3.3. Preparation of Chitosan Hydrogels Modified with GO, rGO and GO-PEG

Polymer solution was prepared by dissolving 2.5 g of CS powder in 30 mL of 5% LAc solution. Water dispersions of GO and GO-PEG were prepared by sonicating appropriate amount of the pastes in 20 mL of distilled water for 2-h. rGO paste was sonicated under alkaline conditions (pH ~10; adjusted by 0.1 M NaOH addition) to secure colloidal stability of the dispersion thanks to electrostatic repulsion forces between rGO sheets. Next, dispersions of GO, GO-PEG or rGO were introduced to the CS solution (final GFM content: 0.5% to CS weight). After stirring for 24 h, TAc (cross-linker, 10% to CS weight) was added and the whole system was stirred for another 24 h. In the same way, hydrogels modified additionally with HAp (10% to CS weight) were prepared.

The obtained hydrogel samples were marked as H0–H6, where H0 was the reference CS-only sample and H1–H6 samples were composites modified with GFM (H1-H3 modified with GO, GO-PEG, rGO, respectively) and both GFM and HAp particles (H4–H6). Their composition was summarized in Table 2.

Composition of the systems used to obtain gradient hydrogels (GO—graphene oxide, GO-PEG—poly(ethylene glycol) grafted graphene oxide, rGO—reduced graphene oxide, HAp—hydroxyapatite; H0: chitosan (CS), H1–H6: CS-based composites).

Three cross-linking systems were tested in search for an appropriate solution to obtain gradient hydrogels with desired shape (Table 3). Initially, sodium tripolyphosphate (TPP) was chosen as it is a commonly used physical cross-linker for chitosan injectable hydrogels or microspheres. Cross-linking was carried out at a low temperature (4 °C) to protect frozen solutions from melting too quickly. However, the use of an aqueous TPP solution resulted in a loss of shape and partial disintegration of the hydrogels. An additional bath in 10% NaOH allowed to obtain layered samples with a cross-linked top layer and a liquid interior (2 h in TPP) or empty shells (4 h in TPP). Chitosan amino groups get protonated in an acidic media. This phenomenon was used to extend the cross-linking time with TPP. Then, sodium chloride was introduced into the TPP solution (5% NaCl and 0.5% TPP) to increase the ionic strength. Electrostatic interactions between NH³⁺ ions from CS and Cl^- ions from NaCl allowed to extend the cross-linking time to 24-h and finally obtain hydrogels with desired shape and gradient structure.

Gelling system compositions: in the first two attempts samples were immersed consecutively in two solutions for different times, in the third gelling solution was just one (TPP—sodium tripolyphosphate).

For cross-linking, the prepared hydrogel solutions (H0–H6) were injected into plastic form (width: 10 mm) and frozen at −20 °C for 24 h. The frozen samples were immersed in the optimized gelling solution (5% NaCl + 0.5% TPP; 24 h) at 4 °C. Finally, the samples were washed with distilled water to remove excess LAc and stored for 24 h before testing.