"Introduction: Ischemic heart disease is a major cause of human death worldwide owing to the heart’s minimal ability to repair following injury. Despite medical advances, current treatments are not able to regenerate the damaged heart tissue. Therefore, alternative strategies are being assayed to identify the proper strategy to induce heart regeneration. In this sense, cardiac tissue engineering aims at obtaining cardiac constructs that mimics the native myocardium. Although major advances have been achieved in this respect, the generation of functional human mature tissue with physiological myocardial architecture and function to native adult myocardium remains a major obstacle. To address this, we have generated a 3D printable design that recapitulates not only the physical myocardial milieu, but importantly, 3D myocyte alignment, which is key to generation of maximum contractile force generation and therefore, maximum therapeutic efficacy.
Methodology: The designs have been 3D printed using Melt Electro Writing, an advanced printing technology uniquely capable of reproducing the properties of the cardiac extracellular matrix. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (CFs) were embedded in a hydrogel (Fibrin or GelMA) and combined with MEW-designs in a composite system. Different ratios of hiPSC-CMs and -CFs (100:0, 95:5, 90:10 and 80:20 for CM:CF), as well as scaffolds fiber thickness (0.20, 0.35 and 0.70 mm) were evaluated for optimal tissue formation. Cardiac engineered tissues were maintained in culture for up to 4 weeks and characterized by histology, staining, ultrastructure, RNAseq, metabolic and electrophysiological analysis.
Results: Overall, contractile human cardiac minitissues can be generated using both fibrin or GelMA hydrogels reinforced with 3D MEW printed structures displaying cardiomyocytes well distributed throughout the scaffold. No statistical differences were found between fibrin-based tissues containing only cardiomyocytes compared with those containing fibroblasts, in terms of beat rate, metabolic activity, gene expression, conduction velocity and activation frequency. However, contractile capacity for fibrin-based engineered tissues was markedly superior. Additionally, a longer remodeling process was required for GelMA-based tissues, whereas fibrin ones displayed an earlier coordinated beating. Also, cell and gel detachment were observed in GelMA-based scaffolds, making necessary the addition of high number of fibroblasts (20%) for optimal tissue formation. Metabolic maturation and transcriptomics analysis also highlighted the differences elicited by the choice of hydrogel. In summary, fibrin-derived tissues exhibited improved biological, structural, and mechanical properties compared with GelMA-based constructs. In vitro findings suggested that the composite fibre-hydrogel system may be a more suitable option for tissue-engineered heart repair.
Conclusion: We are progressing towards the rational development of engineered human cardiac tissues by a precise assessment of the main components, mimicking the unique 3D organization of the native heart architecture. Our results highlight the relevance of the choice of ECM-mimic (hydrogel), and provide and in-depth characterization of their differential effects upon the biology of the resulting tissues."