Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT, United States of America.
These authors have contributed equally to this work.
Biomed Mater. 2020 Sep 12;15(5):055037. doi: 10.1088/1748-605X/ab99d4.
A reactive silk fibroin ink formulation designed for extrusion three-dimensional (3D) printing of protein-based hydrogels at room temperature is reported. This work is motivated by the need to produce protein hydrogels that can be printed into complex shapes with long-term stability using extrusion 3D printing at ambient temperature without the need for the addition of nanocomposites, synthetic polymers, or sacrifical templates. Silk fibroin from the Bombyx mori silkworm was purified and synthesized into reactive inks by enzyme-catalyzed dityrosine bond formation. Rheological and printing studies showed that tailoring the peroxide concentration in the reactive ink enables the silk to be extruded as a filament and printed into hydrogel constructs, supporting successive printed layers without flow of the construct or loss of desired geometry. To enable success of longer-term in vitro studies, 3D printed silk hydrogels were found to display excellent shape retention over time, as evidenced by no change in construct dimensions or topography when maintained for nine weeks in culture medium. Caco-2 (an intestinal epithelial cell line) attachment, proliferation, and tight junction formation on the printed constructs was not found to be affected by the geometry of the constructs tested. Intestinal myofibroblasts encapsulated within reactive silk inks were found to survive shearing during printing and proliferate within the hydrogel constructs. The work here thus provides a suitable route for extrusion 3D printing of protein hydrogel constructs that maintain their shape during printing and culture, and is expected to enable longer-term cellular studies of hydrogel constructs that require complex geometries and/or varying spatial distributions of cells on demand via digital printing.
一种用于在室温下挤出式三维（3D）打印基于蛋白质的水凝胶的反应性丝素蛋白墨水配方被报道。这项工作的动机是需要生产能够使用环境温度下的挤出 3D 打印以复杂形状打印并具有长期稳定性的蛋白质水凝胶，而无需添加纳米复合材料、合成聚合物或牺牲模板。从家蚕中纯化的丝素蛋白，并通过酶促二酪氨酸键形成合成反应性墨水。流变学和打印研究表明，调整反应性墨水中的过氧化物浓度可以使丝素蛋白以纤维的形式挤出并打印成水凝胶结构，支持连续的打印层，而不会使结构流动或失去所需的几何形状。为了能够成功进行更长期的体外研究，发现 3D 打印的丝素水凝胶随着时间的推移显示出极好的形状保留，证据是在培养介质中保持 9 周时，结构的尺寸或形貌没有变化。在打印结构上，Caco-2（肠上皮细胞系）的附着、增殖和紧密连接形成并未受到测试结构几何形状的影响。在反应性丝素墨水中包封的肠肌成纤维细胞在打印过程中的剪切力下存活，并在水凝胶结构内增殖。因此，这项工作为挤出式 3D 打印蛋白质水凝胶结构提供了一种合适的途径，这些结构在打印和培养过程中保持其形状，并且有望能够进行更长期的细胞研究，这些研究需要具有复杂几何形状和/或按需变化的细胞空间分布的水凝胶结构，而无需数字打印。
