Realization of tissue engineered cardiac constructs has progressed with the combination of induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and additive manufactured frameworks for guided repair. Recently, we developed hexagonal 3D microfiber scaffolds with melt electrowriting (MEW). These scaffolds support contracting hiPSC-CMs and promote tissue organization and maturation. However, scaling such constructs includes challenges, such as the need for nutrient and oxygen supply. We hypothesized that control over vascular patterning and deposition of cardiac cells can promote cardiac construct vascularization and native-like tissue formation. This study converges extrusion-based bioprinting and MEW to biofabricate the first anatomically-designed vascularized cardiac patch.
Hexagonal MEW meshes were fabricated with a 3DDiscovery (REGENHU). Cell composition for myocardial and vascular components were tested using varied ratios of hiPSC-CMs, human fetal cardiac fibroblasts and human umbilical vein endothelial cells. Bioinks (myocardial and vascular) comprised of photo-crosslinkable GelMA with the addition of collagen or matrigel were optimized for both cellular performance and bioprintability by use of soluble fraction analysis, immunofluorescent staining, beating rate analysis and qPCR assessment. Viability after bioprinting was assessed using a metabolic assay and live/dead staining, comparing bioprinted to cast constructs. Finally, the myocardial and vascular bioinks were co-printed into the microfibers meshes using an anatomical design, modelled from a branch of the left anterior descending artery. The constructs were analyzed using immunofluorescent stainings.
The myocardial bioink revealed enhanced native-like tissue formation using 6x107 cells/mL with a ratio of 9:1 (cardiomyocytes:fibroblasts). The vascular bioink enabled capillary-like network formations using 3x107 cells/mL with a ratio of 5:1 (endothelial cells:fibroblasts). Bioink optimization revealed that a combination of 5% GelMA with 0.8 mg/mL collagen, photocrosslinked using 0.25/2.5 mM (myocardial) and 0.5/5 mM (vascular) ruthenium/sodium persulfate had an optimal soluble graction and resulting cellular organization for both bioinks. The myocardial bioink exhibited a slowed, more synchronous beating rate pattern following 3 weeks in culture, indicating hiPSC-CM maturation. In addition, the formation of organized tissue-like structures was observed with an enhancement of troponin-T staining, compared to the other hydrogels tested. Bioprinting processes affected the vascular bioink resulting in increased metabolic output, as well as proliferation of the cells. The myocardial bioink showed a significantly reduced metabolic output 1 week following the bioprinting, however no noticeable difference in viability (live/dead assay) was observed between the bioprinted and cast groups. Converged bioprinting into the MEW mesh was achieved by ensuring the gel viscosity that allowed for integration into the hexagonal pores with all pores filled after 2 printed layers. Interfaces between the vascular and myocardial patterned components were visualized using immunofluorescent morphological stainings.
This study has demonstrated potential for patterning anatomically-relevant vascular pathways within a bioprinted myocardial construct, forging an opportunity for scaling up tissue engineered constructs. Our study provides an important step towards the generation of 3D in vitro cardiac models of relevant dimensions with native-like tissue architecture and function.
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