Chondrocyte spheroid-laden microporous hydrogel-based 3D bioprinting for cartilage regeneration

Three-dimensional (3D) bioprinting has brought new promising strategies for the regeneration of cartilage with specific shapes. In cartilage bioprinting, chondrocyte-laden hydrogels are the most commonly used bioinks. However, the dispersion of cells and the dense texture of the hydrogel in the conventional bioink may limit cell–cell/ cell–extracellular matrix (ECM) interactions, counting against cartilage regeneration and maturation. To address this issue, in this study, we developed a functional bioink for cartilage bioprinting based on chondrocyte spheroids (CSs) and microporous hydrogels, in which CSs as multicellular aggregates can provide extensive cell– cell/cell–ECM interactions to mimic the natural cartilage microenvironment, and microporous hydrogels can provide space and channel for the growth and fusion of the CSs. Firstly, we used a non-adhesive microporous system to produce homogeneous self-assembled CSs in high-throughput and evaluated the influence of different CSs preparation parameters (cell number and culture time) on CSs, which aids in the preparation of bioink suitable for cartilage bioprinting. Then, polyethylene oxide (PEO) was introduced into gelatin methacrylate (GelMA) to prepare microporous hydrogel. Finally, the CS-laden microporous hydrogels were printed, and the constructs were implanted into nude mice. The results showed that the CSs with 500 cells cultured for 1 day exhibited better proliferation and growth ability in microporous hydrogels compared to those with more cells and cultured for longer time. In addition, the results also demonstrated that the CS-laden bioink can be successfully printed into predefined lattice-shape constructs with little cell damage and regenerated cartilage tissue in vivo with a structure similar to natural cartilage characterized by typical lacunae structure and abundant cartilage-specific ECM deposition. In summary, our study verified the feasibility and advantages of using CSs as building blocks in cartilage bioprinting, which provides novel strategies for the fabrication and regeneration of patient-specific shaped cartilage.