2.2.3. Biocompatibility of ALIG30@20

ALIPS9 and ALIG30@10 hydrogels (both with a solid concentration of 10%) have been demonstrated to be biocompatible against fibroblasts [17]. Moreover, in the very same study, the in vitro scratch assay proved that the hydrogels were able to accelerate wound closure by favoring fibroblast migration. Nonetheless, the ALIG30@10 hydrogel showed insufficient viscosity, as proven in another study that included a full rheological characterization of ALIPS9 and ALIG30@10 hydrogels [30]. The low consistency of a hydrogel could hinder its topical administration due to excessive fluidity of the formulation. Consequently, the ALIG30@20 hydrogel was prepared and its biocompatibility was evaluated. To do so, the methodology described by García-Villén et al. [17] was used. Normal human dermal fibroblasts (NHDFs, PromoCell GmbH, Heidelberg, Germany) were seeded and cultured in Dulbecco's modified Eagle medium (DMEM, Sigma Aldrich®-Merck, Milan, Italy), supplemented with 10% fetal bovine serum (FBS, Euroclone, Milan, Italy), 200 IU/mL penicillin and 0.2 mg/mL streptomycin (PBI International, I). Once cellular confluence was obtained (area 0.34 cm2/well, density 10<sup>5</sup> cells/cm2), ALIG30@20 was added to the cell culture in concentrations ranging from 1000 to 5 μg/mL and kept in contact with cells for 24 h. Then, the MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed. DMEM phenol red-free and 50 μL of MTT dissolution were added in each well, the final MTT concentration being 2.5 mg/mL. MTT-NHDF contact was maintained for 3 h before the whole supernatant was withdrawn and substituted by 100 μL of dimethyl sulfoxide solution (DMSO, Sigma-Aldrich®-Merck, Milan, Italy) to dissolve formazan. The absorbance of each well was measured at 570 nm with an ELISA plate reader (Imark Absorbance Reader, Bio-rad, Hercules, CA, USA), with the reference wavelength set at 655 nm. Fibroblast viability was calculated with respect to the viability of the corresponding control (fibroblasts cultured in fresh DMEM, abbreviated as GM). MTT tests over ALIPS9, ALIG30@10 and ALIG30@20 were performed after 1 month of hydrogel preparation.
