Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium
Summary
This study introduces a novel κ-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.
Abstract
Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel κ-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the κ-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the κ-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel κ-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.
Introduction
Tissue engineering scaffolds, including electro-spun fibers, porous sponges, and polymer hydrogels, play a pivotal role in the repair and reconstruction of damaged tissues and organs by providing a structural framework supporting cell growth, tissue regeneration, and the restoration of organ function1,2,3. However, traditional scaffolds encounter challenges in accurately replicating native tissue structures, leading to a mismatch between the engineered and natural tissues. This limitation hinders the efficient healing of defective tissues, emphasizing the urgent need for scaffold design advancements to achieve more accurate biomimicry. Three-dimensional (3D) bioprinting is an innovative manufacturing technique that precisely constructs complex biological tissue structures layer by layer using biomaterial inks and cells4. Among various biomaterials, polymer hydrogels emerge as ideal bioinks with their distinctive network that facilitates in situ encapsulation of cells and crucially supports their growth5,6. Nevertheless, many soft and highly hydrated hydrogels tend to induce blurring or rapid collapse of printed scaffold structures during the printing process when used as bioinks. To address this challenge, embedded 3D bioprinting technology employs a microgel bath as a support material, allowing precise soft bioink deposition. Upon gelation of the hydrogel bioinks, refined bionic scaffolds with intricate structures are obtained by removing the microgel bath. Materials like gelatin7,8, agarose9, and gellan gum10,11 have been employed to create microgel baths for embedded 3D bioprinting, significantly advancing the application of soft hydrogels in tissue engineering. However, the micron-level and non-uniform particle size of these particulate gels detrimentally impacts the resolution and fidelity of 3D printing12,13,14. There is an urgent need to fabricate a gel-like suspension float with small and uniformly dispersed particles, offering advantages in achieving high-fidelity bioprinting.
In this protocol, a novel sacrificial granulate κ-carrageenan suspension bath with a uniform sub-micron level is presented for embedded 3D printing. This innovative sub-microgel bath behavior of rapid jamming-unjamming transition facilitates the precise fabrication of biomimetic hydrogel scaffolds with high structural fidelity15. Utilizing this new suspension medium, a series of biomimetic tissue and organ constructs featuring multi-layer tissue structures are successfully printed, employing a composite bioink composed of gelatin methacrylate and silk fibro methacrylate. In this study, we chose the esophagus as the 3D bioprinting biomimetic object mainly because the esophagus not only has a multi-layered tissue structure but also its muscle layer exhibits an internal circular and external longitudinal complex layering structure. Ensuring proper alignment and organization of these layers is essential for functional tissue regeneration. Therefore, we highly desire to replicate the multilayered architecture of the esophagus. More importantly, we utilized κ-carrageenan sub-microgels as the suspension bath and GelMA/SFMA as the bioink to design and construct a biomimetic scaffold for tissue engineering. The printed esophagus can be easily released by repeated phosphate-buffered saline washing. Moreover, the κ-carrageenan sub-microgel bath is free of cytotoxic substances, ensuring high cytocompatibility15. The smooth muscle cells loaded within anisotropic scaffolds exhibit a notable spreading activity. This uniform sub-microgel suspension medium offers a new avenue for the fabrication of complex tissues and organs through embedded 3D bioprinting.
Protocol
Representative Results
Discussion
The preparation of κ–carrageenan sub-microgel suspension baths for use in bioprinting is a carefully orchestrated process that involves several critical steps to ensure the resulting medium exhibits the desired properties for supporting bioinks. Initially, a κ–carrageenan solution is prepared by dissolving the κ–carrageenan powder in deionized water at elevated temperatures, creating a homogeneous mixture. The concentration of th…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by Ningbo Natural Science Foundation (2022J121, 2023J159), Key project of Natural Science Foundation of Ningbo City (2021J256), Open Foundation of the State Key Laboratory of Molecular Engineering of Polymers (Fudan University) (K2024-35), and Key Laboratory of Precision Medicine for Atherosclerotic Diseases of Zhejiang Province, China (2022E10026). Thanks for the technical support by the Core Facilities, Health Science Center of Ningbo University.
Materials
| 3D bioprinter | Custom-designed | ||
| 4’,6-Diamidino-2-Phenylindole | Solarbio Life Science | C0065 | Ready-to-use |
| 405 nm UV light | EFL | XY-WJ01 | |
| Cell Counter | Corning | Cyto smart 6749 | |
| Confocal laser scanning microscope | Leica | STELLARIS 5 | |
| DMEM high glucose | VivaCell | C3113-0500 | High Glucose, with Sodium Pyruvate and L-Glutamine |
| Dynamic rotational rheometer | TA Instrument | Discovery HR-20 | |
| Esophageal smooth muscle cells | Supplied by the Department of Cell Biology and Regenerative Medicine, Health Science Center, Ningbo University | Primary cells from the rabbit esophagus | |
| Fetal bovine serum | UE | F9070L | |
| Fluorescein isothiocyanate labeled phalloidin | Solarbio Life Science | CA1610 | 300T |
| Gelatin methacrylate | EFL | EFL-GM-60 | 60% substitution |
| k-carrageenan | Aladdin | C121013-100g | Reagent grade |
| Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate | Aladdin | L157759-1g | 365~405 nm |
| Live-Dead kit | beyotime | C2015M | |
| Microplate reader | Potenov | PT-3502B | |
| Paraformaldehyde | Solarbio Life Science | P1110 | 4% |
| Penicillin/streptomycin | Solarbio Life Science | MA0110 | 100 ´ |
| Phosphate buffered saline | VivaCell | C3580-0500 | pH 7.2-7.4 |
| Silk fibroin methacrylate | EFL | EFL-SilMA-001 | 39% substitution |
| Triton X-100 | Solarbio Life Science | T8200 | |
| Trypsin-EDTA | VivaCell | C100C1 | 0.25%, without phenol red |
References
- Xu, X., et al. Biodegradable engineered fiber scaffolds fabricated by electrospinning for periodontal tissue regeneration. J Biomater Appl. 36 (1), 55-75 (2021).
- Amann, E., et al. A graded, porous composite of natural biopolymers and octacalcium phosphate guides osteochondral differentiation of stem cells. Adv Healthcare Mater. 10 (6), e2001692 (2021).
- Afjoul, H., et al. Freeze-gelled alginate/gelatin scaffolds for wound healing applications: An in vitro, in vivo study. Mater Sci Eng C. 113, 110957 (2020).
- Hasanzadeh, R., et al. Biocompatible tissue-engineered scaffold polymers for 3D printing and its application for 4D printing. Chem Eng J. 476, 146616 (2023).
- Fu, L., et al. Cartilage-like protein hydrogels engineered via entanglement. Nature. 618 (7966), 740-747 (2023).
- Bertsch, P., et al. Self-healing injectable hydrogels for tissue regeneration. Chem Rev. 123 (2), 834-873 (2023).
- Hinton, T. J., et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv. 1 (9), e1500758 (2015).
- Wang, S., et al. 3d bioprinting of neurovascular tissue modeling with collagen-based low-viscosity composites. Adv Healthcare Mater. 12 (25), e2300004 (2023).
- Sreepadmanabh, M., et al. Jammed microgel growth medium prepared by flash-solidification of agarose for 3d cell culture and 3d bioprinting. Biomed Mater. 18 (4), 045011 (2023).
- Zeng, J., et al. Comparative analysis of the residues of granular support bath materials on printed structures in embedded extrusion printing. Biofabrication. 15 (3), 035013 (2023).
- Terpstra, M. L., et al. Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs. Biofabrication. 14 (3), 034104 (2022).
- Compaan, A. M., et al. Gellan fluid gel as a versatile support bath material for fluid extrusion bioprinting. ACS Appl Mater Inter. 11 (6), 5714-5726 (2019).
- Compaan, A. M., Song, K., Chai, W., Huang, Y. Cross-linkable microgel composite matrix bath for embedded bioprinting of perfusable tissue constructs and sculpting of solid objects. ACS Appl Mater Inter. 12 (7), 7855-7868 (2020).
- Zhang, H., et al. Direct 3D printed biomimetic scaffolds based on hydrogel microparticles for cell spheroid growth. Adv Funct Mater. 30 (13), 1910573 (2020).
- Zhang, H., et al. Cation-crosslinked κ-carrageenan sub-microgel medium for high-quality embedded bioprinting. Biofabrication. 16, 025009 (2024).
- Lee, A., et al. 3d bioprinting of collagen to rebuild components of the human heart. Science. 365 (6452), 482-487 (2019).
- Yao, J., et al. Slightly photo-crosslinked chitosan/silk fibroin hydrogel adhesives with hemostasis and anti-inflammation for pro-healing cyclophosphamide-induced hemorrhagic cystitis. Mater Today Bio. 25, 100947 (2024).
- Senior, J. J., et al. Agarose fluid gels formed by shear processing during gelation for suspended 3d bioprinting. J Vis Exp. (195), e64458 (2023).
- Roche, C. D., et al. Printability, durability, contractility and vascular network formation in 3d bioprinted cardiac endothelial cells using alginate-gelatin hydrogels. Front Bioeng Biotech. 9, 636257 (2021).
- Wang, D., et al. Microfluidic bioprinting of tough hydrogel-based vascular conduits for functional blood vessels. Sci Adv. 8 (43), eabq6900 (2022).
- Shao, L., Hou, R. X., Zhu, Y. B., Yao, Y. D. Pre-shear bioprinting of highly oriented porous hydrogel microfibers to construct anisotropic tissues. Biomater Sci. 9 (20), 6763-6771 (2021).
.