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

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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.