The Effects of Erbium-Doped Yttrium Aluminum Garnet Laser (Er: YAG) Irradiation on Sandblasted and Acid-Etched (SLA) Titanium, an In Vitro Study

The treatment of peri-implantitis implies the decontamination of the surface of the fixture. This study aims to analyze the effect of the erbium-doped yttrium aluminum garnet laser (Er: YAG) on sandblasted and acid-etched (SLA) titanium. 30 titanium SLA disks were divided into three groups. In Group 1, the disks were left intact; on the contrary, both Groups 2 and 3 were irradiated with the Er: YAG laser at different settings, with a pulse duration of 300 μs and a period of 30 s. Group 2 was irradiated at 1 W and 100 mJ/pulse and Group 3 at 4 W and 400 mJ/pulse. The superficial changes at chemical, nano, and microscopical levels were detected through the use of Fourier-transform infrared spectroscopy, atomic force microscopy, and scanning electron microscope. The Kruskal–Wallis test, followed by the Dunn–Bonferroni Post Hoc analysis, detected the presence of statistically significant differences among the groups. The level of significance was p ≤ 0.05. Results showed that Er: YAG irradiation promoted a significant (p < 0.05) increase of oxides and a decrease of microscopical roughness and porosity on SLA disks. However, the protocol tested on group 3 seemed to be too aggressive for the titanium surface, as shown by the presence of micro-cracks and signs of coagulation, melting, and microfractures. In conclusion, Group 2 showed significantly minor surface alterations with respect to Group 3, and the increase of superficial oxide level, the decrease of porosity, and micro-roughness represent a positive alteration that could protect the materials against bacterial adhesion.


Introduction
Dental implants are widely used for the rehabilitation of partially or fully edentulous patients. Today, one in four patients rehabilitated with the implant-supported prosthesis are likely to undergo a peri-implant disease at some point in their life [1]. Peri-mucositis is a pathological reversible condition characterized by the inflammation in the peri-implant tissues, but when the process is associated with a progressive loss of supporting bone, it is defined as peri-implantitis [2]. The etiological factors of

Sample Size Calculation
The sample size was determined based on the study of Kim et al. [28] that calculated a minimum of 3 samples for each group through a preliminary study. However, considering that we had to perform 3 different analyses for each group, Atomic Force Microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR), and Scanning Electron Microscope (SEM), a minimum of 9 disks for each group were necessary.

Titanium Disks
Commercially pure titanium (Isomed, Padova, Italy) disks of 5 mm in diameter and 2 mm in thickness with sandblasted and acid-etched surface were used ( Figure 1A). All the samples were fully packed and sterilized, and the packages were opened just before laser irradiation. In total, 30 disks of titanium grade 4 were used for this research, divided into 3 groups: • Group 1: 10 disks without any treatments: controls • Group 2: 10 disks subjected to Er: YAG laser treatment at 1 W and 100 mJ/pulse • Group 3: 10 disks subjected to Er: YAG laser treatment at 4 W and 400 mJ/pulse Materials 2020, 10, x FOR PEER REVIEW 3 of 12 The aim of this study was to evaluate the effects at the chemical, nano and microscopical level of different powers and durations of Er: YAG laser irradiation on SLA titanium disks in order to find the better clinical protocol.

Sample Size Calculation
The sample size was determined based on the study of Kim et al. [28] that calculated a minimum of 3 samples for each group through a preliminary study. However, considering that we had to perform 3 different analyses for each group, Atomic Force Microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR), and Scanning Electron Microscope (SEM), a minimum of 9 disks for each group were necessary.

Titanium Disks
Commercially pure titanium (Isomed, Padova, Italy) disks of 5 mm in diameter and 2 mm in thickness with sandblasted and acid-etched surface were used ( Figure 1A). All the samples were fully packed and sterilized, and the packages were opened just before laser irradiation. In total, 30 disks of titanium grade 4 were used for this research, divided into 3 groups:


Group 1: 10 disks without any treatments: controls  Group 2: 10 disks subjected to Er: YAG laser treatment at 1 W and 100 mJ/pulse  Group 3: 10 disks subjected to Er: YAG laser treatment at 4 W and 400 mJ/pulse

Laser Device
An Er: YAG laser (Fidelis, Fotona, Ljubljana, Slovenia) with a wavelength of 2940 nm and a sapphire tip has been used in this study. The pulse frequency of 10 Hz and pulse durations up to 300 μs for a period of 30 s were chosen as recommended by the manufacturer.
The laser was applied perpendicular to the disk with a beam that focused on a spot size of 0.4 mm at 1 mm from the application tip. The water flow rate was set manually at 8 mL/min, and the tip was moved with sweeping movements. The laser tip moved from the periphery to the center of the

Laser Device
An Er: YAG laser (Fidelis, Fotona, Ljubljana, Slovenia) with a wavelength of 2940 nm and a sapphire tip has been used in this study. The pulse frequency of 10 Hz and pulse durations up to 300 µs for a period of 30 s were chosen as recommended by the manufacturer.
The laser was applied perpendicular to the disk with a beam that focused on a spot size of 0.4 mm at 1 mm from the application tip. The water flow rate was set manually at 8 mL/min, and the tip was moved with sweeping movements. The laser tip moved from the periphery to the center of the disk in parallel movements with constant velocity ( Figure 1B,C). After laser irradiation, all thirty disks were collected in a sterile and clean container.

Surface Analyses
The surface characterization of the disks was performed using Atomic Force Microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR), and Scanning Electron Microscope (SEM).

Atomic Force Microscopy
The AFM, NX10 Park AFM instrument (Park System, Suwon, Korea) was equipped with 20-bit closed-loop XY and Z flexure scanners and a non-contact cantilever PPP-NCHR 5M, permitted to analyze the surface topography of the disks.
The scan rate was 0.1 Hz, and each disk was analyzed in four different areas of 10 µm × 10 µm, as previously described [29]. The Nanoscope Analysis software (1.5, Bruker, Milan, Itay) was used for measuring the average roughness, Ra, and the root-mean-square roughness of profile, Rq, with the respective standard deviations (SD).

Fourier-Transform Infrared Spectroscopy
The X-ray diffraction (FTIR) analysis and light optical microscope survey were permitted to observe the morphology of the oxide layer of the samples [30]. For the superficial disk microstructure observations, an Olympus/PMG3 optical microscope (Olympus Corporation, Tokyo, Japan) was used.
The area of the disks covered by different colors was recorded by a Sony α330 digital camera (Sony, Konan, Minato-ku, Tokyo, Japan) and then, the NIS-Elements AR software (3.0, Nikon, Minato, Japan) performed the morphometric analysis. The measured parameters were expressed in percentage (Mean ± Standard Deviation).

Scanning Electron Microscopy
The Scanning Electron Microscopy observation, SEM (JSM-6480LV; Jeol, Tokyo, Japan), was used to obtain the surface topography analysis using the instrument's software, as previously described [31].

Statistical Analysis
The data analysis was performed by GraphPad Prism 6 software package (GraphPad Software, Inc., San Diego, CA 92108, USA). The Shapiro-Wilks normality test was performed, and the roughness surface means were evaluated between the study groups by the Kruskal-Wallis test followed by the Dunn-Bonferroni Post Hoc analysis. The level of significance for the analysis was p ≤ 0.05.

Results
The AFM measured the roughness parameters in each of the ten disks and the mean values (±standard deviations) of average roughness (Ra) and root mean square roughness of profile (Rq) were calculated (Figure 2A-C). The field of view was also sufficient to detect the longer-range roughness due to sandblasting. The average Ra (±standard deviations) were 137.71 (±5.317), 57.83 (±3.136), and 45.21 (±3.461) nm for Groups 1, 2, and 3, respectively. The mean Rq (±standard deviations) were 183.92 (±8.391), 72.92 (±4.297), and 59.3 (±2.313) for Groups 1, 2, and 3, respectively. A significant statistical difference was detected for both roughness parameters between controls vs. Group 2 and 3, p < 0.05. A statistical difference (p < 0.05) was also present between Groups 2 and 3 (Table 1 and Figure 3).     . Surface roughness parameters of two differently treated titanium disks. Ra and Rq of Group 1 (no treatment) were significantly higher than Group 2 (1 W and 100 mJ/pulse) and Group 3 (4 W and 400 mJ/pulse) (**p < 0.01). A statistical difference was present between Groups 2 and 3 (*p < 0.05).
The achieved colors by means of FTIR followed a chromatic scale that went from silver-white to golden-yellow, to blue to light yellow, and then to light green and brownish-black ( Figure 4A, B). In the control, Group 1, the color on the surface of the specimen was silver-white (40%), golden-yellow (32% ± 4.2%), and dark violet (25% ± 4.4%), and no microcracks were observed. In the test Group 2, the color on the surface of the specimen was silver-white (18% ± 2.2%), golden-yellow (32% ± 4.2%), and dark violet (28% ± 4.4%), and no microcracks were observed. In the test Group 3, the color on the surface of the specimen was silver-white (15% ± 1.2%), light-yellow (20% ± 2.1%), golden-yellow (20% ± 4.1%), blue (25% ± 2.5%), light-green (5% ± 0.9%), and then brownish-black (15% ± 1.9%). In the test Group 3, it was also possible to observe many microcracks. Such color changes on the samples were closely related to colored titanium oxides in the oxide layer. From the FTIR analysis, we acquired qualitative information about changes in the thickness of the oxide layer as the power laser irradiation. In all groups, the spectroscopic analysis by FTIR showed that absorption in the 400-700 cm −1 range changed with power laser irradiation, while the transmittance was increased after the laser irradiation in the Groups 2 and 3 (Table 2 and Figure 5). According to the described band, the disk surfaces were covered by different thickness of an amorphous TiO2 layer. . Surface roughness parameters of two differently treated titanium disks. Ra and Rq of Group 1 (no treatment) were significantly higher than Group 2 (1 W and 100 mJ/pulse) and Group 3 (4 W and 400 mJ/pulse) (** p < 0.01). A statistical difference was present between Groups 2 and 3 (* p < 0.05).
The achieved colors by means of FTIR followed a chromatic scale that went from silver-white to golden-yellow, to blue to light yellow, and then to light green and brownish-black ( Figure 4A,B). In the control, Group 1, the color on the surface of the specimen was silver-white (40%), golden-yellow (32% ± 4.2%), and dark violet (25% ± 4.4%), and no microcracks were observed. In the test Group 2, the color on the surface of the specimen was silver-white (18% ± 2.2%), golden-yellow (32% ± 4.2%), and dark violet (28% ± 4.4%), and no microcracks were observed. In the test Group 3, the color on the surface of the specimen was silver-white (15% ± 1.2%), light-yellow (20% ± 2.1%), golden-yellow (20% ± 4.1%), blue (25% ± 2.5%), light-green (5% ± 0.9%), and then brownish-black (15% ± 1.9%). In the test Group 3, it was also possible to observe many microcracks. Such color changes on the samples were closely related to colored titanium oxides in the oxide layer. From the FTIR analysis, we acquired qualitative information about changes in the thickness of the oxide layer as the power laser irradiation. In all groups, the spectroscopic analysis by FTIR showed that absorption in the 400-700 cm −1 range changed with power laser irradiation, while the transmittance was increased after the laser irradiation in the Groups 2 and 3 (Table 2 and Figure 5). According to the described band, the disk surfaces were covered by different thickness of an amorphous TiO 2 layer. Materials 2020, 10, x FOR PEER REVIEW 7 of 12       Scanning Electron Microscopy (SEM) analysis revealed a nanoporous network structure on the surface of the titanium disk of Group 1 ( Figure 6A). The topography of the disk showed the typical microroughness imparted by SLA. Delamination and deformation of the surface were present in the Materials 2020, 13, 4174 8 of 12 laser irradiated areas. In particular, a loss of porosity, because of the extensive melting, and a relatively smooth surface were found in the irradiated disks. In particular, the titanium disks of Group 2 showed surface alterations, like exfoliation, melting of the material, and microcracks ( Figure 6B). In Group 3, signs of coagulation, melting, and microfractures were observed ( Figure 6A).
Scanning Electron Microscopy (SEM) analysis revealed a nanoporous network structure on the surface of the titanium disk of Group 1 ( Figure 6A). The topography of the disk showed the typical microroughness imparted by SLA. Delamination and deformation of the surface were present in the laser irradiated areas. In particular, a loss of porosity, because of the extensive melting, and a relatively smooth surface were found in the irradiated disks. In particular, the titanium disks of Group 2 showed surface alterations, like exfoliation, melting of the material, and microcracks ( Figure  6B). In Group 3, signs of coagulation, melting, and microfractures were observed ( Figure 6A).

Discussion
The impact of two different protocols of Er: YAG laser irradiation on SLA titanium disks for the treatment of peri-implantitis has been evaluated in this study. The treatment of peri-implantitis includes granulation tissue removal, the decontamination of exposed implant surfaces, the application of antibacterial agent, and the smoothening of rough implant surfaces, thus rendering those surfaces less attractive for bacterial accumulation [32,33]. Hauser-Gerspachave et al. showed that bactericidal effects of Er: YAG increase with laser dose, but with the risk to surface alterations [15].
Consequently, it is essential to verify and quantify the impact of such therapies on the implant surface, in order to find the safer protocol. Indeed, nano-, micro-, and macro-morphology of titanium are essential for achieving and maintaining osseointegration and stable peri-implant soft tissue conditions. The surface properties have the potential to influence the osteoblast cell proliferation, differentiation, and morphology, and the expression of extracellular matrix components, integrins, and growth factors [34,35]. The outcomes presented here show that Er: YAG laser irradiation on SLA titanium disks can lead to the reduction of the roughness surface with modification of the oxide layer.
A variation of the superficial roughness has the potential to also modify the bacterial adhesion to the implant: it has been shown that by increasing mean Ra above 0.200 µ m, the retention of plaque increases [36]. On the contrary, the presence of nano-roughness on the surface of titanium could exert an antibacterial activity, and some authors proposed a laser nano-texturing process of titanium in order to decrease the biofilm formation [37,38].
In this study, the surface modifications provided by the laser treatment were also visible macroscopically, with the color changing on the samples, that reached a dye that was near to that of the titanium oxides layer. These findings, supplemented by FTIR results, confirmed that our treatment enhanced the deposition of a new oxide layer mainly composed of amorphous TiO2. Moreover, the color of the disks changed significantly with the increase of power laser irradiation. It is well known that titanium is covered in the external layer by titanium oxides, like TiO2, TiO, and Ti2O3 [39]. Our findings suggest that laser irradiation increased the oxide layer of the TiO2 and TiO.

Discussion
The impact of two different protocols of Er: YAG laser irradiation on SLA titanium disks for the treatment of peri-implantitis has been evaluated in this study. The treatment of peri-implantitis includes granulation tissue removal, the decontamination of exposed implant surfaces, the application of antibacterial agent, and the smoothening of rough implant surfaces, thus rendering those surfaces less attractive for bacterial accumulation [32,33]. Hauser-Gerspachave et al. showed that bactericidal effects of Er: YAG increase with laser dose, but with the risk to surface alterations [15].
Consequently, it is essential to verify and quantify the impact of such therapies on the implant surface, in order to find the safer protocol. Indeed, nano-, micro-, and macro-morphology of titanium are essential for achieving and maintaining osseointegration and stable peri-implant soft tissue conditions. The surface properties have the potential to influence the osteoblast cell proliferation, differentiation, and morphology, and the expression of extracellular matrix components, integrins, and growth factors [34,35]. The outcomes presented here show that Er: YAG laser irradiation on SLA titanium disks can lead to the reduction of the roughness surface with modification of the oxide layer.
A variation of the superficial roughness has the potential to also modify the bacterial adhesion to the implant: it has been shown that by increasing mean Ra above 0.200 µm, the retention of plaque increases [36]. On the contrary, the presence of nano-roughness on the surface of titanium could exert an antibacterial activity, and some authors proposed a laser nano-texturing process of titanium in order to decrease the biofilm formation [37,38].
In this study, the surface modifications provided by the laser treatment were also visible macroscopically, with the color changing on the samples, that reached a dye that was near to that of the titanium oxides layer. These findings, supplemented by FTIR results, confirmed that our treatment enhanced the deposition of a new oxide layer mainly composed of amorphous TiO 2 . Moreover, the color of the disks changed significantly with the increase of power laser irradiation. It is well known that titanium is covered in the external layer by titanium oxides, like TiO 2 , TiO, and Ti 2 O 3 [39]. Our findings suggest that laser irradiation increased the oxide layer of the TiO 2 and TiO. TiO 3 is dark-violet while TiO 2 and TiO are respectively white-and golden-tinted [40]. However, the interpretation of the color change on titanium alloys is difficult. The presence and the quantification of the layer of oxidation is very important because it affects the wettability of the implant and consequently, the protein adsorption, the cellular adhesion and spreading, and the ability to promote differentiation of osteoblasts [41,42].
Recently, Ercan highlighted that the distance at which irradiation affects the power density is the principal parameter that influences the surface changes during laser treatment [43]. The laser irradiation of machined Ti at a power setting above 2 W resulted in the increased proportion of oxygen and decreased Ti, due to oxidation, with respect non irradiated disks; on the contrary, anodized titanium irradiated with the same settings resulted in the reduced proportion of O and increased Ti [24].
However, laser settings are also fundamental for the interaction with ti surfaces: the percentage of O in the superficial composition of machined Ti increased from about 2.5% in non-irradiated samples to 5.8%, 12.26%, 24.2%, and 35.0% after Er: YAG irradiation at 2, 3, 4, and 5 W, respectively [24].
Another interesting outcome of the present study is that there was a change in the nano-roughness surface of titanium grade 4. The roughness parameters decreased with the increase of laser settings in acid-etched (SLA) surfaces, and there was also a higher presence of cracks that developed after increasing irradiation at 400 mJ/10 Hz. These results are in accordance with another study [26]. The roughness of titanium is crucial because it is positively correlated to cellular adhesion, functional alterations, and proliferation of both osteoblastic cells and bacteria [34,35,44]. Galli et al. have shown that Er: YAG laser irradiation, 10 s at 200 mj/pulse and 10 Hz, promoted the formation of a melted area on SLA titanium, with the collapse of thin crest to flat, smooth plates [45]. These surface alterations were able to slow down the cellular proliferation, but did not alter the cellular differentiation. On the contrary, Wakim et al. have found no significant alteration on SLA titanium surfaces after multiple Er: YAG treatments with irradiation energy 50 mJ, 30 Hz frequency, 1.5 W power output, and 3.76 J/cm 2 energy density, but in this protocol, a super short pulse of 50 us was applied [46]. As a result, reduction to surface micro-roughness could delay or prevent bone bacterial adhesion. The results of the present research indicated that laser irradiation also changes oxide layers, especially the TiO 2 . This change is significant because it reduces the number of bacteria on the titanium and produces more healthy peri-implant tissues [47].
This study further confirms that the choice of laser parameters, like the power settings, the presence of water cooling, the distance, the inclination, the type of movements of the tip during irradiation, and the time of light exposure, should be made with extreme attention, considering approved and tested protocols, in order not to damage the titanium surface.
The limitations of this study are the absence of cellular and microbiological testing. This is only a preliminary study that aims to evaluate the topographical alterations on SLA titanium disks after Er: YAG irradiations, but the biological consequence of the different protocols should be tested in further in vitro and in vivo experimentations. Moreover, in vivo, the presence of saliva and pH conditions could interfere with the chemical composition of titanium. Another limitation is that the surface roughness statistical analysis performed in this study comprehends only data on the vertical dimension of surface architecture and it should be enhanced by adding other roughness parameters that would allow to describe the texture morphology in terms of height/peaks distribution.

Conclusions
Both tested protocols of Er: YAG laser irradiation of SLA titanium provided some modifications to the topography of the surface of the samples.
However, the treatment at 4 W and 400 mJ/pulse could weaken the titanium surface, as shown by the presence of microcracks and signs of coagulation, melting, and microfractures.