# Cell-Laden Thermosensitive Chitosan Hydrogel Bioinks for 3D Bioprinting Applications
## 3 Results and Discussion
### Effects of Gelling Agents on Chitosan Hydrogels  
The evaluation of the effects of solvents on the chitosan hydrogels was followed by an exploration of the influence of different gelling agents. Figure 2A shows that regardless of the type of gelling agent,the chitosan solution transitioned into the gel state after incubation at 37 \({}^{\circ}\)C. The CS/K\({}_{2}\)HPO\({}_{4}\) group was associated with precipitates of large sizes that might induce clogging of the precision nozzles used in the 3D bioprinting process, since the precision nozzles used to extrude materials are very easily clogged [22]. On the other hand, the CS/\(\beta\)-GP and CS/NaHCO\({}_{3}\) groups showed smaller-sized precipitates, compared to the CS/K\({}_{2}\)HPO\({}_{4}\) group. Figure 2B shows the gelation time of chitosan hydrogels in the presence of different gelling agents. At higher temperatures, the shortest gelation time observed in this experiment was 20 s. The CS/K\({}_{2}\)HPO\({}_{4}\) and CS/NaHCO\({}_{3}\) groups had the fastest and slowest gelation times, respectively. These results indicate that the CS/NaHCO\({}_{3}\) group could decrease the risk of nozzle clogging during the printing because it had the slowest gelation time. With an increase in temperature from 25 \({}^{\circ}\)C to 50 \({}^{\circ}\)C (Figure 2D), all samples exhibited a rapid increase in the storage modulus (G\({}^{\prime}\)) and a decrease in tan(\(\delta\)), showing that the sol-gel phase transition occurred around 37 \({}^{\circ}\)C. Figure 2E shows the microstructure of the chitosan hydrogels. The inner structure has interconnected pores of irregular sizes. Pores larger than 20-100 \(\upmu\)m have been reported to help cell penetration and pores with a size larger than 100 \(\upmu\)m have been found to be associated with neovascularization, since these could provide a large enough space. Therefore, chitosan hydrogels may support cellular dispersions and neovascularization. The CS/K\({}_{2}\)HPO\({}_{4}\) group had a smaller pore size, whereas the CS/\(\beta\)-GP and CS/NaHCO\({}_{3}\) groups had larger pores. Therefore, the CS/\(\beta\)-GP and CS/NaHCO\({}_{3}\) groups might support cell dispersion and neovascularization better than the CS/K\({}_{2}\)HPO\({}_{4}\) group. Figure 2: Influence of gelling agents on chitosan hydrogels. (**A**) Representative images of chitosan sol-gel transitions induced by temperature. (**B**) Gelation time of chitosan hydrogels in the presence of different gelling agents, as estimated by the test tube inversion method. (**C**) Cell viability of PDLSCs via indirect culture using the WST-1 assay. Error bars indicate standard deviation (n = 5 for each group). * Represents statistically significant differences compared to the control group (\(p<0.05\)). (**D**) Rheological analysis of chitosan hydrogels, with temperature-dependent changes in the storage modulus (G\({}^{\prime}\)) and loss modulus (G\({}^{\prime}\)) from 25 \({}^{\circ}\)C to 50 \({}^{\circ}\)C. (**E**) SEM images of the surface of the chitosan hydrogel. Samples were prepared by dipping chitosan gels in distilled water at 37 \({}^{\circ}\)C for 1 day, followed by lyophilization overnight. (**F**) Live and dead assay results of PDLSCs encapsulated in chitosan hydrogels (green: live cells; red: dead cells). PDLSCs, periodontal ligament stem cells; SEM, scanning electron microscopy; WST, water soluble tetrazolium salt. An in vitro assay using PDLSCs demonstrated the cytocompatibility of the chitosan hydrogels (Figure 2C). On day 1, the CS/NaHCO\({}_{3}\) group showed higher cell adhesion rate compared to the other groups. However, on day seven, there was no significant difference among chitosan hydrogels containing different gelling agents. Chitosan hydrogels are, therefore, a potential candidate for bioink production due to their biocompatibility. Figure 2F shows representative images from the live and dead assay. These results demonstrate that cells encapsulated in chitosan hydrogels were viable and well-dispersed, with a round morphology.