## 2 Materials and Methods  
The current study was conducted with blood sampling from a single source: a caucasian male donor with Ethical Committee permission n84/2017 (University of Trieste). The volunteer signed a specific, informed consent for blood sampling and treatment of his blood for scientific purposes. Moreover, the volunteer certified the absence of systemic and/or infectious pathologies, allergies, and did not take medications. Lastly, he committed to not take drugs and/or medications during the study period. Blood did not subdue biochemical manipulation. An amount of 2 cl was used for calculating the angle on the implant surface of specimens (Figure 1) without PRF. Additionally, 10 cl was used for the preparation of the PRF. For this purpose, blood was collected in an empty test tube placed in the appropriate centrifuge (Spectrafuge(r) 6c--Labnet International(r), Inc., Edison, NJ, USA) with 8.5 cm centrifuge radius. In order to obtain PRF, blood centrifugation lasted 10 min at 3000 r.p.m. [9]. An amount of 2 cl of the blood sample was used to calculate the contact angle between the implant surface and blood of the titanium samples after the PRF application and 2 cl of blood sample was used after phosphoric acid (38%) etching. Blood collection was repeated, when necessary, at different times to avoid coagulation. In the current study, a Leica MZ16(r) (Leica(r), Wetzlar, Germany) optical microscope with Leica CDC 320(r) camera (Leica(r), Wetzlar, Germany) with Image ProPlus(r) software Media Cybernetics(r), Inc., Rockville, MD, USA) was used to take profile pictures of titanium samples. Five grade IV titanium disks were used for each surface type: machined, laser-treated, and sandblasted (Figure 2) with a Biohit m1000(r) micropipette (Biohit(r), Helsinki, Finland). Contact angles were then calculated as described in Figure 1.  
In step 2, disks were instrumented as here described: the entire top surface of the titanium disks was instrumented with an Acteon Implant Protect(r) ultrasonic grade IV titanium tip (Acteon India(r)Pvt Ltd., Gurugram Haryana, India) set on a Satelec(r) scaler (Satelec(r)Sas, Merignac, Cedex, France) for a total of 40 strokes by a single operator (FF). The scaler tip was angulated tangentially, and care was taken to exert minimal lateral pressure. Calibrations were performed before the experiment, and the average force applied in this study was 30 g. The back-and-forth movement was performed in the same direction for 40 strokes. Scaler power of 3/10 was applied at 25 to 32 kHz frequency. Samples were  
Figure 1: **(a)** Geometric construction for the determination of the contact angle. These reference points are straight line a, passing across the two points of contact between implant surface and the blood drop, straight line b, which is the radius of the circumference created by the blood drop (constructed with best fit), prolonged to intersect one of the point of intersection between the circumference and the line a, and the line c, perpendicular to line b and tangential to the circumference at the point of intersection with line a. With these reference points, it is possible to calculate the angle between line c and line a, which corresponds to the contact angle between the blood drop and the surface. The smaller is the angle, the greater is the adhesion of the drop, and therefore the greater is the wettability. **(b)** Picture was taken with Leica FDC 320®camera (Leica®, Wetzlar, Germany) set on an optical microscope Leica MZ168(Leica®, Wetzlar, Germany). Reference points in the picture were marked with Image ProPlus® software (Media Cybernetics®, Rockville, MD, USA). Figure 2: 3\(\times\) and 300\(\times\) magnification of brand new titanium surfaces before instrumentation (t\({}_{0}\)) and after instrumentation (t\({}_{1}\)). 300\(\times\) magnification was obtained with a scanning electron microscope (SEM) (Quanta 250 SEM®, FEI Company®, Hillsboro, OR, USA).