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.