Multimodal and dynamic cross-linking of modular thiolated alginate-based bioinks

Abstract

Engineered extracellular matrix-mimicking hydrogels can facilitate 3D cell culture and fabrication of tissue-like constructs and biologically relevant disease models. Processing of cell-laden hydrogels using additive manufacturing techniques further allows for the development of tissue-mimetic structures with higher spatial complexity. Whereas a wide range of printable hydrogels is available, they tend to lack biological functionality and cell compatibility. Here we show an enzymatically mediated thiol-based cross-linking strategy for the design of modular and cytocompatible hydrogel-based bioinks for 3D bioprinting of dynamic multi-material architectures. Alginate is functionalized with cysteines modified with an enzyme-labile thiol protection group. Deprotection using penicillin G acylase (PGA) generates free thiols on-demand, enabling hydrogel cross-linking using thiol-reactive cross-linkers and intermolecular disulfides while avoiding undesired and uncontrolled thiol oxidation. Remaining free thiols can be used for post-printing hydrogel functionalization and lamination of multilayer structures. Moreover, the addition of PGA to a thermo-reversible hydrogel support bath enables the bioprinting of cell-laden 3D structures with high cell viability and excellent shape fidelity. The possibilities to enzymatically generate free thiols during bioprinting facilitate cross-linking and tuning of bioink properties using cytocompatible chemistries and allow for the printing of complex and dynamic cell-laden structures.

Keywords

• Hydrogel
• Alginate
• Bioprinting
• 3D cell culture
• Penicillin G acylase