embedded 3d bioprinting of esophageal muscle-layer analogs for tissue engineering

Avanzamento del bioprinting 3D con il sub-microgel di κ-carragenina

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 &#954;-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 &#954;-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 &#954;-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.

Autore in primo piano: Sviluppo di bagni omogenei di sub-microgel di κ-carragenina per il bioprinting 3D ad alta risoluzione

Bioprinting incorporato di strutture simili a tessuti utilizzando il terreno sub-microgel di κ-carragenina

Bioprinting 3D incorporato di analoghi dello strato muscolare esofageo per l'ingegneria tissutale

embedded-3d-bioprinting-esophageal-muscle-layer-analogs-for-tissue

Research

JoVE Journal

Engineering

Embedded 3D Bioprinting of Esophageal Muscle-Layer Analogs for Tissue Engineering

122 Views. Per iniziare, prendi le cellule muscolari lisce esofagee in coltura una volta che raggiungono l'80% di fluenza. Aspirare il terreno e lavare le cellule muscolari lisce endoteliali con cinque millilitri di PBS. Quindi aggiungere due millilitri di tripsina-EDTA allo 0,2% al pallone e incubare per due minuti per digerire le cellule.Aggiungere due millilitri di DMEM per terminare il processo di digestione e trasferire la sospensione cellulare in una provet...

Video: Bioprinting 3D incorporato di analoghi dello strato muscolare esofageo per l'ingegneria tissutale

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

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 &#954;-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 &#954;-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 &#954;-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 &#954;-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.

This study introduces a novel &#954;-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.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...