Collaborative Research: 4D Bioprinting of Near-infrared Light Responsive Smart Constructs for Pluripotent Stem Cell Derived Cardiomyocyte Engineering

4D生物打印是一种新兴的制造工艺，用于创建智能组织结构，其中细胞行为可以在空间和时间上进行调节。本研究的目的是4D生物打印动态光响应智能构建体，以控制来自人类诱导多能干细胞的心肌细胞（心肌细胞）的功能。 为此，将合成和表征新型光敏油墨材料，并打印一系列智能结构，以研究生物打印过程对结构动态形状变化的影响。最后，将探索光触发4D结构对调节心肌细胞生长、分化和跳动的影响。通过4D生物打印过程操纵心脏细胞行为将扩大我们对潜在心脏工程应用的心脏细胞功能的理解。此外，这项合作研究将为下一代4D生物打印平台奠定基础。教育和推广活动将涉及乔治华盛顿大学和马里兰州大学帕克分校之间的合作，以分享研究经验，改进现有课程，并纳入本科生和K-12学生，在研究中广泛代表性不足的少数民族，帮助教育未来的生物工程劳动力。这项研究的目的是4D生物打印可重新编程近红外光（NIR）本发明的目的在于构建响应性智能构建体，并发现4D动态效应对控制人诱导多能干细胞衍生的心肌细胞（iPSC-CM）功能和搏动行为的影响，假设这些结构将被成功创建，并且4D动态效应将极大地改善iPSC-CM功能和搏动行为。第一项关键创新专注于创造新一代光敏智能墨水，具有精确控制的多响应4D效果和生物功能。 人类良性NIR敏感部分将在合成的4D墨水中用作模型智能开关。与紫外/可见光相比，长波近红外可以有效地穿透打印的生物材料，并且不会伤害周围的细胞。 选择iPSC-CM是因为它是一种机械响应细胞系，非常适合研究基础和转化心血管研究的4D动态效应。 该细胞系还提供了几乎无限的心肌细胞供应以及具有高再生能力的关键优势。该项目的目标将在三个目标下实现。 第一个目标是配制和表征具有三个关键组分的新型智能墨水：将用作墨水的可印刷基质的天然的基于三聚氰胺的单体，作为发挥可重编程特性的关键功能组分的液晶聚合物和具有光可聚合双键基团的NIR部分。反应和分子结构将通过傅里叶变换红外光谱（FTIR），核磁共振（NMR）和差示扫描量热法（DSC）进行表征。4D油墨将通过改变上述组分的比例来配制，以实现所需的可打印流变性能。 第二个目标是生物打印智能结构，并探索生物打印过程对4D动态形状变化的影响。定制设计的立体光刻（SL）生物打印机，能够控制关键的生物打印参数（打印速度，打印层高度和激光强度），将用于打印合成的油墨材料。建立了印刷参数与光敏智能结构4D形状变化之间的关系。第三个目的是研究近红外响应结构的动态形状变化对iPSC-CM功能的调节。iPSC-CM与NIR响应结构的相互作用将被彻底研究; iPSC-CM在生物打印构建体上的生长将被确定;生物打印构建体的4D变化对iPSC-CM的钙瞬变、肌生成和基因表达的影响将通过Fluo-4 AM、免疫细胞化学和基因分析进行定性和定量检查。这项研究将首次探索近红外调节的4D结构和心肌细胞行为之间的基本相互作用。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

4D bioprinting is an emerging manufacturing process to create smart tissue constructs where cellular behaviors can be regulated in both space and time. The objective of this study is to 4D bioprint dynamic light-responsive smart constructs to control the functions of cardiomyocytes (heart muscle cells) derived from human induced pluripotent stem cells. For this purpose, novel light-sensitive ink materials will be synthesized and characterized, and a series of smart structures will be printed to study the effect of the bioprinting process on the structure's dynamic shape changes. Finally, the effects of light-triggered 4D structures on regulating cardiomyocyte growth, differentiation, and beating will be explored. The manipulation of cardiac cell behaviors through 4D bioprinting processes will expand our understanding of cardiac cell function for potential cardiac engineering applications. In addition, this collaborative research will lay the foundation for next-generational 4D bioprinting platforms. Educational and outreach activities will involve a collaboration between the George Washington University and University of Maryland, College Park for sharing research experiences, improvement of existing courses, and inclusion of undergraduate and K-12 students, with broad representations of underrepresented minorities in research, to help educate the future bioengineering workforce.The objective of this study is to 4D bioprint reprogrammable near-infrared light (NIR) responsive smart constructs and to discover 4D dynamic effects on controlling human induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) function and beating behaviors, hypothesizing that these structures will be successfully created and the 4D dynamic effect will greatly improve iPSC-CM functionality and beating behaviors. The first key innovation focuses on creating a new generation of light-sensitive smart inks with precisely controlled multi-responsive 4D effects and bio-functionality. A human benign NIR sensitive moiety will be used in the synthesized 4D ink as a model smart switch. The long-wavelength NIR can efficiently penetrate through the printed biomaterials compared to ultraviolet/visible light and will not harm the surrounding cells. The iPSC-CM is selected because it is a mechanically responsive cell line that is perfect for studying 4D dynamic effects for basic and translational cardiovascular research. This cell line also offers the key advantages of being in virtually unlimited cardiomyocyte supply, as well as having a high regenerative capacity. The project's objectives will be accomplished under three aims. The FIRST Aim is to formulate and characterize a novel smart ink with three key components: a natural triglyceride-based monomer that will serve as the printable matrix of the ink, a liquid crystal polymer that is a critical functional component to exert the reprogrammable property and a NIR moiety with a light polymerizable double bond group. The reaction and the molecular structures will be characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). The 4D inks will be formulated by varying the ratios of the above components in order to achieve a desired, printable rheological property. The SECOND Aim is to bioprint smart structures and explore the effect of the bioprinting process on 4D dynamic shape changes. A custom designed stereolithography (SL) bioprinter, which is capable of controlling key bioprinting parameters (printing speed, printing layer height and laser intensity), will be used to print the synthesized ink materials. The relationship between the printing parameters and the 4D shape change of the light-sensitive smart structure will be established. The THIRD aim is to investigate the dynamic shape change of NIR responsive structures on regulating iPSC-CM functions. The interaction of iPSC-CMs with the NIR responsive structures will be thoroughly studied; the growth of iPSC-CMs on the bioprinted constructs will be determined; the effect of the 4D variations of the bioprinted constructs on calcium transience, myogenesis, and gene expression of iPSC-CMs will be qualitatively and quantitatively examined by Fluo-4 AM, immunocytochemistry, and gene analysis. This study will for the first time explore the fundamental interactions between NIR regulated 4D structures and cardiomyocyte behaviors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.