Department of Health Science and Technology, ETH Zürich, Switzerland.
Department of Health Science and Technology, ETH Zürich, Switzerland.
Acta Biomater. 2018 Sep 1;77:182-190. doi: 10.1016/j.actbio.2018.07.020. Epub 2018 Jul 10.
The bacterial ligase Sortase A (SA) and its mutated variants have become increasingly popular over the last years for post-translational protein modifications due to their unparalleled specificity and efficiency. The aim of this work was to study SA as a cross-linking enzyme for hydrogel-based tissue engineering. For this, we optimized SA pentamutant production and purification from E. coli to achieve high yields and purity. Then using hyaluronan (HA) as a model biopolymer and modifying it with SA-substrate peptides, we studied the cross-linking kinetics obtained with SA, the enzyme stability, cytocompatibility, and immunogenicity, and compared those to state-of-the-art standards. The transglutaminase activated factor XIII (FXIIIa) was used as the reference cross-linking enzyme, and the clinical collagen scaffold Chondro-Gide (CG) was used as a reference biocompatible material for in vivo studies. We found SA could be produced in large amounts in the lab without special equipment, whereas the only viable source of FXIIIa is currently a prescription medicine purified from donated blood. SA was also remarkably more stable in solution than FXIIIa, and it could provide even much faster gelation, making it possible to achieve nearly-instantaneous gel formation upon delivery with a double-barrel syringe. This is an interesting improvement for in vivo work, to allow in situ gel formation in a wet environment, and could also be useful for applications like bioprinting where very fast gelation is needed. The cytocompatibility and lack of immunogenicity were still uncompromised. These results support the use of SA as a versatile enzymatic cross-linking strategy for 3D culture and tissue engineering applications.
Enzymatic crosslinking has immense appeal for tissue engineers as one of the most biocompatible methods of hydrogel crosslinking. Sortase A has a number of unique advantages over previous systems. We show an impressive and tunable range of crosslinking kinetics, from almost instantaneous gelation to several minutes. We also demonstrate that Sortase A crosslinked hydrogels have good cytocompatibility and cause no immune reaction when implanted in vivo. With its additional benefits of excellent stability in solution and easy large-scale synthesis available to any lab, we believe this novel crosslinking modality will find multiple applications in high throughput screening, tissue engineering, and biofabrication.
近年来，由于其无与伦比的特异性和效率，细菌连接酶 Sortase A（SA）及其突变变体已成为翻译后蛋白质修饰的热门选择。本研究旨在研究 SA 作为水凝胶基组织工程中的交联酶。为此，我们从大肠杆菌中优化了 SA 五突变体的生产和纯化，以获得高产率和高纯度。然后，我们使用透明质酸（HA）作为模型生物聚合物，并通过 SA 底物肽对其进行修饰，研究了使用 SA 获得的交联动力学、酶稳定性、细胞相容性和免疫原性，并将其与最先进的标准进行了比较。转谷氨酰胺酶激活因子 XIII（FXIIIa）被用作参考交联酶，临床胶原蛋白支架 Chondro-Gide（CG）被用作体内研究的生物相容性材料参考。我们发现，SA 可以在没有特殊设备的情况下在实验室中大量生产，而 FXIIIa 的唯一可行来源目前是从捐赠血液中纯化的处方药物。SA 在溶液中的稳定性也明显优于 FXIIIa，并且可以提供更快的凝胶化速度，使得在使用双筒注射器输送时可以实现几乎即时的凝胶形成。这是体内工作的一个有趣改进，可以允许在潮湿环境中就地形成凝胶，对于需要非常快速凝胶化的应用，例如生物打印，也可能非常有用。细胞相容性和免疫原性仍未受到影响。这些结果支持将 SA 用作 3D 培养和组织工程应用的多功能酶交联策略。
酶交联作为水凝胶交联最具生物相容性的方法之一，对组织工程师具有巨大的吸引力。Sortase A 相对于以前的系统具有许多独特的优势。我们展示了令人印象深刻且可调谐的交联动力学范围，从几乎即时凝胶化到几分钟。我们还证明，Sortase A 交联水凝胶具有良好的细胞相容性，并且在体内植入时不会引起免疫反应。由于其在溶液中稳定性好的额外优势，以及任何实验室都可轻松进行大规模合成，我们相信这种新型交联方式将在高通量筛选、组织工程和生物制造中得到广泛应用。
