## 4 Discussion
In this respect, this measure can be considered an assessment of the \"biological cleaning\" of the implant. In the aforementioned paper from [PERSON] et al., the 22 different types of clinically available implants evaluated exhibited wide variation of adherent endotoxin, evoking a significantly different device-induced macrophage activation. Among tested implants, nine exhibited an expression of IL-6 that was 10 times higher than that of the control, including three implants with an expression greater than 40 times, and one implant with an expression greater than 100 times. The amount of adherent endotoxin can overwhelm the contribution of other implant surface characteristics (including chemical composition and topography) in affecting bone regeneration at the bone-implant interface and could be responsible for the so-called \"aseptic loosening\" of orthopedic prostheses and possibly of the breakdown of osseointegration at dental implants [2; 4; 20]. In addition to IL-6, the contact between inflammatory cells and surfaces triggers the production of a large number of other cytokines and chemokines influencing the peri-implant biological environment. The study of the biochemical pathways that they can potentially influence is very complex. Nitric oxide (NO) is a powerful gas molecule that acts as neurotransmitter and vasodilator; in addition, it is produced by immune cells as an effector molecule against tumor cells and pathogens. In particular, macrophages produce high levels of NO after being stimulated by various cytokines or by endotoxins such as bacterial lipopolysaccharide-ride (LPS) that, after binding to CD14 receptor, activates the NF-\(\kappa\)B signaling pathway. The activation of transcription factors such as NF-\(\kappa\)B leads to the transcription of inducible genes involved in inflammatory response, such as iNOS (NOS 2) and cyclooxygenase 2 (COX-2). iNOS is expressed by macrophages after a pro-inflammatory stimulus, and it determines the production of high levels of NO, creating an effective defense mechanism. In our experiment using RTqPCR, no increase in NOS expression was observed; on the contrary, a general decrease in NOS was evident in implant-incubated cells in comparison to polystyrene. This decrease was statistically significant for Shard implants (\(p<0.05\)). After their activation, macrophages also produce MMPs, a protease that is able to degrade extracellular matrix components. MMPs are also required for the correct process of cellular migration to the site of infection. Among MMPs, MMP-8 is one of the main collagenases found in periodontal tissues affected by periodontitis and peri-implantitis. Its correlation with the entity of tissue damage and to pathology degree has been proven and has led to its identification as a marker of active inflammation. The evaluation of MMP-8 expression induced by the analyzed implants showed a mild increase in all the implant types in comparison to polystyrene, but without a statistically significant difference. MMP-9 is another metalloproteate that is thoroughly expressed in tissues affected by periodontal disease, and, such as other MMPs, it has a regulating role in the inflammatory process thanks to its protease activity, which allows transforming a target substrate activating or inactivating it. Since MMPs can carry out destructive actions if not controlled, their expression and function are strictly regulated both at transcriptional and post-translational levels. A statistically significant mild increase (\(p<0.05\)) in MMP-9 expression was found in Prama(r) implants, in comparison with 3i T3(r) implants and to polystyrene reference sample. Molecules such as TIMPs act as MMP antagonists, inactivating them, and through this inhibition they play a key role in regulating an insult-induced inflammation. TIMP-3, in particular, can silence several MMPs, and it is produced by various cells, such as macrophages.