Methods to 3D print alginate-based scaffolds
During the first month of 2022, we focused on alginate hydrogels used in tissue engineering and drug delivery applications. As it has been discussed in the first publication of this topic, alginate is commonly used as bioink in 3D bioprinting to produce 3D structures (scaffolds) containing cells or bioactive molecules depending on the application. A particular feature of alginate solutions is their ability to quickly crosslink (have their molecules bonded together) in the presence of ions (e.g., Ca2+) resulting in a cohesive hydrogel that can be prepared in different shapes. And their final mechanical properties can be tuned (at least to a certain extent) by varying the components used in their preparation.
In the second publication of January 2022, alginate hydrogels were crosslinked with CaCl2 solution at different concentrations and incremental timing. Understanding the behavior of a specific formulation during crosslinking such as initiation time and speed of gelation, and potential swelling and shrinking related to this gelation process is essential to better design experiments especially for 3D printing.
For extrusion 3D printing technique, numerous methods have been developed to print alginate 3D structures. Alginate solution has a relatively low viscosity and to be able to print it pure and post crosslink it, a laborious optimization of the protocol is required. For example, bioink’s manufacturers do have detailed protocols that were developed for printing their bioinks at a specific concentration within their suggested range of printing speed, extrusion pressure, nozzle size, temperature, among others. One example is the alginate used in one of our published application notes. Cellink developed a detailed protocol for the printing of their bioink in their extrusion 3D printers.
When a protocol for printing pure alginate solution is not readily available or challenging to obtain, different methods were also developed to overcome its low viscosity. Some reported methods include (Section A in Figure 1) the pre crosslinking of the alginate solution in the syringe barrel with CaCl2 solution for up to 30 min before printing [1,2], (Section B in Figure 1) the use of a coaxial needle with alginate and CaCl2 solution in the shell and core, respectively, for rapid crosslinking as soon as they come out from the needle during the printing process , and very common, (Section C in Figure 1) the mixing with other biomaterials such as gelatin, agar, PEG, among others to improve the immediate printability .
In addition, a relatively new printing method recommended for improving printing fidelity of soft biomaterials, known as Freeform Reversible Embedding of Suspended Hydrogels (FRESH), can also be used to print pure alginate structures . In this technique (Section D in Figure 1), the bioink is extruded in a supporting bath composed of a sacrificial hydrogel that holds the bioink in place until it gelifies. In the case of alginate, the support bath should also contain calcium chloride for triggering the crosslinking. Altogether, the FRESH printing provides an unmatched combination of capabilities in terms of bioinks, resolution, size and 3D geometric complexity that can be used and/or achieved.
Being pure or mixed with other biomaterials, alginate provides to the scaffold an inherent biocompatibility, relatively low cost, low toxicity, mild and tunable gelation with the addition of divalent cations that are important factors for the encapsulation of cells or bioactive molecules.
 Freeman, F. E., & Kelly, D. J. (2017). Tuning alginate bioink stiffness and composition for controlled growth factor delivery and to spatially direct MSC fate within bioprinted tissues. Scientific reports, 7(1), 1-12.
 Daly, A. C., Critchley, S. E., Rencsok, E. M., & Kelly, D. J. (2016). A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage. Biofabrication, 8(4), 045002.
 Zhang, Y., Yu, Y., Chen, H., & Ozbolat, I. T. (2013). Characterization of printable cellular micro-fluidic channels for tissue engineering. Biofabrication, 5(2), 025004.
 Mallakpour, S., Azadi, E., & Hussain, C. M. (2021). State-of-the-art of 3D printing technology of alginate-based hydrogels—An emerging technique for industrial applications. Advances in Colloid and Interface Science, 102436.
 Shiwarski, D. J., Hudson, A. R., Tashman, J. W., & Feinberg, A. W. (2021). Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication. APL bioengineering, 5(1), 010904.