Proteolytic remodeling of 3D bioprinted tumor microenvironments.

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 a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

Dynamic peptide-folding mediated biofunctionalization and modulation of hydrogels for 4D bioprinting.

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.