(E-166) 3D Bioprinting-based Tissue Assembly to Create Transmurally Oriented Myocardial Fibers on Chamber-like Structure

Introduction:: The myocardial fibers in the left ventricle (LV) of the heart display a unique arrangement wherein the fiber orientation gradually shifts from the epicardium to the endocardium. Comprising uniaxially aligned cardiomyocytes, each fiber generates contractile forces along the alignment, contributing to the twisting contraction of the LV and enhanced ejection fraction [1]. The advent and development of biofabrication technology allow researchers to investigate cardiac physiology, and there have been many efforts to induce and regulate cardiomyocyte alignment to understand the fundamental mechanisms governing the structure and function of the heart. Traditional top-down tissue engineering strategies, such as seeding cells on highly aligned structures or encapsulating cells in extracellular matrix (ECM)-based hydrogels and subjecting them to tensile stress using posts [2] and wires [3], have proven to be effective in inducing cellular alignment. However, these strategies lack the flexibility to modify the cellular alignment once it has been established. Thus, the combination of top-down and bottom-up approaches has emerged for generating multi-axial fiber orientation with its improved flexibility [4, 5, 6]. In this study, we suggest bioprinting-based tissue assembly, a method of fabricating tissue modules using bioprinting and assembling them to generate a higher-order structure, for producing chamber models with myocardial fiber orientation (MFO).






Materials and Methods:: To prepare bioink, we encapsulated induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts within the heart tissue-derived decellularized ECM developed in our lab previously. An EHT module, a structural and functional unit, was generated by an extrusion-based 3D bioprinter. We then characterized the cardiac functions of EHT modules (e.g., contractility and electrophysiology (EP)) and confirmed the cellular alignment using immunofluorescence (IF) imaging to decide the optimal time point for inducing sufficient cellular alignment. We generated an assembly platform that secured the EHT modules in specific locations to induce tissue fusion and control their orientations. The fusion between EHTs was assessed by quantifying the newly formed area, and its mechanisms were investigated by observing the expression of a cardiac-specific gap junction protein. Furthermore, we demonstrated the structural and functional synchronization and directional modulation of assembled EHT modules by force measurement and EP analysis. Finally, we fabricated a chamber-like structure assembled using three helical and three circumferential EHT modules. Afterwards, the deformation and torsional movement according to the MFO were observed using a digital microscope.






Results, Conclusions, and Discussions:: We successfully generated uniaxially well-oriented EHT modules that were capable of recapitulating key aspects of cardiac physiology, including contractility and electrophysiological properties. Furthermore, we developed a pin-hole-based assembly platform using a 3D printer and established an assembly process, demonstrating the versatility and flexibility of this technique through the generation of various shapes of assembled EHT. To observe structural and functional changes via assembly, we employed parallel assembled and twisted assembled EHTs in subsequent experiments. Within 6 hours after assembly, the synchronized beating was observed, and the formation of new tissue between the two tissues via fusion was observed. Upon detailed observation of the tissue interface, it was confirmed that the fusion between the two tissues takes place via the formation of junctions among the cardiomyocytes rather than through cell migration. Furthermore, we confirmed that the EHT assembly can control the direction while maintaining the pre-existing cell alignment, which was demonstrated to control the direction of cardiac functions such as contractility and action potential propagation. Based on these results, we developed an EHT module and assembly platform to create a MFO on a chamber-like structure. Strip- and ring-shaped EHT modules were utilized to produce helical and circumferential fibers, respectively, and were assembled into transmurally oriented myocardial layers consisting of deep, middle, and outer layers. We observed that each layer exhibited synchronized beating, demonstrating functional integration. Furthermore, detailed observation of the long-axis view revealed deformation at the apical and basal regions, while rotation was observed in the short-axis view. In conclusion, we confirmed the versatility and flexibility of 3D bioprinting-based tissue assembly, enabling the creation of a complex structure such as the MFO on a chamber-like structure. Despite the challenges posed by the polymer structure-based assembly method, which limits the simulation of the ejection fraction in the model, we were able to observe structural changes in the LV. The limitations can be overcome by integrating other approaches, such as adhesive materials. This method can be utilized for disease modeling by incorporating disease modules or for organ-organ axis modeling by assembling multiple organ modules.






Acknowledgements (Optional): :



This research was supported by the Korean Fund for Regenerative Medicine, funded by the Ministry of Science and ICT and the Ministry of Health and Welfare (21A0104L1, Republic of Korea), and supported by the NRF grant funded by the Korean Government (NRF-2019-Global Ph.D. Fellowship Program).










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