Hydrogels are the primary choice for many 3D bioprinting approaches. While they have many advantages on the biological side, especially their biocompatibility, tuneable stiffness and a wide range of possible modifications , , they are not well suited for additive layer manufacturing, due to their low viscosity and fluidic nature , . An increase of the hydrogel concentration to counteract the low viscosity is problematic, because it would lead to stiffer hydrogels after gelling . Therefore a very soft environment cannot be produced by additive layer manufacturing of pure hydrogels. Several studies showed that there are applications where such soft environment is needed, e.g. the directed differentiation of cells , . Additionally with an ongoing increase of complexity in artificial grafts and a high demand for regenerative medicine, simple printing techniques are suited to strike market first. To retain flexibility and at the same time be as simple as possible, we developed a new method for production of alginate scaffolds with high spatial resolution based on a temperature controlled environment. Thus we are able to 3D print unmodified biopolymers without any additives in a viscosity range below 3 Pa*s, which is below the viscosity of maple syrup .
2 3D printing of hydrogels in a temperature controlled environment
We employed the 3D Bioscaffolder (GeSiM) for 3D printing of hydrogels. It offers two cartridges for sequential printing of two different materials. Pressures can be adjusted in 1 kPa steps. All scaffolds were produced using a 0.2 mm syringe opening at the end of cartridges and applying 30 kPa, 40 kPa or 50 kPa pressure. To control temperature we designed a metal container for dry ice, on which the scaffolds were printed. After surface reached a temperature between −15°C and −20°C (referred to as sub-zero temperatures in this study) scaffold production is started. We use ultra-high viscosity (UHV) alginate, a blend of biopolymers harvested from two brown algae species (Lessonia nigrescens and Lessonia trabeculata). These biopolymers exhibit high biocompatibility and are solidified through ionotropic gelling . Along with alginate concentration, the type and concentration of ions are responsible for the stiffness in the final hydrogel construct, therefore offering a high flexibility in terms of mechanical features . For this study we used 0.7% alginate solution (ALG1) with a viscosity of 2.8895 ± 0.0374 Pa*s (20°C). Alginate solutions in the 3D printer cartridges are kept at room temperature (20°C) and freeze rapidly only after being printed on the cooled stage. Imaging has been performed with FitC labeled Poly-l-lysine (Sigma Aldrich) stained scaffolds on a SP8 confocal laser scanning microscope (Leica).
3 Comparison between printing in sub-zero and room temperatures
For direct comparison of additive layer manufacturing either at sub-zero or room temperatures, we decided to print grids, because they show complex characteristics of the printing process. Particularly, they incorporate alternating patterns of overlaying and uncovered struts and additionally precision of strut deposition can be easily observed.
Printing of ALG1 utilizing conventional printing led to melting of struts orthogonal to the printing axis. This flow depends on the wettability of the surface and the volume of alginate deposited. These two factors combined with surface tension and viscosity of the alginate determine the final geometry. In our setup the resulting thickness is lower than 100 μm (Figure 1B).
Furthermore, strut width is around 1 mm despite printing with a 0.2 mm syringe opening (Figure 1A and 2). Macropores, being the openings between printed struts, are also minimized owing to the outflow, especially from the crossing points, leading to a diminished accuracy. We have also observed a complete sealing of some macropores in each scaffold when printing at 50 kPa under room temperatures. Additionally, there seems to be almost no difference in height between overlaying areas and single printed struts, further underlying the effects of surface tension and wettability.
In contrast, printing under sub-zero conditions is beneficial for a higher control over the printing process, higher printing pressures led to much lower differences in strut width, compared to non-freezing. Indicating that the difference is caused by higher material deposition in z-axis and thus creating a higher volume (Figure 2). Likewise, no increase in strut diameter variability is visible when applying higher pressures (Figure 2, 50 kPa).
Therefore, ALG1 printed in a sub-zero environment does not only result in a lower strut diameter in xy axis, it also enables the alginate to be printed with high strut diameter in z-axis (Figure 3A) The outflow is minimized, leading to a higher printing precision.
4 Freezing effects on alginate
Freezing and vitrification have been shown to effect alginate morphology , . Although we do not reach such deep temperatures as with liquid nitrogen, we still have observed structural changes of printed hydrogels. The surface is covered with microscale structures (Figure 4), compared to alginate printed at room temperature, which has an even surface (Figure 1). Pores have been observed, reaching as deep as 70 μm into the scaffold (Figure 2C). We speculated that these pores are the result of ice crystallization that penetrates the water rich hydrogels, as shown in other studies . Microscale structures are known for their effect on cell attachment and behavior and could further enhance biomimicry of a cell niche , . Additionally, they represent statistically random distributed structural patterns just as extra cellular matrices in physiological tissues do .
Printing in sub-zero environments enables the user to create hydrogel scaffolds with high spatial resolution. The possibility to print very low viscosity polymers facilitates the creation of very soft microenvironments. This is achieved without any modification of the used biopolymers. In conclusion, we designed a 3D printing method which has the capability to print soft and stiff parts in one scaffold. While 3D printing of soft and hard materials has been realized before, to our knowledge, none has achieved this with such liquid materials and high spatial resolution using only non-modified hydrogels.
Research funding: The author state no funding involved. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The conducted research is not related to either human or animal use.
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