Optimization of Gelatin-alginate Ink for 3D-Printing and Non-toxic Cross-linking

Objectives: The 3D-printing of hydrogels has provided numerous opportunities in biomedical research. In this study, the composition of gelatin and alginate, 3D-printing parameters, and the concentration of CaCl₂ were optimized for tissue engineering applications.
Methods: Three concentrations of gelatin (2, 3.5, and 7% w/v) were selected based on the previous experiments in our research group. The proportion of gelatin to alginate was approximately 2:1 and kept constant for all the experiments. At first the appropriate amount of gelatin was dissolved in deionized water at 60 ^oC and then sodium alginate was added and stirred for 24 h. After that, the inks were 3D-printed using Envision-Tech 3D Bio-plotter. All the printing parameters including temperature, pressure, and speed of printing were also optimized. The 3D-printed scaffolds were cross-linked using various concentrations of CaCl₂ solution (5 and 10% wt) at room temperature for 3 h. Then, the scaffolds were thoroughly washed with copious amount of deionized water. The morphology of the scaffolds were characterized using SEM and 3D-laser microscope. The biological responses of scaffolds were also evaluated with dental pulp stem cells (DPSCs).
Results: The results showed that the optimum concentration of gelatin and sodium alginate for 3D-printing would be 7 and 3% w/v, respectively. The optimization of 3D-printing parameters revealed that the temperature, pressure, and speed of printing should be set on 27-29^oC, 1.0 bar, and 22-25 mm/s, respectively. The imaging analyses demonstrated that the proposed ink possessed excellent printability for fabricating various constructs. Ultimately, using CaCl₂ solution with the concertation of 10% wt showed that the presence of sodium alginate can be extremely useful due to the elimination of other toxic cross-linking agents such as EDC and NHS. The fabricated scaffolds showed proper cell attachment and proliferation as well.
Conclusions: The combination of gelatin and sodium alginate not only improves the printability of hydrogel but also introduces a non-toxic cross-linking agent as a suitable substitute for toxic cross-linking agents while keeping good biological responses.