*2.5. Characterization Methods*

A polarized optical microscope (OM) (Zeiss Axioscop, Zaventem, Belgium) was used to detect electrospun fibers directly collected on glass slides during electrospinning. Scanning electron microscope (SEM) observations were performed 24 h after mat fabrication at an acceleration voltage of 15 kV. Samples were mounted on a stub with double-side adhesive tape and sputter-coated with gold before observation. The distribution of fiber diameters (average and standard deviation) was measured on the SEM images of about 50 fibers by means of ImageJ software. Wide-angle X-ray diffraction (WAXD) analysis was carried out using a PANalytical powder diffractometer (Almelo, Netherlands) endowed with a fast X'Celerator detector. The radiation was generated from a CuKα (λ = 0.15418 nm) source (40 mA, 40 kV). WAXD data were obtained from 2θ values from 5◦ to 60◦, where θ is the incidence angle of the X-ray beam on the sample. Fourier transforms infrared (FTIR) spectroscopy was carried out on a Nicolet 380 FTIR spectrometer (Thermo Scientific, Waltham, MA, USA) using ATR Golden Gate. 32 scans were performed with a resolution of 4 cm−<sup>1</sup> in the range 4000–800 cm<sup>−</sup>1. Spectra were smoothed using the Savitzky–Golay function and second-derivative spectra of the amide I region were used at peak position guides for the curve fitting procedure, using OriginPro 9.1 software.

#### *2.6. Crosslinking Extent*

The crosslinking extent was measured according to the method of Panzavolta and coworkers [8]. Briefly, an UV assay of uncrosslinked ε-amino groups was performed on differently treated mats and on fish gelatin as reference. After the reaction with 0.5% TNBS, gelatin was hydrolyzed with 6 M HCl and extracted with diethyl ether. The solution's absorbance was measured against a blank at 346 nm. The moles of free ε-amino groups per gram of gelatin were calculated by the following Equation (1):

$$\text{Moles of } \varepsilon \text{-amino groups/g of galactic} = \frac{2 \cdot A \cdot V}{\varepsilon \cdot b \cdot \chi} \tag{1}$$

where *A* is the sample absorbance, *B* is the final sample volume (L), ε is the TNP-lys molar absorptivity (1.46 <sup>×</sup> <sup>10</sup><sup>4</sup> L mol−<sup>1</sup> cm<sup>−</sup>1), *<sup>b</sup>* is the cell path length (cm), *<sup>x</sup>* is the sample weight (g).

The cross-linking extent (CE) was determined from the ratio between the moles of crosslinked ε–amino groups of treated gelatin mats (obtained as a difference between uncrosslinked groups before and after crosslinking) with respect to ε–amino groups measured in fish gelatin.
