EAGER: 4D Bioprinting of Smart Complex Tissue Constructs

4D生物打印是一种高度创新的添加剂制造工艺，可以制造预先设计的、能够随时间改变形状的自组装结构。然而，目前基于4D生物打印的添加剂制造技术由于缺乏先进的智能“墨水”、细胞活力低以及人工组织结构中细胞功能调节不力而受到阻碍。这一早期概念探索性研究(AGUGER)奖支持基础研究，以解决4D生物打印中的这些障碍。这项研究旨在创造具有形状记忆行为和4D生物打印复杂组织结构的智能生物墨水。研究结果有助于将4D生物打印作为一种有用的快速原型工具带入生物医学的前沿。这可能会使许多患有各种组织缺陷的患者受益。本项目的研究目标是(1)建立打印参数(激光功率、打印速度和打印距离)与4D生物打印复杂组织结构的形状记忆效应之间的关系；(2)了解4D动态形状变化对神经干细胞活性、生长和分化的影响。为了实现第一个目标，将合成一种具有高活性可打印基团和功能片段的新型智能脂质大分子，并将其4D生物打印成智能结构。三个打印参数将变化如下：激光功率5,000-10000赫兹，打印速度1,000-3,000毫米/分钟，打印距离1-3毫米。生物打印构件的微观结构将通过扫描电子显微镜观察，构件的抗拉强度将使用单轴机械测试仪测量，构件的形状记忆效应将通过形状记忆测试来确定。为了达到第二个目标，生物打印的结构将被固定在一个临时的形状，并通过不同的恢复过程和恢复速度恢复其原始形状。4D生物打印构建物中的神经干细胞活性将通过活/死活性测定来量化；神经干细胞的生长、轴突延伸和分化将通过细胞增殖试验和免疫细胞化学染色来测量。

4D bioprinting is a highly innovative additive manufacturing process to fabricate pre-designed, self-assembly structures with the ability to change their shape over time. However, current 4D bioprinting based additive manufacturing technologies are hindered by the lack of advanced smart "inks", low cell viability, and poor modulation of cellular function within manufactured tissue constructs. This EArly-concept Grant for Exploratory Research (EAGER) award supports fundamental research to address these obstacles in 4D bioprinting. The research aims to create smart bioinks with shape memory behavior and 4D bioprint complex tissue constructs. Research results can help bring 4D bioprinting to the forefront of biomedicine as a useful rapid prototyping tool. This can potentially benefit numerous patients with various tissue defects. The research objectives of this project are (1) to establish the relationship between printing parameters (laser power, printing speed, and printing distance) and the shape memory effect of 4D bioprinted complex tissue constructs, and (2) to understand the effects of 4D dynamic shape change on neural stem cell viability, growth, and differentiation. To achieve the first objective, a novel smart lipid macromer with highly reactive printable groups and functional segments will be synthesized and 4D bioprinted into smart constructs. Three printing parameters will be varied as follows: laser power from 5000 to 10000 Hz, printing speed from 1000 to 3000 mm/min, and printing distance from 1 to 3 mm. The microstructure of the bioprinted constructs will be observed via scanning electron microscopy, the tensile strength of the constructs will be measured using a uniaxial mechanical tester, and the shape memory effects of the constructs will be determined with shape memory tests. To achieve the second objective, the bioprinted constructs will be fixed at a temporary shape and restore their original shape with varied recovery process and recovery speed. Neural stem cell viability in the 4D bioprinted constructs will be quantified using a live/dead viability assay; neural stem cell growth, axonal extension, and differentiation will be measured via a cell proliferation assay and immunocytochemistry staining.