3D Bioprinting of Sugar Beet Pectin through Horseradish Peroxidase-Catalyzed Cross-Linking

Horseradish peroxidase (HRP)-mediated hydrogelation, caused by the cross-linking of phenolic groups in polymers in the presence of hydrogen peroxide (H₂O₂), is an effective route for bioink solidification in 3D bioprinting. Sugar beet pectin (SBP) naturally has cross-linkable phenols through the enzymatic reaction. Therefore, chemical modifications are not required, unlike the various polymers that have been used in the enzymatic cross-linking system. In this study, we report the application of SBP in extrusion-based bioprinting including HRP-mediated bioink solidification. In this system, H₂O₂ necessary for the solidification of inks is supplied in the gas phase. Cell-laden liver lobule-like constructs could be fabricated using bioinks consisting of 10 U/mL HRP, 4.0 and 6.0 w/v% SBP, and 6.0 × 10⁶ cells/mL human hepatoblastoma (HepG2) cells exposed to air containing 16 ppm of H₂O₂ concurrently during printing and 10 min postprinting. The HepG2 cells enclosed in the printed constructs maintained their viability, metabolic activity, and hepatic functions from day 1 to day 7 of the culture, which indicates the cytocompatibility of this system. Taken together, this result demonstrates the potential of SBP and HRP cross-linking systems for 3D bioprinting, which can be applied in tissue engineering applications.