2.2. Characterization of the hydrogels
Hydrogels Scanning Electron Microscopy (SEM). Surface morphology and porosity of the hydrogels were investigated using SEM imaging techniques. SEM images were recorded with a Carl Zeiss – Merlin, field emission scanning electron microscope, equipped with a Gemini column and integrated high efficiency In-lens and SE2 detectors, for high spatial/depth-of-field resolution secondary electrons (SE) imaging of surface structure and topography. The microscope was used in high vacuum and high-resolution acquisition mode and the images were acquired at an accelerating voltage of 5 kV and a few seconds frame-integration time, in order to minimize charging effects and sample damages. The hydrogels were cut, frozen and lyophilized prior to be imaged. The pore size was determined by taking 50 random pores followed by statistical analysis using ImageJ software (NIH, United States).
Stability test . The analysis was carried out keeping the hydrogels either in PBS or DMEM at 37 °C, and at determined time points (up to 7 days) the weight was measured. The stability of the hydrogels was then measured as residual weight percentage:
\begin{equation} Rw\%=\frac{w-w_{0}}{w_{0}}100\%\nonumber \\ \end{equation}
where w0 is the starting weight of the hydrogel at t = 0, and w is the weight at each time point. The measure was repeated three times and the data are reported as the average of the three measures.
Swelling Behaviour . The swelling property of the hydrogels was determined by using a conventional gravimetric method (N. Gallo et al., 2020). Briefly, the dry weight of hydrogels was recorded prior to immerse them in PBS at 37 °C. Then, the swelled weight of hydrogels was taken at various time points up to 48 h. The swelling behaviour was estimated as the swelling ratio percentage using the following equation:
\begin{equation} Sr\%=\frac{w_{s}-w_{d}}{w_{d}}100\%\nonumber \\ \end{equation}
where wd is the dry weight of the hydrogel and ws is the wet weight after hydration in PBS.
Collagen Release via BCA assay . The A-C hydrogels were kept in PBS at 37 °C and at determined time intervals the volume of buffer (equal to 1 mL) was collected to estimate the protein content via BCA assay (Terzi et al., 2018). A calibration curve was set using collagen solutions at known concentrations. For each time interval, a blank sample (i.e. the PBS collected from agarose hydrogels prepared without collagen) was measured too.
Fourier-transformed infrared (FTIR) spectroscopy . The measurements were performed on the A-C hydrogels at the different collagen ratios (0.125%, 0.25%, and 0.50%) directly deposited on the ATR crystal. FT-IR spectra were recorded in transmittance mode on a Jasco 6300 spectrometer (Jasco Corp., Tokyo, Japan) between 4000 and 500 cm-1 with 40 scans and a resolution of 4 cm-1 and analyzed with the Spectra Manager software (Jasco). The spectrum of each sample was acquired against a background obtained with the crystal without any sample. All analyses were carried out at room temperature.