Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, a de novo designed helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show how integrated 3D bioprinted structures with distinctly different responses to proteolytic activity can be utilized for systematic investigation of proteolytic remodeling of the extra cellular matrix and the impact of stromal cells on protease driven processes. Bioprinted structures combining non-degradable and degradable hydrogels were designed and demonstrated to be selectively degraded by proteases allowing for protease-mediated material reorganization with high spatial resolution. Bioprinting of tumor microenvironments combining bioinks with different susceptibilities to proteolytic degradation shows that breast cancer cell proliferation, migration into stromal compartments, and spheroid size are significantly increased in protease degradable hydrogels, but only in the presence of fibroblasts. Proteolytic remodeling of the tumor microenvironment has a significant effect on tumor progression and is drastically influenced by the intimate crosstalk between fibroblast and breast cancer cells.

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 intramolecular 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.

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