Introduction: The lack of variety in printable hydrogel systems has recently been identified as one major drawback in Biofabrication[1]. For rational development of tailorable bioinks, there are strategies developed in neighboring disciplines such as supramolecular chemistry with principles that can be transferred to 3D printing[2]. Here we present recombinant spider silk proteins as novel bioink completely relying on such interactions but nonetheless allowing for printing of cell loaded constructs of more than 10 layers without the need for chemical cross-linking[3].
Materials and Methods: Hydrogel were prepared from recombinant spider silk proteins (eADF4(C16)) based on the repetitive core sequence of dragline silk fibroin 4 (ADF4) of the European garden cross spider (Araneus Diadematus) by storing a 3 wt% solution. Robotic dispensing was performed with a 3D Discovery Bioprinter (RegenHU). Gels were characterized using standard methods, with special attention to conformational changes of the peptides during gelation using FT-IR. Human dermal fibroblast cells were cultured for cell printing and cell seeding. Cell viability was evaluated after printing using live/dead staining and monitored using confocal fluorescence microscopy.
Results and Discussion: Hydrogels were obtained from eADF4(C16) through storage of a 3 wt% solution in an incubator at 37°C overnight. FT-IR analysis showed a doubling of beta-sheet content in the peptides during gelation, indicating intermolecular beta-sheet interaction as main gelation force. These gels could directly be used for 3D printing due to the rapid reversible nature of these supramolecular interactions that are characterized by a fast regain of the initial structure after relaxation of shear. Dispense plotting of more than 10 layers was possible purely relying on supramolecular beta-sheet mediated interaction between the peptides (Fig. 1). For printing of cell loaded constructs, cells were mixed into the gels before the overnight gelation. A quantification of cell viability 48 h after printing showed an average viability of 70.1 ± 7.6%, which is identical to non-printed gels from the same material, revealing that the printing process did not negatively influence cell viability.
Fig. 1: Dispense plotting (A), eADF4(C16) printed construct (B), live/dead staining of embedded cells (C), CAD file (D) and printed result (E) of a eADF4(C16) ear.
Conclusion: Using recombinant spider silk proteins we demonstrate that physical crosslinking by β-sheet structures is a promising strategy for bioink development, especially due to the rapid regain of structure after release of shear stress. This allows for 3D printing of stable constructs without the need of thickeners or crosslinking additives.
We thank the European Union (FP7) for funding under grant agreement no. 309962 (project HydroZONES).
References:
[1] Malda, J. et al. Adv. Mater. 2013, 25, 5011-5028.
[2] Jüngst, T. et al. Chem. Rev. accepted.
[3] Schacht, K. et al. Ang. Chem. Int. Ed. 2015, 54, 2816-2820