3D bioprinting with self-healing, photocurable supramolecular hydrogels without photo-initiators

Abstract:
Introduction
Hydrogels are three-dimensional networks of polymers that can absorb and store large amounts of water. Conventional hydrogels are based on static covalent crosslinks, which limits their adaptability to varying mechanical loads and environmental conditions. In supramolecular hydrogels the chains in the network are connected by reversible physical bonds that confer viscoelastic properties to the hydrogels. These hydrogels can be synthesized to include both covalent and dynamic bonds, enhancing their mechanical adaptability. Self-healing, shear-thinning and photocurable hydrogels can be synthesized by forming inclusion complexes between cyclodextrin (CD) host molecules and benzophenone and/or adamantane guest molecules. The inclusion complex formed between CD and benzophenone facilitates the formation of covalent bonds upon UV irradiation, eliminating the need for photoinitiators and resulting in robust hydrogels. This study describes the synthesis of these novel hydrogel materials using dextran as the backbone and provides a detailed evaluation of their physicochemical and biological properties for 3D bioprinting (Figure 1)

Methods
The chemical structures of the modified dextrans were validated by NMR and FT-IR spectra. Hydrogels were formed by mixing the modified dextran solutions and UV irradiation at a wavelength of 365 nm. The rheological properties were systematically evaluated using a rheometer, varying the parameters of chain length, anchor point ratio, solution concentration and hydrogel composition. Printability was tested using an extrusion bioprinter and examined using optical microscopy. Cytotoxicity was determined using a fluorometric assay.

Results
The chemical structures of the modified dextrans were confirmed by characteristic peaks in FT-IR spectra. The degree of substitution, calculated from NMR spectra, was used to determine the extent of modification of the dextran chains, which directly affected the properties of the hydrogels. The viscosity of the hydrogels increased drastically after mixing the different components. The hydrogels exhibited dynamic rheological properties, which could be efficiently adjusted by varying different parameters. The hydrogels were completely cured within 10 seconds by 365 nm UV treatment. The printability of the hydrogels was satisfactory, as shown by tests with a bioprinter. In contrast to GelMA, bioprinting did not require temperature control or a photoinitiator. In vitro tests showed negligible cytotoxicity of the hydrogels, indicating good biocompatibility.

Discussion
The novel supramolecular hydrogels exhibit tunable mechanical properties, making them suitable as a matrix for bioinks to modulate mechanical properties based on different cell types. Since these hydrogels do not require a photoinitiator, they also demonstrate enhanced biocompatibility. In conclusion, these hydrogels have significant potential for bioprinting applications in tissue engineering.

Acknowledgments
This project is supported by the Independent Research Fund Denmark (nr. 2035-00275B) and by the European Union (Horizon Europe Grant Agreement nr. 101191695).