High-Resolution Bioprinting of Soft Hydrogel Biomaterials through Enzyme-Triggered Bulk Softening

Introduction:: 3D printing has revolutionized tissue engineering through rapid and flexible customization of tissue scaffolds from a variety of biomaterials [1]. Despite these advances, ongoing challenges lie in the dichotomy between printing resolution and material biocompatibility: stiffer printed materials afford higher structural resolution with sustained spatial architecture while softer materials promote greater proliferation, remodeling, and self-organization of living cells embedded in the printed tissues. To address this challenge, we have developed and characterized a new biomaterial platform that prints “stiff” materials at high resolution but can be softened on-demand in the presence of cells through bioorthogonal chemistries.

Materials and Methods:: State-of-the-art methods to soften biomaterials have exploited photolabile bonds embedded into material crosslinkers [2]. While these approaches are well suited for bulk material softening, the photodegradable bioinks are typically incompatible with high-resolution bioprinting methods involving stereolithographic photopolymerization. To address this limitation, we have developed a novel bio-ink formulation that can be photopolymerized at high resolution and then uniformly and rapidly softened via exogenous enzyme treatment. Such a material would uniquely permit culture of living human cells in biomaterials with intricately specified structure.

Engineered variants of the sortase transpeptidase enzyme have been used to selectively cleave distinct peptide sequences nearly absent from the mammalian proteome [3]. Thus incorporating these particular peptide sequences into a crosslinked hydrogel network has enabled sortase to act as a bioorthogonal tool for bulk hydrogel degradation. With this tool in hand we have found new applications to now 3D print with these degradable materials as bio-inks.

We have fully characterized the sortase-cleavable hydrogel system and have shown that this method can completely degrade with vast tunability and control while remaining a viable material for 3D cell culture [3]. If a biomaterial is partially crosslinked with such a degradable linker, enzymatic treatment yields discrete material softening as opposed to complete material degradation. Photocrosslinking of gelatin methacrylate (GelMA) hydrogels partially doped with this enzymatically degradable crosslinker yields hydrogels with exogenously controlled softening. This system now enables high-resolution printing and subsequent softening with sortase to a modulus suitable for hosting human cells, such as hepatocytes of the liver.


Results, Conclusions, and Discussions:: We found that treating hydrogels with evolved sortase in the presence of primary mammalian cells had little impact on their transcriptome and cells exhibited high viability in the sortase-degradable networks decorated with an RGD cell adhesion motif [3].

By optimizing the range of moduli for high-resolution 3D printing and maximal cytocompatibility, we hope for incorporation and retention of large-scale vasculature networks in the hydrogel construct. We are forming vascular networks using anatomical maps of blood vessels in human tissues such as the liver which are sourced from datasets of cleared, immunostained, and computationally reconstructed organs. The bioprinted hydrogel will access physiologic scale architecture to produce vessels < 50 μm in diameter with high resolution that is maintained after softening. This will enable us to seed endothelial cells in these networks and further determine if these vessels maintain perfusion and viable endothelial lining in each condition using 3D immunostaining.



This new approach to create bioprinted hydrogel materials with tunable mechanical properties will yield high resolution, cytocompatible, and implantable biomaterials to treat injured liver tissue as a bridge or alternative to transplantation. This work will be transformative for future tissue engineering applications.


Acknowledgements (Optional): :

References (Optional): : [1] Grigoryan, B et al., Science, 364(6439) : 458-464 (2019)

[2] DeForest, C.A et al., Nature Chemistry, 3, 925-931 (2011)

[3] Bretherton, R. C. et al, Adv. Mater. 2209904 (2023)