Effect of a Er, Cr:YSGG laser and a Er:YAG laser treatment on oral biofilm-contaminated titanium

Abstract Implant surface decontamination is a challenging procedure for therapy of peri-implant disease. Objective: This study aimed to compare the effectiveness of decontamination on oral biofilm-contaminated titanium surfaces in Er:YAG laser, Er, Cr:YSGG laser, and plastic curette. Methodology: For oral biofilms formation, six participants wore an acrylic splint with eight titanium discs in the maxillary arch for 72 hours. A total of 48 contaminated discs were distributed among four groups: untreated control; decontamination with plastic curettes; Er, Cr:YSGG laser; and Er:YAG laser irradiation. Complete plaque removal was estimated using naked-eye and the time taken was recorded; the residual plaque area was measured and the morphological alteration of the specimen surface was observed by scanning electron microscopy. The total bacterial load and the viability of adherent bacteria were quantified by live or dead cell labeling with fluorescence microscopy. Results: The mean treatment time significantly decreased based on the treatment used in the following order: Er:YAG, Er, Cr:YSGG laser, and plastic curettes (234.9±25.4 sec, 156.1±12.7 sec, and 126.4±18.6 sec, P=0.000). The mean RPA in the Er, Cr:YSGG laser group (7.0±2.5%) was lower than Er:YAG and plastic curettes groups (10.3±2.4%, 12.3±3.6%, p=0.023). The viable bacteria on the titanium surface after Er, Cr:YSGG laser irradiation was significantly lower compared to the decontamination with plastic curette (P=0.05) but it was not significantly different from the Er:YAG laser irradiation. Conclusion: We found that Er:YAG laser and Er, Cr:YSGG laser irradiation were effective methods for decontaminations without surface alterations.


Introduction
Current implant dentistry focuses on the maintenance of long-term stability of peri-implant tissue. As the popularity of dental implant therapy has increased, so have the reported cases of peri-implant diseases. 1 Peri-implant mucositis is a reversible inflammatory reaction of soft tissues around functioning implants, whereas peri-implantitis is a nonreversible inflammatory process related to the loss of the supporting bone around functioning implants. 2 According to a review of Lee, et al. 3 (2017) peri-implant mucositis occurs in 46.83% of patients receiving dental implant therapy and in 29.48% of functioning implants, whereas peri-implantitis has been found in 19.83% of patients and in 9.25% of functioning implants.
As peri-implant infection is caused by biofilm formation on implant surfaces, it is mandatory to decontaminate the implant surfaces for treatment or maintenance of peri-implant tissue. 4 Removal of all calcified deposits and plaque from the implant surface is challenging because of macroscopic structures and specific micro-surface topography that hampers the cleansing procedure. 5 Several conventional instruments and approaches including plastic or titanium curettes, ultrasonic and air abrasive devices have been commonly used for removing biofilms from the dental implant surfaces. 6 However, as these conventional approaches cannot remove bacteria stuck to the implant surfaces, adjunctive chemical irrigation agents have been clinically examined and they have been shown to improve post-treatment healing. 7 Patianna, et al. 8 (2018), in an in vitro study, showed that the use of 14% doxycycline gel efficaciously decontaminated both machined and sandblasted acid etched implants surface.
Apart from these conventional approaches, the choice of several lasers has been proposed to treat peri-implant diseases. Lasers with several wavelengths may have different clinical applications depending on the tissue affinity and the degree of penetration. 9 According to the research of Mailoa, et al. 10 (2014), some effective lasers for periimplantitis treatment are still inadequate, the CO 2 and Er:YAG lasers are the most studied lasers due to their high bactericidal ability, and lasers could be an adjunct in the treatment of peri-implantitis. Nd:YAG (Neodymium-doped Yttrium Aluminum Garnet) laser and CO 2 lasers are not suitable, for they generate heat while treating peri-implant infection, altering or damaging the implant surface. 11 Recently, diode laser, which are increasingly used in dentistry due to their excellent versatility and being cheaper than garnet laser, are employed as an adjunctive tool of non-surgical mechanical therapy or even used itself as a valuable tool for treating peri-mucositis and periimplantitis, [12][13][14][15][16] Er,Cr:YSGG (erbium, chromium-doped: yttrium, scandium, gallium, garnet) lasers, at a wavelength of 2,780 nm, have also been reported to improve the decontamination of bacterial deposits from the implant. 25 According to Schwarz, et al. 26 (2006)

In vivo biofilm formation
Acrylic splints for the maxillary arch including eight titanium discs (Kobe Steel, Japan, commercially pure titanium grade II, diameter 6 mm thickness 2 mm), were fabricated for the collection of plaques ( Figure   1a). The surface roughness (Ra) of titanium disc specimens was determined using a three-dimensional (3D) optical profiler (MV-E1000, NANO SYSTEM, Korea). Measurements were taken from an average of five different spots in each specimen (Ra=0.66 mm).
Acrylic splints with 8 mm x 30 mm rectangular hole of 3 mm depth on the left and right palatal side were manufactured and sterilized with ethylene oxide gas.
After the titanium discs sterilization in a high pressure steam turbine, discs were attached in acrylic splints with flowable resin (G-aenial Flo, GC Co., Japan) with 1 mm distance to the palate, according to John, et al. 28 (2014). By maintaining this distance, soft tissues and tongue influence could be excluded while ensuring a moist and nutritious environment.
Participants wore the splints for 72 hours, except when they manually brushed their teeth. Participants followed their regular diet during this period.
Immediately after biofilm formation in vivo, the titanium discs were removed from the splint, waterwashed, and dyed with disclosing solution (FD&C Red #28, Sultan Healthcare, USA). The disclosing solution was used to dye oral-biofilms growth in titanium surfaces ( Figure 1d).

Materials
Contaminated titanium discs were collected and each splint was divided into four sections with two titanium discs each, and they were assigned to the following groups (  (4) Er,Cr:YSGG laser irradiation (n=12).

Treatment procedure
The following treatments were applied in all samples to completely remove the biofilm stained by disclosing the solution visible to the naked eye. In the plastic curette group, stained biofilms were removed using a plastic curette (Hu-Friedy Mfg. Co., USA) with saline irrigation. In the Er:YAG laser (wavelength of 2,940 nm, Anybeam E, BnB system, Korea) group, laser parameters were set at 50 mJ/pulse (8.92 J/cm 2 ), 30 Hz, 150 μsec, water 30%, air 70%. Laser irradiation was performed in non-contact mode at a distance of 0.5 ~ 1 mm from the disc surface by a pain-ended cylindrical tip with a diameter of 850 µm to avoid any mechanical damage caused by contact mode according to Matsuyama, et al. 29 (2003). In Er,Cr:YSGG laser (wavelength of 2,780 nm, Waterlaser MD, Biolase, USA) group, irradiation was performed at a setting of 2.5 W average power at 25 Hz, pulse duration 140 μsec (35.7J/ cm 2) , water 30%, air 30%. 26 The laser beam was collimated by a cylindrical glass tip with 600µm diameter at 1 mm distance perpendicularly to the disc surface with non-contact mode. Irradiation was performed in a zigzag pattern. The operator was trained to perpendicularly irradiate the titanium surface via a cone-shaped fiber tip placed 1 mm above the surface. All treatments were conducted by a single    Fewer total live and dead bacteria were covered in the two lasers than in plastic curette group (P=0.05 for both comparisons). The viable bacteria on the titanium surface after Er,Cr:YSGG laser irradiation was significantly lower compared to the decontamination with plastic curette use (p=0.05) and the difference was not significant after Er:YAG laser irradiation.  This agrees with previous studies investigating biofilms formation on different specimens, which showed a mature and homogenous biofilm of inserted disks after 24 hours. 30,31 The splints used in this study were made similarly to that described in an aforementioned study. 30 Surfaces of titanium discs in acrylic splints were positioned toward the palate at a distance of 1 mm for nutritious moist circumstances to provide favorable conditions for in vivo biofilm formation.

Total bacteria
All decontamination methods used in this study decreased the residual plaque area. After cleansing, mean residual plaque area in the plastic curette group was higher than that in the Er:YAG laser and the Er,Cr:YSGG laser group. The results of previous investigations examining the effectiveness of different types of lasers as plaque removal methods are similar to ours: Er:YAG laser: 5.8±5.1%, Er,Cr:YSGG laser: 9.8±6.2%. 26,30 However, in our study, the mean residual plaque area after decontamination with plastic curette was lower than previous study outcomes. 29,31 According to the results from previous experiments, mean RPA after decontamination with a plastic curette ranged between 58.5±4.9% and 61.1±11.4%. 30,32 This discrepancy is due to complete removal of bacterial plaque using disclosing agent without any time restrictions in our study. Furthermore, titanium surfaces without any structures (e.g. threads) enable easier access to a plastic curette.
The mean treatment time from the experiments in decreasing order: Er:YAG laser, Er,Cr:YSGG laser, and plastic curette group. The Er:YAG laser had the longest treatment time, concurring with previous study results. 30 The treatment time for decontamination with an Er:YAG laser was longer than other methods 336±72s). The treatment time in this study was shorter compared to that study 30 for the smaller sample size, with an area of 0.28 cm 2 compared to 0.7 cm 2 . Furthermore, because of the smooth titanium surface, the area that can be removed at once with a plastic curette is larger. This explains why the plastic curette group presented the shortest treatment time.
Treatment time in Er,Cr:YSGG laser was shorter than Er:YAG laser. One could explain that Er,Cr:YSGG laser was used at higher power settings in this study, and it has an ablative hydrokinetic process that enables more efficient decontamination and debridement. 25 The quantification of adherent microorganisms based on biofluorescence are simple, precise, and reproducible. These systems are more convenient and reliable than the traditional methods of microbial quantification. 33 The visual differences presented in this study between living and dead bacteria using live/dead staining techniques was significant in this investigation. 34 SYTO9 and propidium iodide staining has shown to be better than other assays by providing an obvious difference between dead and active microorganisms without interfering with the background fluorescence. 35 The use of fluorescence microscopy approaches enables the visualization of bacteria. 36,37 In this study, anti-adherence activity of three treatments on biofilm contaminated titanium surface was examined by estimating the total bacterial load.
The total bacteria on the titanium surface after two process of laser irradiation were significantly lower than after decontamination with plastic curette. This difference occurred because of the water source, which comes directly from the laser apparatus in irradiation, compared with passive irrigation in hand scaling. Additionally, the bactericidal effectiveness of laser irradiation was determined by estimating the percentage of dead to total bacteria after 72 hours of biofilm formation and decontamination procedure.
All methods of treatment were capable of inactivating adhered microorganisms. We found that there was a higher ratio of dead to total bacteria in the laser group, which suggests that the laser had a bactericidal effect. The results of this experiment agree with previous study. 18 According to the microbiological and microscopic result of previous study, Er:YAG laser is likely to have higher bactericidal possibility on implant surfaces. 18 In our study, supragingival biofilm adhered on the titanium surfaces after a period of 72 hours was early non-mineralized plaque. Microbial composition might be different from subgingival plaque in the crevice of peri-implantitis site. Giannelli, et al. 23 (2017) in their study used a bacterial plaque originated from the subgingival margin of diseased implants with sterile curettes and smeared on the surface of disks.
Therefore, this issue about true pathogenic biofilm in peri-implantitis must be developed in further study. Matsuyama, et al. 29 (2003) suggested that for periodontal debridement, the radiant energy below 50mJ/pulses has usually been used, and, Er:YAG laser debridement of dental implant surfaces may be feasible without damaging their surfaces in the low irradiation setting. Furthermore, 30mJ/pulse, 30 Hz with water irrigation enabled effective removal of bacterial deposit and regions of calcification on the implant abutments without damaging their surfaces. Strever, et al. 38 (2017) showed that an Er,Cr:YSGG laser effectively removes single-species biofilms on disks without cognizable physical injury, using clinically relevant power setting.
As long as we know, we are the first to report the effectiveness of Er.Cr:YSGG laser in compared to Er:YAG laser in removing in vivo biofilms on titanium surface by comparing quantitative or qualitative values obtained in vitro with conventional cleansing methods.
A limitation of this study is that biofilms were not formed in real rough surface of an implant but on a smooth titanium surface. Investigation of Rimondini, et al. 31 (1997) showed that the surface roughness of specimens is the key factor in early in vivo plaque accumulation. According to Quirynen, et al. 39 (1993) the surface roughness acts as a threshold in bacterial colony formation, preventing large bacteria from adhering to the surface with roughness lower than Ra=0.2 µm. We also found that the value of surface roughness (Ra=0.66 µm) of titanium disk surface was higher than the threshold even though it was machined surface, it did not prevent the bacterial colonization. In clinical situations, defect morphology around implants or poor restorations hamper the access to the region of interest. In fact, plastic curette is suitable for cleansing the platform site and upper structures of the implant, but if the tip width is larger than the distance between screw pitches of the implant, it is difficult to remove the biofilm, and may be left fine plastic remnants when used on rough-surface implants. Therefore, further research is necessary to clarify bacterial adhesion and method of decontamination on rough surface implant surfaces in clinically simulated situations. Considering that the laser energy at target may differ from the set in the control panel, especially for laser devices using optic fibers, direct measurement of beam energy of Effect of a Er,Cr:YSGG laser and a Er:YAG laser treatment on oral biofilm-contaminated titanium 2020;28:e20200528 9/10 both Er:YAG and Er.Cr:YSGG lasers are necessary to perform by a power meter. It is important to provide reliable information on the delivered energy to achieve optimal decontamination without inducing unwanted alterations of implant surface. Also, an infrared thermal camera could also be helpful to monitor temperature at the targeted surface during laser irradiation.