Cacl2

Lixiviation tests were conducted according to a normalized protocol (AFNOR, NF ISO15181). Immersion and test procedures were carried out as previously described [32 (link)]. Synthetic seawater was composed as following: NaCl (24.5 g/L, Fisher), MgCl₂·6H₂0 (5.20 g/L, Acros), Na₂SO₄ (4.09 g/L, Aldrich), CaCl₂ (1.16 g/L, Carlo Erba), KCl (0.695 g/L, Fisher), NaHCO₃ (0.201 g/L, Carlo Erba), and H₃BO₃ (0.027 g/L, Carlo Erba, Val de Reuil, France). Surrounding water was analysed at each sampling dates and changed in order to avoid the risk of medium saturation. The releases of zinc pyrithione and copper thiocyanate were quantified by atomic absorption spectroscopy.

For the preparation of polymeric solutions the following materials were used: Deacetylated gellan gum (GG, Gelrite^®, Kelco Division of Merck & Co., Rahway, NJ, USA), Kolliphor P407 poloxamer (P407, BASF SE, Ludwigshafen, Germany), poly (ethylene oxide) of high molecular weight (h-PEO, MW = 4000 kDa, Colorcon, Dartford, UK), poly (ethylene oxide) of low molecular weight (l-PEO, MW = 600 kDa, Sigma Aldrich, Milan, Italy) and glycine (Gly, Sigma Aldrich, Milan, Italy). GG-based NFs cross-linking was performed using anhydrous calcium chloride (CaCl₂) and absolute ethanol (EtOH; Carlo Erba Reagents S.r.l., Milan, Italy).
Reagents and solvents for RC-33 synthesis were purchased from Merck (Milan, Italy).

Cells were seeded in a 6-well plate in 1,5 mL serum-free DMEM. The serum-free conditioned medium was harvested after 24 h for zymography. Similarly, the culture medium in the bioreactor was replaced with 6 ml of serum-free medium for 24 h and then collected for zymography. Zymography was carried out as described by Frankowski and colleagues⁵⁶ . Briefly, 1% gelatine (J.T. Baker) was added to the 12.5% polyacrylamide gel (Bio-Rad). After the development, the gel was washed in 2.5% Triton X-100 (Bio-Rad) for 1 h and then incubated in a development buffer containing 100 mM CaCl2 and 0.2% NaN3 (Carlo Erba Reagents) overnight. Finally, it was stained in a Coomassie brilliant blue R-250 solution (Bio-Rad, art). Zymography band quantification was performed using Gel Analyzer function in Fiji software^{57 (link)}.