## 2 Materials and Methods  
### Preparation of CGF and LPCGF  
Blood samples (8 mL) were taken by puncture of a vein from five donor patients, non-smokers, and those in good general health. The five donors involved in the study (three men and two women) were aged between 25 and 45 years, with a BMI (Body Mass Index) between 21 and 23 points. The remote and pathological anamnesis were negative. Patients were not on therapy with any type of drug and the blood samples were taken separate from the main meals, on empty stomach. Informed consent was obtained from the patients included in this study. Tubes of blood were processed by a device (Medifuge MF200; Silfradent srl, Forli, Italy) to obtain CGF, following the manufacturer's instructions. The resulting CGF was then inserted into dedicated implant ampoules (Figure 1), so that the coating procedure could be carried out in a closed field; each contained an implant (Immediateload(r), Swiss dental implants, diameter 4 mm and height 8 mm). To incorporate the CGF onto the implant surface, these tubes were inserted into a second device, Round Up (Silfradent srl, Forli, Italy), and centrifuged for 5 s, following the manufacturer's instructions (Figure 2). Evaluation of the quality of the CGF was done on two fractions--the so-called white fraction and the red fraction. The white cup-tubes allowed the obtainment of a fraction, known as LPCGF (liquid phase of CGF), that comprised non-polymerized liquid fibrin. It was isolated by placing the centrifuged blood in a test tube, with a white lid; this was completely smooth within and contained no additives. It produced the material in a liquid state; this would polymerize at room temperature (RT), over the subsequent 15 min. The red cup-tubes allowed the obtainment of a fraction, known as CGF polymerized fibrin, which was isolated using a test tube with a red lid and textured inner walls, to promote polymerization, through an exclusively physical process. The resulting fraction had a thicker consistency than the fraction obtained by the white cup-tube, a gelatinous appearance, and a higher cell concentration of the non-polymerized fibrin. Figure 1: Concentrated Growth Factor (CGF) isolation after using the Siffradent device (Medifuge MF200: Siffradent srI, Forfi, Italy). Figure 2: Centrifugation of the test tubes with Immediateload implants along with CGF or the Liquid Phase of CGF (LPCGF) by Round Up (Siffradent srI, Forfi, Italy). By using an innovative device, we evaluated the potential incorporation of CGF on the surface of the titanium implant. We verified the interaction between the CGF and the titanium implant surface, by SEM analysis, revealing that the CGF actually permeated the surface of the implant (Figure 4). Having shown that, in our experimental conditions the CGF was able to adhere to the implanted surface forming a fibrin network, we evaluated whether the CGF-permeated implant also allowed the release of growth factors, in particular VEGF, from the implant towards the medium--PBS. As shown in Figure 5, the implants permeated with CGF or LPCGF were able to release VEGF in PBS medium. At time 0, in the permeated implant, as well as in the system without implants, the concentration of VEGF was five times higher in CGF than in LPCGF. However, contrary to the CGF or LPCGF without implants, VEGF released from the CGF or the LPCGF-permeated implant, increased on time. In particular, the VEGF levels from the LPCGF-permeated implant increased in a time-dependent manner, until the second day and then remained constant. Figure 4: (a) SEM photomicrograph of the CGF-permeated implant. (b) Large magnified image of the CGF-permeated implant detail. Figure 3: ELISA quantification of human vascular endothelial growth factor (VEGF) into phosphate buffer saline (PBS) from CGF and LPCGF at various times (0–3 days). Values are means \(\pm\) SD, n = 3. \({}^{*}\)\(p<\) 0.05 denotes the statistically significant differences between VEGF released by CGF or LPCGF.