2.1. Materials

Olive leaves extract (OLE) preparation was carried out according to the previously reported procedure from Olea europaea var. Olivastra seggianese cultivar [19]. The leaves from which OLE was extracted were collected at CNR-IVALSA, Follonica (GR), Italy. Briefly, the freshly harvested olive leaves were freeze-dried (Freeze Dryer Modulyo, Edwards, Latina, Italy), then grounded in the presence of dry ice. The powder was dispersed in deionized water (1:5 w/v) and homogenized (IKA-ULTRA-TURRAX T25, Milan, Italy) for 5 min at 8000 rpm. Then the mixture was sonicated (Digital Ultrasonic Cleaner, Argolab 40 KHz, Milan, Italy) for 20 min at 35 °C, 40 KHz. The resulting suspension was centrifuged for 5 min at 4000 rpm and the supernatant was filtered through a 0.45 µm cellulose acetate filter. The filtrate was lyophilized (VirTis adVantage-53, Stereoglass, Perugia, Italy. Freezing −35 °C, 180 min; drying −30 °C, 360 min; −10 °C, 240 min; 25 °C, 180 min) and kept in the freezer until use.

Gallic acid and Folin–Ciocalteu reagent, as well as all the inorganic salts were purchased from Sigma (Milan, Italy). Hyaluronic acid (HA) was purchased from Contipro (Dolní Dobrouc, Czech Republic), had a molecular weight of 454.8 ± 103.6 kg/mol (determined by Debye plot method, zetasizer, Malvern). Commercial chitosan, minimum 90% deacetylated from shrimp shell (Chito-clear FG90, Primex, Drammen, Norway) and average viscometric molecular weight of 590 kDa [20] was converted into a chitosan·HCl powder (Cs). Cs was prepared by making an aqueous chitosan suspension (12 g in 2000 mL) to pH 4.7 with 1 N HCl (about 43.5 mL) and lyophilizing the resulting solution after filtration.

Murine embryonic fibroblasts BALB/3T3 clone A-31 cell line was purchased from the American Type Culture Collection LGC standards (ATCC HTB-37, Milan, Italy) and propagated as indicated by the supplier. Dulbecco’s modified Eagle medium (MEM), complete Dulbecco’s modified Eagle medium (DMEM), non-essential amino acid, 0.01 M pH 7.4 Dulbecco’s phosphate-buffered saline (DPBS), phosphate buffered-saline free of calcium and magnesium (PBSA), fetal bovine serum (FBS), and Hank’s balanced solution were purchased from Sigma (Milan, Italy). Cell proliferation reagent (WST-1) was provided by Roche diagnostic (Milan, Italy). Staphylococcus aureus 33591 and Pseudomonas aeruginosa 27853 were purchased from ATCC (Milan, Italy). Tryptone Soy Broth (TSB) and Tryptone Soy Agar (TSA) were purchased from Oxoid (Basingstoke, UK).