(692c) Engineering a Highly Elastic Protein-Based Bioink for Printing Complex Soft Tissues

Bioprinting has shown remarkable promise for fabrication of complex tissues and organs, and has tremendous potential to overcome issues with organ transplant shortage, drug testing and screening, and the study of biological phenomena such as tissue morphogenesis [1]. The most common hydrogel-based bioinks are biodegradable hydrogels, including those made of naturally derived polymers, such as fibrin, gelatin, hyaluronic acid, alginate, and agarose; synthetic polymers, such as poly(ethylene glycol) diacrylate (PEGDA) [2]; or natural−synthetic composites [3-7]. However, many of these hydrogels are brittle and lack elasticity due to the nature of the polymer backbones, which limits their ability to mimic the mechanical behavior (e.g. softness, stretchability, and elasticity) of human tissues, such as skin, lung, skeletal muscle, and cardiovascular tissues. Elastin-based proteins have been shown to exhibit enhanced elasticity as compared to many other polymers, as well as high tunability of the polymer backbone using recombinant technologies. In this study, we developed a highly elastic bioink for 3D printing by combining a photocrosslinkable elastin-based protein and silicate nanoparticles (Laponite XLG). This novel bioink can be used for fabrication of complex elastic tissues with exquisitely defined architectures, such as lung, muscle, vascular, and cardiac tissues.

Materials and methods

The elastic protein-based bioink was synthesized using a composite composed of elastin-based proteins obtained via recombinant technology [8, 9] and Laponite, which impart liquid polymer solutions with enhanced shear thinning properties. The polymer solution and nanoparticles were mixed in a solution containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (0.5% w/v) in distilled water at 4 °C. The resulting solution was kept at 4 °C prior to bioprinting to prevent coacervation and aggregation of the elastin-based protein. For experiments with cells, a concentrated solution of cells was added to the bioink using a static mixing device (CELLINK). The mechanical and rheological properties of the composite bioinks were characterized based on procedures described elsewhere [10, 11]. After careful mixing, the bioink was quickly transferred to a 3 mL syringe cartridge and stored in a custom cooled printhead maintained at 4 °C during the printing process. A 27 Gauge printing nozzle (210 μm inner diameter) was fixed onto the cartridge before printing. Printing pressures and speeds were adjusted depending on ink flow properties, and gels were typically printed at pressures ranging from 40-100 kPa at printing speeds between 1-10 mm/s. Different geometric shapes were printed by extruding the elastin protein/Laponite solution through a 27 Gauge nozzle and then photopolymerized for 120 sec under exposure to visible light (395−405 nm, 10 mW/cm²).

Result and discussion:

The mechanical and rheological properties of hydrogels with varied concentrations of elastin protein and Laponite were evaluated prior to printing. The optimal composition was chosen for its high elasticity and shear-thinning properties. Various structures relevant to elastic human tissues were printed, including a blood vessel, a left ventricle, a bronchial tree, and a simple lattice structure that mimics the aligned syncytium of muscle cells. The resolution could be adjusted by varying the printing pressure or printing speed, where increased speed and decreased pressure provided smaller printed lines. Cyclic mechanical testing of printed structures demonstrated that the constructs exhibited high recovery of their original shape with low energy loss and hysteresis. We printed hydrogels loaded with cells to determine the practicality of using this material for bioprinting cell−laden constructs. High cell viability was achieved in the printed constructs over the course of a week. These results suggest that the elastin-based bioink could potentially be used to print a wide range of different human elastic tissues.

Conclusion

In this study, we developed a visible light crosslinkable and highly elastic bioink based on an elastin-based protein and Laponite. To our knowledge, this is the first time that elastin-based composites have been used to bioprint complex tissue-like structures. The engineered bioprinted constructs showed high biocompatibility, slow degradability, and easily tunable mechanical properties. In addition, the resilience of the engineered constructs can provide a novel material platform for soft tissue engineering, especially elastic tissues such as lung, arteries and heart.

Acknowledgements

The authors would like to acknowledge the support from the American Heart Association (AHA, 16SDG31280010)­­­­ and National Institutes of Health (R01- EB023052; R01HL140618).

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