## 4 Discussion
Recently, the anti-inflammatory properties of olive products have also been investigated in relation to dietary food [39]. Chronic inflammation is linked to ROS production causing oxidative damage and antioxidant depletion. As such, some studies have investigated the influence of oleuprein on macrophages, the key cells involved in inflammatory processes by producing ROS and other pro-inflammatory cytokines, but further able to support healing by releasing anti-inflammatory cytokines, if timely and properly differentiated [40; 41; 42]. In some studies, OLE is reported to show direct antimicrobial activity, which was primarily attributed to its main polyphenols [43]. However, the findings available on the antibacterial properties of OLE are still debated, as they may depend on extraction methods, polyphenol composition and quantity, including dose, on selected bacteria; as such, combined technologies including for example OLE and plasma can be needed [44]. In our study, we assayed the release of three main components of OLE, namely, oleuprein, luteolin-7-O-glucose and apigenin-7-O-glucose from PHA fiber meshes. Such polyphenols were almost totally released after 24 h, reaching oleuprein about 90% and the two glucosides about 80% in 6 h. Unlike using systemic or even local injections, the scaffold can promote a better confined delivery at the wound site by avoiding the dispersion of the active principle through the blood vessels, thus keeping the drug concentration locally higher [45]. In addition to OLE release, it is expected that the fiber meshes would provide a suitable support for tissue regeneration. Since re-epithelialization is a leading phenomenon in skin repair, we investigated the viability of HaCaT cells by measuring their metabolic activity on the different meshes, with or without OLE, including using OLE alone. We observed that, when cultured on the fibrous scaffolds for 24 h, HaCaT cell metabolic activity was very similar, independently of the type of fiber or OLE loading, whereas it was higher when pristine OLE was added at the same amount. The difference of plain OLE effect on cell metabolic activity can be explained by the subitaneous availability of the bioactive ingredients with respect to a slower release operated by the fibers. However, it is frequently observed that the presence of a three-dimensional substrate affects cell response at early time-points, as adhesion phenomena and cell-cell interactions are more complex events than in bi-dimensional tissue culture plastics [46; 47]. In addition to macrophages, also epithelial cells, including skin keratinocytes, play a fundamental role in innate immunity, being primarily exposed to injuries and inflammatory stimuli. As such, epithelial cells contribute to healing by secreting several cytokines and defenses [17]. As well as for metabolic activity, the administration of OLE alone or mediated by PHA fibers after 24 h gave different outcomes in terms of cytokine expression at mRNA level. Our results showed that all the PHA fiber samples were endowed with immunomodulatory properties. In fact, they were able to downregulate the pro-inflammatory cytokines at 6 and 24 h, thus showing a suppressive function towards inflammation. Many studies have reported that cell-biomaterial surface interaction is central in biocompatibility as it rules the inflammatory process, including anti-fouling and antibacterial aspects [48]. Differently from the fiber samples, pure OLE displayed a role in the wound healing process, by modulating the expression of the pro-inflammatory cytokines, which were upregulated at 6 h and downregulated at 24 h. Wound healing is a complex process occurring via a cascade of events such as inflammation, reepithelial, matrix formation, tissue repair and remodeling. Two main phases drive this process: the first phase starts with the production of pro-inflammatory cytokines. TNF-\(\alpha\) and IL-1 represent the primary cytokines of pro-inflammatory response, which are immediately released by keratinocytes upon wound damage.