# Development of Injectable Thermosensitive Chitosan-Based Hydrogels for Cell Encapsulation
## 4 Discussion  
Although other studies reported the obtainment of thermoresponsive chitosan-based hydrogel by using BGP as gelling agent [21] and the addition of SHC to increase the mechanical properties of the CH+BGP system [31], here, we combined for the first time CH with SHC solution as GA and studied the influence of the BGP and ARG addition on the CH+SHC hydrogel properties. Hydrogel solutions flowing freely at RT were successfully obtained after mixing CH solution with SHC, BGP and ARG solutions. The hydrogel solutions turned into a solid state at 37 \({}^{\circ}\)C due to the decrease of chitosan chain polarity and the increase of BGP hydrophobic interactions with CH [42]. The hydrogel solutions could all be easily injected into a PBS solution at 37 \({}^{\circ}\)C. A sol-gel transition was observed as the temperature increased, showing the hydrogel thermo-responsiveness (see Videos SI, S2, S3 in SI). The inversion tube test results, in agreement with literature, indicated that the gel state of CH-SHC systems is reached after a few minutes of incubation at 37 \({}^{\circ}\)C [43]. The addition of an organic salt, BGP, and L-Arg, did not induce any change in the thermoresponsive behavior of the CH+SHC system. One of the most important properties of hydrogels for 3D cell culture is their ability to incorporate large amounts of water, conventionally evaluated by measuring their swelling capability. The SR was almost equal for the different hydrogel formulations, with average values in the range 600-1000%, in perfect agreement with literature results [18], clearly indicating a superabsorbent behavior. As expected, the SR of the herein proposed hydrogel systems was lower than SR values reported for pure CH  
Figure 7: Representative live/dead (green/red) images for SH-SY5Y cells encapsulated in CH+BGP+SHC hydrogel for 10 min (**a**) and 24 h (**b**). Scale bar, 100 \(\upmu\)m.  
hydrogel, confirming that BGP, SHC and Arg interact with CH chain limiting CH swelling capability [40]. Moreover, though all the three hydrogels presented very high SR values, CH+SHC+BGP+ARG SR after 30 min was significantly lower, suggesting a stronger initial interaction among chitosan chains due to the presence of three GAs. Degradation behavior is another key parameter for the design of 3D hydrogel systems, as it provides an indication of their short and long-term stability during cell culture. The tested hydrogel formulations presented a similar degradation profile, showing a faster weight loss percentage in the first 24 h, without any further increase up to 4 days. The maximum weight loss settled in the range of 25-30%, in agreement with other studies [44]. Samples with SHC+BGP without ARG, however, presented weight loss after 48 h of \(28.5\pm 1.2\%\) significantly higher than those of other two hydrogel formulations, indicating the possibility of tuning the degradation profile according to the need, by simply varying the GA solution components. It is well known that the mechanical properties of cell microenvironment influence cell behavior and functions [45]. From adhesion to differentiation, the most relevant cellular events from early embryogenesis onwards are all, in some way, affected by tissue stiffness [46]. To evaluate the hydrogel bulk stiffness, compression tests have been performed and the results indicated that the addition of BGP induced an increase in the Young modulus in comparison to CH hydrogels with only SHC, likely due to stronger interactions among chitosan chains occurring in the presence of BGP. Conversely, the presence of ARG does not change the hydrogel mechanical properties in compression mode even if a stabilizing effect has been underlined by swelling test. Interestingly, the three hydrogel formulations provide different Young values in compression at low strain values according to the GA composition but always in the range of most of \"soft\" biological tissues, i.e., 1-10 KPa [41; 42; 43; 44; 45; 46]. Preliminary biological tests have been performed to assess the hydrogel biocompatibility.