## 2 Materials and Methods  
### Study Design  
Grade 4 titanium disc samples \"FINOFNAME TI-4\" (FINO-Gmbh, Mangesfeld 18, 97708 Bad Bocklet, Germany) were used in our study because this is the most commonly used grade of titanium to produce dental implants [22]. The sample was divided into 4 equal slides, as shown in (Figure 1). One slide was not treated and considered as a control (a). Three slides were treated with the lasers 1064-nm Nd-YAG (b), 980-nm diode (c), or 1064-nm Q-switch Nd:YAG nanosecond pulse (d), through the parameters described in Table 1. All of the lasers used were produced by the Italian company DMT Srl. (20851--Via Nobel 1 Lissone (MI)-Italy). Since the oral environment could influence the effects on peri-implants, a bioreactor simulating the saliva and titanium interaction was designed (Figure 2). The bioreactor was able to mimic the saliva composition, humidity, pH, and temperature characteristics of the oral environment. Essentially, a thermostatic bath with a recirculation pump (Grant model ST 12; Grant Instruments Cambridge, SG8 6 GB, Cambridgeshire, United Kingdom) was filled with distilled water to keep the temperature constant and contain a cubic glass tank (15 cm for side) immersed. A stub was then glued to the bottom of the cubic glass tank by a two-component adhesive (Tintoke 3M, Saint Paul, Minnesota, USA) to support the titanium disc. Fifty cetilitres of a salivary substitute solution (0.103 g/L of CaCl, 0.019 g/L MgCl-6H2O, 0.544 g/L KH2 POA, 2.24 g/L KCl, and TCP-KOH buffer, final pH = 7.0) was added [23]. The pH of the artificial saliva was balanced by measuring it with the Five Easy Plus pH Meter FP20-Std-Kit, (Mettler Toledo, Columbus, OH, USA). The thermostatic tank with a recirculation pump was activated to induce a temperature of 37.5 \({}^{\circ}\)C, which was measured with the double probe K thermocouple thermometer, HI 935002 digital thermometer, K probe, 2 inputs (HANNA INSTRUMENTS Italia srl--Viale Delle Industrie, 11--35010 Ronchi di Villafranca Padovana, Italy). Additionally, the humidity was maintained at around 93 Kg/m\({}^{3}\) and measured with the HI9564 digital thermo-hygrometer (HANNA INSTRUMENTS Italia srl) [24; 25]. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**LASER**} & **1064 nm Nd-Yag** & **980 nm Diodo** & \begin{tabular}{c} **1064 nm Q-Switch** \\ **Nd-Yag** \\ **d** \\ \end{tabular} \\  & **b** & **c** & **d** \\ \hline WAVELENGTH & 1064-nm & 980-nm & 1064-nm \\ PRIMARY POWER & 10 W & 7 W & 1.5 W \\ POWER PEAK & 3500 W & 15 W & 25 KW \\ FREQUENCY & 200 Hz & 1000 Hz & 500 Hz \\ PULSE DURATION & 50–500 us & 1 ms—CW & 3 ns \\ CLASS MEDICAL & \begin{tabular}{c} IIB \\ **EQUIPMENT** \\ **CLASS OF THE** \\ **LASER** \\ \end{tabular} & \begin{tabular}{c} IIB \\ **IV** \\ **IV** \\ **1.5 W** \\ **50 us pulse** \\ **10 Hz** \\ **3500 W power peak** \\ \end{tabular} & \
\begin{tabular}{c} IIB \\ **IV** \\ **IV** \\ **1.5 W** \\ **10 ms Ton** \\ **10 ms Toff** \\ **320 um fiber** \\ \end{tabular} &
\begin{tabular}{c} IIB \\ **IV** \\ **IV** \\ **20,000 Hz** \\ **25,000 W power peak** \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Laser specifics and parameters of irradiation. Parameters were chosen according to the therapies for peri-implant disease programs that each company of laser device set. Figure 1: Design of the treated area on a titanium grade 4 disc: (**a**) no-treated area (control); (**b**) area treated with 1064 nm Nd-YAG; (**c**) area treated with 980 nm Diode; (**d**) area treated with 1064 nm Q-Switch Nd-YAG. The structure was placed on the top of an orbital shaker (SLA-OS-200. Scitech LabApp Ltd., Micklefield, Leeds, LS25 9 BP, UK) set at 20 rpm to allow the oscillating artificial saliva to touch the titanium surface of the samples. The titanium discs were pre-incubated inside the bioreactor for 7 days before the experiments took place.