Poster T11 - 3D Bioprinting-assisted Tissue Assembly to Create Transmurally Oriented Myocardial Fibers on Chamber-like Structure

Introduction: The left ventricular myocardial fibers show a distinctive arrangement wherein the orientation gradually changes from the epicardium to the endocardium. This structural feature of the left ventricle (LV) contributes to enhance blood pumping function by allowing the LV to twist [1]. Many efforts have been dedicated to creating engineered heart tissue (EHT) that reproduces the structure and functions of the LV. Specifically, various bioprinting strategies have been developed to simulate LV structures by using FRESH bioprinting [2] or photoinitiators [3]. Currently, chamber-like scaffolds with helical fibers have been achieved using fiber jet spinning [4] and shear-induced bioprinting [5]. The cardiomyocytes (CMs) seeded on the scaffolds formed a helical alignment and exhibited alignment-dependent functional changes. Still, no models have been developed that recreate the multiple layers and orientations mimicking native LV structure.
In this study, we propose bioprinting-assisted tissue assembly (BATA), a combinational approach to bioprinting and tissue assembly, to reproduce the hierarchical structure of LV. We established the EHT modules as building blocks representing uniaxial structures and cardiac functions (e.g., contractility and electrophysiological properties). We developed an assembly strategy to integrate multiple EHT modules and simultaneously control their orientation. Afterwards, the BATA was utilized to create chamber-like structures with fiber orientation changes. The chamber-like structure showed synchronized functions, resulting in a LV twist with the opposite rotation between the apical and basal regions. Moreover, this approach was employed to produce a variety of EHT models by developing and assembling various types of EHT modules, such as vascularized and fibrotic EHTs.

Materials and

Methods: To fabricate EHT modules, bioink was prepared by encapsulating induced pluripotent stem cell-derived CMs and cardiac fibroblasts into heart tissue-derived extracellular matrix (hdECM) hydrogel. Then the bioink was extruded onto PEVA constructs to generate EHT modules, which show uniaxial structure and cardiac functions. The EHT modules were assembled through physical contact at fixed positions and orientations using the EHT assembly platforms. Tissue integration was assessed by measuring contractility, electrophysiological properties, and observing new tissue formation between the assembled EHT modules. For a chamber-like structure, proper EHT modules and an assembly platform were developed and assembled to form three different layers and orientations in a chamber-like structure. Afterwards, the successful fabrication was validated by measuring structural and functional integration and twisting motion.

Results, Conclusions, and Discussions: Uniaxial cell alignment was formed over approximately 5 days of tissue maturation and confirmed using immunofluorescence (IF) imaging. Then, cardiac functions such as contractile force and electrophysiological properties were identified. The EHT assembly was performed to control orientation and induce tissue integration using assembly platforms. The synchronized beating and new tissue formation between the EHT modules were observed, indicating functional and structural integration, respectively. The contributions of each cell type to the assembly process were confirmed using IF imaging. CMs contribute to functional integration by producing junctional proteins between the EHT modules, while CFs contribute to structural integration by creating new tissues between the EHT modules. In addition, the capacity of orientation control was confirmed by observing the cell alignments, which were followed by the guidance of EHT assembly platforms. Moreover, the modulation of cardiac function directionality is also demonstrated by changes in longitudinal contractile forces and action potential propagation directions. Given its capacity to induce tissue integration and control orientation, BATA was utilized to fabricate a chamber-like structure with a hierarchical LV structure. We used six EHT modules to create the chamber-like structure, which displayed three layers and distinct orientations. The synchronization of EHT modules was identified by measuring beating motion and calcium propagation throughout the tissue construct. Consequently, the LV twist of this structure was confirmed by measuring opposite rotations in the apical and basal regions. Moreover, the scalability of the BATA was demonstrated by assembling different types of EHT modules, including vascularized and fibrotic EHT modules. To summarize, the BATA was proven to reproduce the hierarchical structure of LV and its resulting functions, which has not been achieved previously. This approach can be further utilized for multi-organ constructs by developing multiple types of organ structures and advancing assembly strategies.

Acknowledgements (Optional): This work was supported by the Korea Institute for Advancement of Technology (KIAT) and the Ministry of Trade, Industry & Energy(MOTIE) of the Republic of Korea (No. P0021109) and supported by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (21A0104L1, Republic of Korea).