Printability, Durability, Contractility and Vascular Network Formation in 3D Bioprinted Cardiac Endothelial Cells Using Alginate-Gelatin Hydrogels.

Carmine Gentile Gentile

doi:10.3389/fbioe.2021.636257

Abstract

Background3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural self-organisation into networks.MethodsWe evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches.ResultsPrintability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200–700 μm line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10–27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures.ConclusionAlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a degree of tissue maturation but before many patches start to lose integrity. We quantify AlgGel printability and present novel factors which reduce AlgGel patch durability (layer number, movement, and the addition of AlloECM) and factors which had minimal effect on durability (bioprinting system and cellular patch content).

Highlights

The latest developments in three-dimensional (3D) bioprinting technology have led to the hope that viable 3D bioprinted cardiac tissues could be generated to promote myocardial regeneration (Noor et al, 2019; Roche et al, 2020)
For AlgGel hydrogel patches, resolution was similar compared to GelMA; printing accuracy was higher for AlgGel with 78/176
Correcting for differences in print accuracy on day one, the 28-day shape fidelity rate was 72/78 (92%) for AlgGel and 51/53 (96%) for GelMA (p = 0.36; χ2 test; n = 131); for extrudability, AlgGel dripped hydrogel from the nozzle and GelMA did not and AlgGel had zero nozzle blockages per six patches compared to one per six patches for GelMA (AlgGel tended toward more flow than input software instructions and GelMA tended toward less flow)

Summary

Introduction

The latest developments in three-dimensional (3D) bioprinting technology have led to the hope that viable 3D bioprinted cardiac tissues could be generated to promote myocardial regeneration (Noor et al, 2019; Roche et al, 2020). During this post-printing phase, the bioink can promote tissue maturation, with durability in culture being an important characteristic to predict hydrogel disintegration (Bishop et al, 2017; Roche and Gentile, 2020), the optimal moment to transplant after a period in culture has not previously been confirmed During this phase, cardiomyocyte contractility should be permitted and endothelial cells within the bioprinted tissue should be permitted to organise into networks, as one of the major challenges in 3D bioprinting of cardiac tissues is the fabrication of a hierarchical vascular system within tissues (Gentile, 2016; Ong et al, 2017b; Polonchuk et al, 2017; Cui et al, 2019; Polley et al, 2020; Roche et al, 2020; Xu et al, 2020). We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural selforganisation into networks

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