## 4 Discussion
For instance, IL-1 ra and IL-10 have well known anti-inflammatory functions [40; 41]; IL1 ra, physiologically secreted by the anabolic macrophage phenotype M2 (with anti-inflammatory and pro-regenerative phenotype) and by the recruited stromal stem cell contributes to the formation of a fibro-vascular granulation tissue ultimately resulting in local bone formation [42]. IL-2 is involved into both the induction and the termination of inflammatory immune responses and play a key role in favoring wound healing [43; 44]. IL-12 is a pro-inflammatory factor that amplifies downstream inflammatory signals and impairs MSC-mediated bone regeneration [45]. Conversely, IL-17, released by \(\gamma\delta\) T cells after bone damage, enhances osteogenic differentiation of resident MSCs [46]. In addition, basic FGF has a crucial role for the maintenance of stem cell features, as well as for bone regeneration [47]. In bone regeneration there is not a dominant cytokine, but modulation drive matrix deposition and wound healing. In vivo, in peri-implant sites their release can be influenced by certain factors: age, diabetes, smoking habits, oral hygiene, preoperative and postoperative antibiotics administration, some surgical aspects (flap technique, drilling protocols, insertion torque) [48]. The relationship between the surface parameters (roughness, wettability, chemical composition) and their ability to influence the cytokines release aimed to promote the osseointegration process is unclear. Our results showed that BFP-MSCs release detectable levels of a large variety of cytokines, chemokines and growth factors and that the implant surfaces do not interfere with their secretory functions. For example, no differences were found in the release of IL-5, involved in eosinophil functions [49], of IL-9, implicated in inflammatory diseases and in guarding immune tolerance [50], and of IL-15, required for osteoblast function and bone mineralization [51]. Nevertheless, in presence of Kontact S surface (D2-chemically etched surface), cells released a lower amount of IL-1 ra, IL-2, IL-10, IL-12, IL-17, G-CSE, and basic FGF compared to cells plated on D3 (sandblasted surface). Interestingly, in presence of D4 (blasted/etched surface) BFP-MSCs released significantly higher concentrations of G-CSF, compared to cells plated without disk and to cells plated on D1 and D2. G-CSF is a glycoprotein that is used therapeutically for its ability to mobilize hematopoietic, mesenchymal and vascular stem cells in the systemic circulation. It has a proliferative effect on osteoporosis and enhances bone regeneration via revascularization and osteogenesis [52; 53]. As result, the increased levels of this growth factor may have a crucial role for the improvement of peri-implant bone healing. The Kontact N surface, for its roughness, adhesivity capacity and grow factors stimulation, seemed to assure the best outcomes in comparison to the others examined implant surfaces. In future, research should clarify the correlation between the biological response and the implant surface characteristics; it would be the ideal situation from an industrial standpoint and the development of a clear and simple ISO standard. The strength of this study is represented by the use of primary mesenchymal stem cells obtained from the Bichat fat pad and by the evaluation of a large number of cytokines, chemokines and growth factors. Indeed, beside cell viability, the MSC secretory pattern is an important parameter to consider in order to achieve tissue regeneration. Thus, the determination of an MSC secretory phenotype may represent a novel and relevant functional assay. The main limitation is represented by the limiting number of not reusable disks, which did not allow measuring cell proliferation as a function of time and by the lack of a straight methodology for direct visualization and counting of cells. However, the use of crystal violet staining and of an automated cell counter are largely used methods to visualize as well as to evaluate cell number and viability.