This study was performed following an ex vivo design to overcome the drawbacks of previous studies. One particular difference in this study was the use of a commercially available screw-shaped implant. As the implant shape and design have rather complicated macro- and microstructures compared with titanium disks or different forms of titanium commonly used in experimental studies, previous results could not be easily interpreted and extrapolated to the clinical setting [14, 19]. However, the use of genuine implants allowed us to evaluate the cleansability of each method on contaminated implant surfaces. Without any limitations of accessibility and visibility, the efficacy of each decontamination method could be evaluated in a limited time frame.
Another critical difference in this study was the evaluation of dental plaque on implant surfaces collected in the mouth of participants rather than a single bacterial species [14, 20] or artificial biofilms [21, 22]. Dental plaque comprises 700–1000 bacterial species and is significantly different to a single bacterial colony or artificial biofilms. By assessing CFU counts via culture technique, the ability of each method to physically disrupt oral biofilms on contaminated implant surfaces could be evaluated. Conversely, a limitation of this study is that biofilms that cause peri-implantitis differ from those evaluated in this study. The biofilms in peri-implantitis form in anaerobic deep submucosal areas . However, in this study, only supragingival oral biofilms could accumulate on the mounted implant surfaces. It seems difficult to reproduce the same quality of submucosal biofilms around implants, which are thought to be an etiological factor of peri-implant disease in the laboratory . To date, there are a few studies that have tried to reproduce submucosal biofilms [22, 25], but such systems have not yet been completely established. This limitation of the present study should be kept in mind.
To the best of our knowledge, there is only one study that has used a similar experimental design to that of this study. Augthun et al.  examined the cultivability of mouse fibroblasts after cleansing machined or plasma-splayed surface implants carried on acrylic plates that had been contaminated with supragingival plaque from individuals. A plastic hand scaler and an air-abrasive system with sodium bicarbonate powder were employed in their study. A similar number of viable fibroblasts were observed after cleansing the implant with the air abrasive as the non-contaminated control implant. However, the number of viable cells was significantly reduced on the implant cleansed with the plastic scaler. This study had two drawbacks. First, they did not employ a quantitative analysis to evaluate the cleansing effect. Second, the SEM analysis used to evaluate the cleansing effect was too low (10- to 100-fold). In the present study, the presence of residual biofilms after instrumentation was determined using higher magnification SEM analysis (up to 5000-fold) and CFU counts. In this context, our findings may provide more accurate evidence than that demonstrated by the aforementioned study.
Based on the results of the SEM qualitative analysis, gauze soaked in saline and the rotary stainless steel instrument consistently showed good cleansability on rough and machined surface implants compared with the other methods. Conversely, the Er:YAG laser showed inferior cleansability to all other methods especially on rough surface implants. The ultrasonic scaler and air abrasive exhibited fair to good cleansability on both surface implants. Generally, the cleansability of each method appeared to be better on machined surface implants than rough surface implants.
The cleansability of gauze soaked in saline has previously been evaluated with and without antiseptics in vitro and in vivo [6, 9, 23, 26]. Charalampakis et al.  examined the efficacy of mechanical and chemical decontamination methods using titanium disks contaminated intraorally for 4 days. They employed four decontamination methods: gauze in saline, chlorhexidine, delmopinol, and an essential oil mixture. The SEM analysis demonstrated that three different rough surface disks harbored complex and firmly attached biofilms after gauze scrubbing irrespective of which antiseptic or saline was used. However, the disks with a turned surface hosted fewer biofilm clusters after scrubbing. This finding is in line with our result showing the better cleansability of gauze soaked in saline on the machined surface implants compared with the rough surface implants. The ultrasonic scaler, air abrasives, and Er:YAG laser have also been well investigated and used for the treatment of peri-implantitis [7, 27]. Schmage et al.  revealed the high cleansability of air abrasives and considerable cleansability of ultrasonic scalers and Er:YAG laser on titanium disks contaminated by a biofilm layer of Streptococcus mutans. The cleaning score of the air abrasives was the highest, and two types of ultrasonic scaler with a non-metal tip and the Er:YAG laser showed medium cleaning scores but better cleansability than non-metal curettes or a prophylaxis brush/cup. In the present study, the ultrasonic scaler displayed modest results. The tip used in this study was specially designed for cleansing contaminated implants with complicated macro- and microstructures. As the tip dimension was small in order to cleanse very narrow spaces, such as the valley of micro- or macrothreads, good cleansability was expected to be seen in such areas. This method could remove biofilms from small areas, but not in their entirety and not from the valley of microthreads. Moreover, the overall effect of biofilm removal did not appear impressive. One possible explanation for this result is that a treatment time of 1 min was not sufficient to use this small tip effectively. If more time was given to the ultrasonic scaler group, it might be possible to eliminate more biofilms, especially from microstructured areas of the implant.
Regarding the air abrasives, the cleansing effect in the SEM analysis was also as considerable as that achieved by the ultrasonic scaler in the present study, in contrast to the results of the aforementioned study. Louropoulou et al.  also stated in their systematic review that an air-powder abrasive system with sodium bicarbonate powder could cleanse contaminated rough/smooth implant surfaces without losing biocompatibility compared with a plastic scaler, metal curette, rotating titanium brush, and ultrasonic scaler. In the present study, the air abrasive showed fair to good cleansability with glycine powder but did not achieve the best result among the tested decontamination methods. The reason for this difference may be associated with the different experimental conditions (e.g., cleaning time, powder, power setting, and nozzles). Although free access to the genuine implant surface in the present study allowed us to evaluate the efficacy of each decontamination method, glycine powder as an air abrasive may not have the best cleansing potential among the tested methods. In the present study, the Er:YAG laser generally showed inferior cleansability. Er:YAG lasers have also been used in non-surgical  and surgical  peri-implantitis treatment. It was previously reported that implant surface decontamination by Er:YAG lasers demonstrated good cleansability of the contaminated implant surface compared with other decontamination methods [31,32,33]. The reason for the inferior cleansability of the Er:YAG laser observed in this study compared with the other decontamination methods is discussed below; however, dense biofilms remained on rough surface implants in particular after decontamination by the Er:YAG laser.
A rotary stainless steel instrument has a small head composed of stainless steel that allows clinicians good accessibility to deep intrabony defect areas. To the best of our knowledge, no study has clarified the cleansability of this rotary stainless steel instrument. In the present study, it was shown that it might be useful for cleansing contaminated implant surfaces. However, the rotary stainless steel instrument created numerous shallow scratches, especially on machined surface implants. John et al.  compared the supragingival plaque cleansability of a rotary titanium instrument to that of a stainless metal curette on contaminated titanium disks. The residual biofilm area left on implant treated with the rotary titanium instrument was significantly lower than in the stainless metal curette, and the surface alteration of the titanium disks could not be shown in SEM analysis. Although the cleansability of the rotary stainless steel instrument in the present study is superior and advantageous, the downside of the surface alteration is an issue to consider.
It has been previously stated that the alteration of the implant surface during cleansing may attenuate biocompatibility . However, several clinical studies revealed the considerable treatment effect even though there was certain expected damage on the implant surface [7, 34]. Therefore, it is assumed that the most important consideration for treating peri-implantitis in the clinical setting should be to improve the cleansability of any instrumentation to effectively remove biofilms irrespective of implant surface alteration.
Analysis of bacterial CFU count
In the present study, the gauze soaked in saline, rotary stainless steel instrument, and air abrasive demonstrated significantly greater cleansability to remove biofilms compared with the ultrasonic scaler on rough and machined surface implants. Generally, gauze soaked in saline appeared to possess the best cleansability among all the tested decontamination methods although there was no significant difference among the three methods with the greatest cleansability (G, Rot, Air). In the analysis between the two surfaces, surface characteristics significantly influenced total CFU counts between rough and machined surface implants when testing the control and gauze soaked in saline and ultrasonic scaler. Overall, machined surface implants tended to show lower CFU counts than rough surface implants apart from those treated with the Er:YAG laser.
Charalampakis et al.  examined the effectiveness of mechanical and chemical decontamination methods using titanium disks contaminated intraorally. They employed four decontamination methods: gauze in saline, chlorhexidine, delmopinol, and an essential oil mixture. The authors discovered there was no significant difference in CFU counts among the four methods. In the present study, our findings were in line with their report regarding the difficulty of removing biofilms from contaminated titanium surfaces. Even mechanical decontamination with a chemical agent did not yield any significant difference in CFU counts in their study. It has also been revealed that chemical agents in conjunction with mechanical debridement on contaminated implants could not augment a significant treatment effect . This is one of the reasons why we focused on mechanical decontamination methods to cleanse the contaminated implant surfaces.
Sahrmann et al.  tested three instruments (ultrasonic scaler, Gracey curette, and air abrasive device with glycine powder) on rough surface implants stained with indelible ink used as artificial plaque. There was a statistically significant difference in terms of stain removal rate. The air abrasive device showed the best result among the tested instruments. The result of this study is in line with our result showing the superiority of the air abrasive compared with the ultrasonic scaler.
Widodo et al.  evaluated the efficacy of different methods used to cleanse titanium disks contaminated by S. aureus biofilm in vitro. They used the following methods: (i) rinsing with phosphate-buffered saline, (ii) rinsing with chlorhexidine digluconate 0.2%, (iii) application of photodynamic therapy (iv), use of a cotton pellet, (v) use of a titanium brush, and (vi) the combination of a titanium brush and photodynamic therapy. The results showed that the use of a titanium brush with/without photodynamic therapy was more effective in reducing the bacterial load on both polished and rough titanium implant surfaces than the other methods. Our results are also in accordance with their results in terms of the high cleansability of the rotary metal instrument. In addition, the cotton pellet showed moderate cleansability among the tested methods, but the cleansing time for the cotton pellet (60 s) was shorter than that of the titanium brush with (120 s + 60 s)/without (120 s) photodynamic therapy. If adjusting the difference of cleansing time, the cotton pellet might show equivalent cleansability to the titanium brush.
In contrast to the past in vivo and in vitro studies [35, 36], the Er:YAG laser demonstrated an inferior cleansability on the contaminated implant surfaces. The Er:YAG setting (60 mJ/pulse, 10 pps) in the present study was within the normal recommended range for cleansing an implant surface without causing damage to the implant surface or the peri-implant tissue cells [37,38,39] and to ensure the safety of peri-implant tissue . Kreisler et al. [11, 37] used the same setting to cleanse a contaminated implant surface but without water coolant and demonstrated a good result. The reason why we could not achieve the same result might be associated with the water coolant used for further safety reasons in our study. In the clinical setting, the Er:YAG laser has been applied to treat peri-implantitis [27, 30, 40]. However, one report cautioned that the use of Er:YAG laser treatment as a non-surgical therapy had previously led to trauma of the peri-implant soft tissue, thereby causing unnecessary recession of the peri-implant mucosa . In this context, when the Er:YAG laser is applied to the treatment of peri-implant disease, water coolant should be considered for safety. There are many aspects that contribute to the efficacy of the Er:YAG laser (e.g., setting, coolant, tip distance from the tip to the contaminated implant surface). Such differences should be investigated in future studies.
Through SEM analysis and CFU counts, it was demonstrated that, except for the Er:YAG laser, decontamination of the machined surface implant was easier than that of the rough surface implant regardless of decontamination method. Gauze soaked in saline and the ultrasonic scaler demonstrated a statistically significant difference in CFU counts between the two surfaces. In this context, a machined surface implant may be advantageous for recovering biocompatibility after cleansing the contaminated implant surface. In a randomized controlled trial, Carcuac et al.  demonstrated greater treatment success in a machined surface implant group than a modified surface implant group. The present study may support this clinical result, and the application of gauze soaked in saline may be regarded as a gold standard technique to cleanse a machined surface implant.