Freeform reversible embedding of suspended hydrogels (FRESH) is a layer-by-layer extrusion-based technique to enable three-dimensional (3D) printing of soft tissue constructs by using a thermo-reversible gelatin support bath. Suboptimal resolution of extrusion-based printing limits its use for the creation of microscopic features in the 3D construct. These microscopic features (e.g., pore size) are known to have a profound effect on cell migration, cell–cell interaction, proliferation, and differentiation. In a recent study, FRESH-based 3D printing was combined with freeze-casting in the Freeze-FRESH (FF) method, which yielded alginate constructs with hierarchical porosity. However, use of the FF approach allowed little control of micropore size in the printed alginate constructs. Herein, the FF methodology was optimized for 3D printing of collagen constructs with greater control of microporosity. Modifications to the FF method entailed melting of the FRESH bath before freezing to allow more efficient heat transport, achieve greater control on microporosity, and permit polymerization of collagen molecules to enable 3D printing of stable microporous collagen constructs. The effects of different freezing temperatures on microporosity and physical properties of the 3D-printed collagen constructs were assessed. In addition, finite element (FE) models were generated to predict the mechanical properties of the microporous constructs. Further, the impact of different micropore sizes on cellular response was evaluated. Results showed that the microporosity of 3D-printed collagen constructs can be tailored by customizing the FF approach. Compressive modulus of microporous constructs was significantly lower than the non-porous control, and the FE model verified these findings. Constructs with larger micropore size were more stable and showed significantly greater cell infiltration and metabolic activity. Together, these results suggest that the FF method can be customized to guide the design of 3D-printed microporous collagen constructs.

中文翻译：

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微孔胶原蛋白结构 3D 打印冷冻新鲜方法的优化

悬浮水凝胶的自由形式可逆嵌入 (FRESH) 是一种基于逐层挤出的技术，可通过使用热可逆明胶支撑浴来实现软组织结构的三维 (3D) 打印。基于挤出的打印分辨率不佳，限制了其在 3D 结构中创建微观特征的用途。已知这些微观特征（例如孔径）对细胞迁移、细胞间相互作用、增殖和分化具有深远影响。在最近的一项研究中，基于 FRESH 的 3D 打印与 Freeze-FRESH (FF) 方法中的冷冻铸造相结合，产生了具有分级孔隙率的藻酸盐结构。然而，使用 FF 方法几乎无法控制打印的藻酸盐结构中的微孔尺寸。在此，FF 方法针对胶原蛋白结构的 3D 打印进行了优化，可以更好地控制微孔率。对 FF 方法的修改需要在冷冻前熔化 FRESH 浴，以实现更有效的热传输，更好地控制微孔性，并允许胶原蛋白分子聚合，从而实现稳定的微孔胶原蛋白结构的 3D 打印。评估了不同冷冻温度对 3D 打印胶原蛋白结构的微孔性和物理特性的影响。此外，还生成了有限元（FE）模型来预测微孔结构的机械性能。此外，还评估了不同微孔尺寸对细胞反应的影响。结果表明，3D 打印胶原蛋白结构的微孔率可以通过定制 FF 方法来定制。微孔结构的压缩模量显着低于无孔对照，有限元模型验证了这些发现。具有较大微孔尺寸的构建体更稳定，并且表现出显着更大的细胞浸润和代谢活性。总之，这些结果表明，可以定制 FF 方法来指导 3D 打印微孔胶原蛋白结构的设计。