3D Printing of Enzymatically Softening Hydrogel Biomaterials.

Purpose: 3D printing has accelerated tissue engineering by enabling rapid fabrication of bioprinted tissues from a variety of soft biomaterials. Yet, an ongoing challenge is that for many bioprinting technologies, the materials (bioinks) need to be printed "stiff" (i.e., G' > ~ 15 kPa) so that the fabricated tissue constructs retain high resolution and shape fidelity. Conversely, softer materials tend to generally be more supportive of cellular phenotype and function. To bridge this gap, we sought to develop a hydrogel system that would expand bioprinting access to softer materials, while retaining the resolution of fabricated spatial features. Methods: We developed a photopolymerizable copolymer hydrogel system consisting of nondegradable synthetic and proteolytically degradable natural polymers. Varying the overall polymer content, as well as the ratio between the poly(ethylene glycol) and gelatin species, we generated a library of lithographically printable hydrogel formulations with differing initial stiffnesses that could be further variably softened following enzymatic treatment using collagenase. Results: Varying the copolymer composition and overall concentration resulted in the creation of gels whose initial stiffness ranged from 82 to 2 kPa and could be subsequently softened up to 20-fold upon enzymatic treatment. When 3D-printed via digital light processing (DLP), softened gels maintained higher structural integrity than those with matched initial stiffness. Softened gels supported greater endothelial cell perfusion-based seeding compared to those untreated while maintaining high cell viability. Conclusion: Our material system presents a simple solution to the ongoing challenge of 3D-printing soft materials with high resolution. Future Work: In future studies, we will develop post-print softening materials with bio-invisible stimuli to expand applications to in vivo softening of biomaterial tissue mimics. Lay Summary: 3D-printing has become popular in tissue engineering applications, but printing complex, organ-like structures with soft materials remains challenging. We created a material that can hold patterned shapes and small printed structures using a post-print softening technique with a degrading enzyme. We found that different formulations of this hydrogel material offer varying stiffness levels (G' = 2 kPa-82 kPa) and can soften up to 20-fold with enzymatic treatment. Notably, this material retains the structure of 3D-printed open channels even after significant softening, and cells respond well when seeded in these channels. This demonstrates the promise of post-print softening to create soft 3D-printed materials.