Roche Christopher David, Sharma Poonam, Ashton Anthony Wayne, Jackson Chris, Xue Meilang, Gentile Carmine
Northern Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, Sydney, NSW, Australia.
Front Bioeng Biotechnol. 2021 Feb 26;9:636257. doi: 10.3389/fbioe.2021.636257. eCollection 2021.
3D 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.
We 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.
Printability 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; < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); = 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.
AlgGel-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).
用于心外膜移植的3D生物打印心脏补片是心肌再生的一种很有前景的方法。但仍存在一些挑战，如量化可打印性、确定理想的移植时机以及促进生物打印补片内的血管生成。我们旨在评估3D生物打印心脏补片的可打印性、培养中的耐久性、细胞活力以及内皮细胞在网络中的结构自组织能力。
我们使用藻酸盐/明胶（AlgGel）水凝胶和三种挤出式生物打印机（REGEMAT3D、INVIVO、BIO X）评估3D生物打印双层补片。生物墨水包含新生小鼠心脏细胞球体或游离的（非球体形式的）人冠状动脉内皮细胞与成纤维细胞，与AlgGel混合。为测试对耐久性的影响，一些补片仅打印为单层，在最小运动条件下培养或添加了成纤维细胞衍生的细胞外基质水凝胶（AlloECM）。对照包括无细胞AlgGel和甲基丙烯酸明胶（GELMA）补片。
各生物打印机的可打印性相似。与GELMA相比，AlgGel的分辨率相似（线直径200 - 700μm），打印精度分别为45%和25%（AlgGel的精度高1.7倍；P < 0.05），形状保真度分别为92%（AlgGel）和96%（GELMA）；P = 0.36。对于耐久性，AlgGel补片在培养中的中位存活时间总体为14天（四分位间距：10 - 27天），不受生物打印系统或补片中细胞成分的显著影响。我们确定了三个降低培养中耐久性的因素：（1）打印单层深度的补片（而非两层）；（2）培养基中补片受到运动干扰；（3）向AlgGel中添加AlloECM。生物打印后细胞在培养28天后仍有活力，所有BIO X生物打印的小鼠心脏细胞球体补片在生物打印后第7至13天开始呈现收缩活性。在第28天，水凝胶中的内皮细胞显示出组织形成内皮网络样结构。
基于AlgGel的3D生物打印心脏补片可实现心肌细胞收缩性和内皮细胞结构自组织。生物打印后，移植前在培养中成熟2周可能是最佳的，这允许一定程度的组织成熟，但在许多补片开始失去完整性之前。我们量化了AlgGel的可打印性，并提出了降低AlgGel补片耐久性的新因素（层数、运动和添加AlloECM）以及对耐久性影响最小的因素（生物打印系统和补片中的细胞成分）。
