and Group A (p < 0.0001), while the difference between LX3 and the control group was not statistically significant. The decontamination of the implant surfaces with a low-energy Er:YAG laser with three passages, appeared to be an encouraging approach.


Introduction
Peri-implantitis is an inflammatory process that takes place in the soft tissue, with a bone loss around an osseointegrated implant in function [1,2]. Diagnosis is based on the bleeding and the probing depth of the peri-implant pockets, along with the suppuration and gradual loss of bone height around the implant [3][4][5]. The etiology of the implant infection includes many factors, such as implant design,

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Group A: Thirty sterile implants served as the control group. The implants of this group were kept in their own sterile packages, until their examination through EDX and SEM. • Group B: Sixty contaminated implants were collected from failed cases of implants. The implants were retrieved from patients previously diagnosed with peri-implantitis. Their removal was not, in any of the cases, related to our study (severe peri-implantitis and bone resorbtion, loss of osteointegration, etc.). Before experimentation and for the purpose of standardization, all contaminated implants were preserved in sterile saline liquid of 0.9% NaCl, at a temperature of 37 • C, for the simulation of the intra-oral in vivo conditions; the solution was changed every 24 h, until experimentation. To assess the efficacy of the laser irradiation, in our study we compared the carbon percentage of the contaminated implant surfaces, before and after the laser irradiation. At the baseline, the sixty implants of Group B were all evaluated (eight points were randomly analyzed per sample), using energy dispersive X-ray analysis (SEM-EDX). After that, the implants were randomly assigned into two equal samples-LX1 and LX3-for the laser irradiation. Sample LX1 was irradiated by one passage and sample LX3, by three passages. Afterward, a second analysis of the carbon content of both samples was done.

Decontamination
Laser surface preparation: The contaminated implants of Group B were irradiated using a custom-designed device ( Figure 1). The custom made machine was driven by a stepper motor, controlled by a software, and was connected to a computer through a USB. Such a device was used for the fixation of the laser handpiece, in a standard manner, for the irradiation of the implant, at a constant speed and time. The machine allowed for the standardization of all laser irradiations, thus, allowing an accurate comparison between the different treatment protocols. It should have been able to: (1) Standardize the angulation of the laser beam.
(2) Standardize the distance between the tip of the handpiece and the implant surface.
(4) Have a semi-adjustable base on which an implant, connected to an abutment, is attached to a Plexiglass ® plate.
An Er:YAG laser (Fotona; 2940 nm, Ljubljana, Slovenia) was used in our experiments, with an irradiation energy of 50 mJ, frequency of 30 Hz, output power of 1.5 W, and an energy density of 3.76 J/cm 2 . The energy was chosen, based on the literature review; higher energy was shown to damage the titanium implant surface [38]. A sapphire tip with a length of 8 mm was used in the respective handpiece (H14) and not in contact with the surface of the titanium, with concomitant water spray irrigation, under air 6 and water spray 4, the irradiation angle was 90 degrees, at a focal Dent. J. 2018, 6, 66 4 of 14 distance of 2 mm, the spot size diameter was 1.3 mm; Super short pulse mode (SSP) was of 50 µs; the contaminated implants of Group B were irradiated as:

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Thirty contaminated implants received one passage of laser (LX1). • Thirty contaminated implants received three passages of laser (LX3).
As the irradiation speed was 2 mm per second, the time of irradiation for each implant, depended on its dimension, with an average of 40 s in the LX1 group, for an implant of 4 mm diameter and length of 10 mm, while the irradiation took three times longer for the LX3 group. After the laser irradiation, all fixtures were returned to sterile packages, for testing. An Er:YAG laser (Fotona; 2940 nm, Ljubljana, Slovenia) was used in our experiments, with an irradiation energy of 50 mJ, frequency of 30 Hz, output power of 1.5 W, and an energy density of 3.76 J/cm 2 . The energy was chosen, based on the literature review; higher energy was shown to damage the titanium implant surface [38]. A sapphire tip with a length of 8 mm was used in the respective handpiece (H14) and not in contact with the surface of the titanium, with concomitant water spray irrigation, under air 6 and water spray 4, the irradiation angle was 90 degrees, at a focal distance of 2 mm, the spot size diameter was 1.3 mm; Super short pulse mode (SSP) was of 50 μs; the contaminated implants of Group B were irradiated as:

•
Thirty contaminated implants received one passage of laser (LX1).
As the irradiation speed was 2 mm per second, the time of irradiation for each implant, depended on its dimension, with an average of 40 s in the LX1 group, for an implant of 4 mm diameter and length of 10 mm, while the irradiation took three times longer for the LX3 group. After the laser irradiation, all fixtures were returned to sterile packages, for testing.

Scanning Electron Microscopy
Through SEM (Seron technologies AIS2100, Uiwang, South Korea) or EDX (EDAX Apollo detector) we analyzed the effects produced by the Er:YAG laser in one and in three passages, by evaluating the proportion of the carbon on the implant surfaces, for the sterile (control-Group A) (Figure 2), and the contaminated implant surfaces, before ( Figure 3) and after the irradiation ( Figures  4 and 5). EDX, which is the standard technique for the local determination of the chemical composition, was used to measure the presence of carbon on the implant surfaces. The area of an EDX peak, of an element in a sample, is directly proportional to the abundance of the elements in the sample.
The implants were carefully removed from their container, using sterile tweezers, in order to prevent any contamination. Then, they were placed on a sample holder and fixed by a double-faced

Scanning Electron Microscopy
Through SEM (Seron technologies AIS2100, Uiwang, South Korea) or EDX (EDAX Apollo detector) we analyzed the effects produced by the Er:YAG laser in one and in three passages, by evaluating the proportion of the carbon on the implant surfaces, for the sterile (control-Group A) (Figure 2), and the contaminated implant surfaces, before ( Figure 3) and after the irradiation (Figures 4 and 5). EDX, which is the standard technique for the local determination of the chemical composition, was used to measure the presence of carbon on the implant surfaces. The area of an EDX peak, of an element in a sample, is directly proportional to the abundance of the elements in the sample.
The implants were carefully removed from their container, using sterile tweezers, in order to prevent any contamination. Then, they were placed on a sample holder and fixed by a double-faced conductive tape on one side, while the other sides were freely facing the electron beam of the SEM. No special sample preparation was used, since the implants were already metallic and conductive, which canceled the need for any sputtering or metallization. A high-vacuum SEM was used with an acceleration voltage of 20 kV and a working distance of 25 mm, the take-off angle for the EDX was 23 degrees. The alterations on the implant surface were evaluated by inspecting the SEM images

Statistical Analysis
Statistical analyses were performed by a blind statistician, using the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA, USA). Means and standard deviations (SD) of the carbon mass (%) on the implant surfaces, were reported for each group. Data showed normal and

Statistical Analysis
Statistical analyses were performed by a blind statistician, using the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA, USA). Means and standard deviations (SD) of the carbon mass (%) on the implant surfaces, were reported for each group. Data showed normal and homogeneous distributions and were submitted to one-way ANOVA, followed by post-hoc Newman-Keuls test, for pairwise comparisons. A p-value of less than 0.05 was considered to be statistically significant.

Statistical Analysis
Statistical analyses were performed by a blind statistician, using the GraphPad Prism program (GraphPad Software, Inc., San Diego, CA, USA). Means and standard deviations (SD) of the carbon mass (%) on the implant surfaces, were reported for each group. Data showed normal and homogeneous distributions and were submitted to one-way ANOVA, followed by post-hoc Newman-Keuls test, for pairwise comparisons. A p-value of less than 0.05 was considered to be statistically significant.

Analytical Results
At baseline, the contaminated Group B had a mean carbon mass (%) of 37.18 ± 15.31. This value decreased to 6.17 ± 1.45 after one laser passage and to 1.43 ± 0.41 after three laser passages ( Table 2). The mean difference between the three groups (contaminated, LX1, and LX3) was statistically significant (p < 0.0001), as shown in Table 2. For the comparison between LX1, LX3, and the control Group A (sterile); the mean carbon mass (1.86 ± 0.68) analysis showed a significant difference between LX1 and Group A (p < 0.0001), while the difference between LX3 and the control group was not statistically significant (Table 3). Results are further illustrated in Figure 6. Thus, the null hypothesis was accepted, when comparing the group irradiated by three passages versus the control group of the sterile implants (p value > 0.05).

SEM Observations
The SEM examination of the implant surface after laser irradiation, in comparison with a sterile and not-treated surface, showed what has been presented in Figures 7 and 8. There was no presence of any cracks or melted surface in all figures. When we used 50 mJ by three passages, the implant surface did not get affected and the rough surface was similar to the one of the sterile implant.

SEM Observations
The SEM examination of the implant surface after laser irradiation, in comparison with a sterile and not-treated surface, showed what has been presented in Figures 7 and 8. There was no presence of any cracks or melted surface in all figures. When we used 50 mJ by three passages, the implant surface did not get affected and the rough surface was similar to the one of the sterile implant.

SEM Observations
The SEM examination of the implant surface after laser irradiation, in comparison with a sterile and not-treated surface, showed what has been presented in Figures 7 and 8. There was no presence of any cracks or melted surface in all figures. When we used 50 mJ by three passages, the implant surface did not get affected and the rough surface was similar to the one of the sterile implant.

Discussion
Numerous procedures have been proposed for the clinical use of Er:YAG laser, in dentistry, ranging from the removal of tooth decay and cavity preparation, to many soft and hard tissue surgical procedures [35,[42][43][44][45][46][47][48]. Along with these, there are other benefits, such as ablation of the target tissues and the ability to reduce bacterial contamination. Many studies have already validated the bactericidal effect of laser irradiation on the surface of the contaminated dental implants. Due to its unidirectional light and the side-firing tips, the laser beam allowed access to all threads of the implant surface, compared to the mechanical debridement by curettes which are not able to reach all parts of the surface [38].
Kreisler et al. [36] evaluated the use of Nd:YAG, Ho:YAG, Er:YAG, CO2, and GaAlAs lasers for the decontamination of implant surfaces; they concluded that Er:YAG and CO2 lasers may be used at limited powers. Several researches have studied the cleaning efficacy of the Erbium laser beam on a titanium surface, but to date there is no consensus on the appropriate parameters of lasers that are to be used during decontamination [49]. Er:YAG pulses of 300 mJ/10 Hz produce alterations to the SLA (sandblasted and acid-etched) surfaces and 500 mJ/10 Hz pulses alter the polished surfaces [36]. Galli et al. investigated the Er:YAG irradiation at two levels: 150 and 200 mJ/pulse at 10 Hz; the results of the study indicated that Er:YAG laser at these energy levels, could alter the surface profile of titanium implants and subsequently, may negatively affect the viability and the activity of the osteoblastic cells [50]. Several in vitro and in vivo studies had investigated the use of Er:YAG laser at a pulse energy of 100 mJ, with a frequency of 10 Hz, and had found it effective for the decontamination of implant surfaces [51,52]. However, it decreases the surface roughness and increases the wettability of the SLA and hydroxyapatite titanium surfaces, after one minute of irradiation [44]. Nevertheless, another study concluded that no surface alteration was detected after irradiation at an intensity of 100 mJ/pulse, at 10 Hz, for 1 min, while the titanium's roughness was affected after 1.5 min of Er:YAG irradiation [41]. This result was already concluded by Kim et al., who recommended the application settings of 100 mJ/pulse, at 10 Hz and for less than two minutes, to detoxify the implant surface without causing any surface modifications [53]. The energy level of 50 mJ has shown efficiency in removing plaque and calculus on the implant abutments, without injuring their surfaces [38]. When the pulse energy and irradiation time increased, greater surface alterations, including surface flattening and microfractures were observed [41]. In the present study we used the same low-level energy of Er:YAG (50 mJ) on a rough implant surface, to test it by a single and multiple passages. The results were promising as Group LX3 (3 passages) was almost perfectly cleaned, in comparison to the negative control group, in which no contamination technique was used. The value of carbon mass decreased after one laser passage but analysis showed a significant difference between LX1 and the control group (p < 0.05), while the difference between LX3 and the control group was not statistically significant (Table 3). Therefore, irradiation by one passage, with the mentioned parameters (50 mJ), did not show efficiency in the implant surface decontamination. A great difference was noted between LX1 (one passage) and LX3. Reduction in the presence of carbon in LX3, in comparison to LX1, was probably caused by the multiple passages of laser beam. Thus, multiple irradiations with Er:YAG laser produced the removal of all particles.
The control of the energy density is a key factor of success for any treatment by laser irradiation [54]. Taniguchi et al. used a pulse energy of 30-50 mJ/pulse at a repetitive rate of 30 Hz. They had concluded that Er laser irradiation at pulse energies below 30 mJ/pulse and 30 Hz, is effective for debriding microstructure surfaces and the fluency being 10.6 J/cm 2 [55], which was greater than the value used in our study. These findings suggest that in our experiment, the reduction of carbon presence on the implant surface was rather due to the multiple passages of laser.
No surface change was seen in the irradiated groups ( Figure 6); this result was in agreement with a previous study that revealed no distinct morphological alterations under 50 mJ. Due to a serious concern regarding implant overheating, the laser beam was used with concomitant water spray irrigation under air 6 and water 4, to minimize thermal damage, by keeping the irradiated area moist [56]. We cannot completely rule out the effect of water irrigation on removing some contaminants, but it is impossible to get a surface as clean as a sterile implant's surface. Although, Park et al. concluded in their study that the use of a dental water jet didn't show any efficacy in the dental implant decontamination [57]. The impact of the laser tip on heat generation, during implant decontamination, has received little attention. The tip used in our study was a sapphire tip. Romanos et al. have studied the influence of two laser tips (sapphire chisel and radial firing perio) on temperature change after laser irradiation; they concluded that sapphire may be preferable for implant debridement [8].
In addition, one of the key factors that regulate the regime and efficacy of laser decontamination is the laser pulse duration. The VSP (very short pulse) Er:YAG laser can be operated at adjustable pulse duration, from super short pulses (SSP) that are ideal for the precise ablation of hard tissue, to very long pulses (VLP) for more coagulative soft tissue procedures. This technological solution provides nearly square-shaped power pulses, the duration of which can be conveniently controlled over a wide range of pulse durations [58]. In our experiment, the beneficial effect of the Er:YAG laser in implant decontamination was particularly pronounced when the laser was set to operate at super-short pulses (SSP, 50 µs) and a low-pulse energy, which provided an extremely precise laser ablation with minimal thermal effect, since there was a very short duration to escape the ablated volume [59].
Folwaczny et al. concluded that the efficiency was maximal at an angle of 90 degrees, during the removal of a root substance [60]. In this study, for maximum energy application, the irradiation angle was 90 degrees at a focal distance of 2 mm, however, the implant design was not a smooth titanium plate but a dental implant thread surface, and the laser illumination angle limitation caused by the threads could make the clinical application different from in vitro experiments. Our study has shown that the decontamination of implants is possible with Er:YAG, under certain conditions, particularly, the condition of angulation of 90 degrees. Having a 90 • angle may not always be respected, in situ, therefore, there is probably a risk of reduced effectiveness in cleaning the implant. Accordingly, it is interesting to use the adapted Er:YAG tips allowing the delivery of a beam at 90 • into the pitch of the threads, for effective decontamination.
The previous studies focused on the activity of the Er:YAG laser, against planktonic bacteria [15,35,61]. Our in-vitro study has evaluated the efficiency of Er:YAG laser to remove carbon and to get a surface composition comparable to the uncontaminated implant surface with decreased amounts of carbon.
The percentage mass of carbon present on the implant's surface is a measure of its contamination. The lower the percentage, the cleaner was the implant. The results of our study showed similarity in the count of carbon between the sterile and laser-irradiated implants (by three passages), in comparison with contaminated ones and one passage lasing. Er:YAG was efficient to clean the implant surfaces without altering the titanium surface, by an irradiation of 50 mJ/pulse at 30 Hz, for three passages. In addition, this procedure was very fast since an implant may be cleaned in about one minute, depending on the exposed area.
Further studies should be directed to evaluate the biocompatibility and the cellular adhesion of the titanium surfaces, after their cleaning using similar Er:YAG irradiation conditions.

Conclusions
Our results suggest that the Er:YAG laser could be considered an effective tool for the decontamination of implant surfaces when used with an energy of 50 mJ, frequency of 30 Hz, in the super short pulse mode (SSP), and a fluency of 3.76 J/cm 2 . When the irradiation speed was 2 mm/s, the laser irradiation by three passages was very efficient.