Design and Fabrication of Mature Engineered Pre-Cardiac Tissue Utilizing 3D Bioprinting Technology and Enzymatically Crosslinking Hydrogel

Abstract

The fabrication of mature engineered cardiac tissue is one of the major challenges in cardiac tissue engineering. For this purpose, we attempted to apply the 3D bioprinting approach. Aiming to construct an oriented tissue, a fine fiber-shaped scaffold with a support structure was first designed using CAD software. Then, a 3D bioprinter and cell-adhesive bio-inks were utilized to fabricate this structure. The cell-adhesive bio-inks were synthesized by combining sodium alginate and gelatin with tyramine, respectively, to form pre-gel materials that allow enzymatic crosslinking by horseradish peroxidase. By absorbance measurements, we confirmed that the tyramine modification rate of each polymer was 0.535 mmol/g-alginate and 0.219 mmol/g-gelatin. The width of the fiber-shaped scaffold was 216.8 ± 24.3 μm for the fabricated scaffold, while the design value was 200 μm. After 3D printing and adhesion-adding treatment of the scaffold with these bio-ink materials, cardiomyocytes were seeded and cultured. As a result, the cells spread onto the scaffold, and the entire pre-tissue contracted synchronously by day 6 of culture, showing a greater pulsatility than in the early days. Video analysis showed that the beating rate of pre-myocardial tissue on day 6 was 31 beats/min. In addition, we confirmed that the cardiomyocytes partially elongated along the long axis of the fiber-shaped scaffold in the pre-tissue cultured for 15 days by staining actin, suggesting the possibility of cell orientation. Furthermore, treatment with adrenaline resulted in a 7.7-fold increase in peak beating rate compared to that before treatment (from 6 beats/min to 46 beats/min), confirming the responsiveness of the pre-tissues to the drug. These results indicate that 3D bioprinting effectively produces mature cultured myocardial tissue that is oriented, contracts synchronously, and is responsive to drugs.