(L-476) Evaluation of Alginate Based 3D Printing Flow and Cross-Linking Rates

Introduction:: This study focuses on the development of extrusion based 3D Bioprinting using an alginate gel and calcium chloride cross-linker. Specifically this project focuses on the evaluation of extrusion rates, part thickness, an settling times on the quality of the printed gels. Developing a better understanding of the dynamics of extrusion, cross-linking, support, and extraction of 3D printed hydrogels will diversity the geometries and materials suitable for Bioprinting. The long-term goal of this study is to develop cheap and robust hardware and protocols to facilitate the teaching of Bioprinting concepts in K-12 and undergraduate curriculum, and to contribute to the general body of knowledge around Bioprinting techniques.

Materials and Methods:: A Prusa 3-D MK3 and an Ender-3 were modified by replacing the original hot ends with new components for extrusion from a 10ml syringe with a 20-gauge blunt tip. A combination of custom (hand written) and modified g-code from the Cura slicer and the Pronterface interface were used to control the printers.

To investigate the impact of print thickness and crosslinking, 2 replicates of, 2 triangular prisms (1.66mm² and 3.32mm² cross-sectional area), with alginate mixtures of 2%, 3%, and 4%, with CaCl₂ concentrations of 0.25% and 0.50%, and post-print settling times of 0min, 10min, and 30min, and a gelatin slurry of 4% (all percentages by weight). A total of 72 samples were compared.

The FRESH (Freeform Reversible Embedding of Suspended Hydrogels) protocol, modified for expediency and simplicity was employed to create the support substrate. Extraction was achieved by melting the gelatin support bath out from around the print and suctioning off the liquid.

To investigate the effect of print speed and air space in the syringe on extrusion rates, syringes were prepared with a known air space and a 3% alginate gel and used to print with constant rate of plunger movement, followed by stopping the plunger while continuing to move the cross-head at constant speed. The amount of alginate extruded was then measured as a function of distance of movement of the cross-head.

The extruder code was then modified to include both a ‘priming’ and ‘retraction’ phase to attempt to reduce extrusion ramp-up as well as post extrusion leakage.


Results, Conclusions, and Discussions:: Many factors are involved in producing a high-quality print from a bioprinter, including the properties of the substrate, bioink, crosslinker, and the printer settings. Based on these experiments we determined that:

• Smaller cross section in general allowed adequate time for crosslinking at both low and high concentrations of CaCl₂


• Larger cross sections either did not fully cross link or resulted in deformation of the shape (bulging) during printing.


• The 3% alginate bio-inks produced the best results overall. The larger sized 2% geometries usually collapsed due to lower viscosity and the larger 4% prints produced mostly globular masses- although they did appear more elongated at lower CaCl₂ concentrations.


• Crosslinking seems to be faster at higher CaCl₂ concentrations, with post-print settling times having a smaller impact for higher CaCl₂ concentrations.


• For all concentrations of alginate and CaCl₂, a longer post-print settling time produced at sharper, more solid result as the crosslinker had more time to penetrate the into the solid architecture.


• Modifications to printer G-code can be used to compensate for syringe air-space. Further development of procedures to modify code based on desired print characteristics is needed.

Additional work is needed to classify the effect of print geometry and print quality, and to develop methodologies to decrease print quality dependence on print geometry and material flow rates.

Acknowledgements (Optional): : We would like to thank the Florida Gulf Coast University, Bioengineering Program Foundation for supporting this research.

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