# Development of Injectable Thermosensitive Chitosan-Based Hydrogels for Cell Encapsulation
###### Abstract
Moreover, the three-dimensional and porous structure of such hydrogels make an ideal network, which holds the cells in place while allowing the transport of nutrients, wastes, and other essential molecules [18]. Although several synthetic polymers, such as PVA, PEG and PLA [19; 20], have been proposed in this context for hydrogel preparation, natural polymers have shown higher biocompatibility and physical match to the ECM compared to the synthetic ones [21]. Polymers such as hyaluronic acid [22], chitosan [23], gelatin, fibrinogen [24], collagen [25], and alginate [26] can be further modified to induce specific and desired biological reactions Among the above-mentioned natural polymers, chitosan-based hydrogels are presently being widely used due to their well-documented biocompatibility, low toxicity and degradability by human enzymes [27]. Chitosan is a linear polysaccharide derived from chitin, extracted from the crustacean exoskeleton [28], and characterized by marked biocompatibility and biodegradability properties. Chitosan temperature-controlled gelation can be easily obtained by adding beta-glycerol-phosphate to the system (BGP) [21; 22; 23; 24; 25; 26; 27; 28; 29]. BGP prevents the precipitation of chitosan and induces its sol-gel transition when the temperature is increased in the range 32-37 \({}^{\circ}\)C, i.e., compatible with cell viability. [29]. BGP also allows to obtain a gel with a physiological pH, and therefore suitable for cell encapsulation. Moreover, such hydrogel systems are softer and less cytotoxic compared to gels formed by chemical modification [2]. Few studies have shown that chitosan can also thermogel in the presence of other weak bases, such as sodium hydrogen carbonate (SHC) [30], but the obtained hydrogels did not present rapid gelation and appropriate mechanical properties. [PERSON] et al., however, demonstrated that a very interesting synergic effect can be reached combining SHC with BGP, obtaining hydrogels with rapid gelation and improved mechanical properties, despite even lowered salt concentrations as compared to CH:BGP hydrogels [31]. In the present study, SHC and L-arginine (ARG), a dibasic amino acid fundamental in cellular physiology [32], were introduced in a BGP hydrogel system with the aim of enhancing the in vitro performance in terms of stability and mechanical properties of thermoresponsive chitosan-based hydrogels with a sol-gel transition at 37 \({}^{\circ}\)C and at physiological pH, tested as 3D cell culture systems. L-Arg is a conditionally essential amino acid, involved as a precursor in many important biochemical pathways in cellular physiology (i.e., nitric oxide pathway involved in nerve regeneration) [33; 34; 35], and therefore an enhancer of some key cell processes, e.g., collagen synthesis, T-cell mediated responses, and also the release of pituitary hormones [36]. For these reasons, in our previous studies [37; 38; 39], we used it as a non-toxic ionic cross-linking biomolecule for chitosan structures, hopefully eliciting enhanced biological responses. The results showed that ARG was able to improve the in vitro stability of chitosan 3D structures, such as scaffolds and electrospun membranes. In the present work, we aim at verifying if ARG could give a similar contribution even in chitosan hydrogel systems. The hydrogel systems have been characterized in terms of injectability and gelation, in vitro swelling ratio and stability and mechanical properties. Finally, preliminary biological tests have been performed to investigate the suitability of the developed hydrogels for encapsulation of a neuronal-like cell type.