Dynamic 3D Printing With In Situ Depolarization: A New Biomanufacturing Paradigm for Guided Cell-Cell Communication

在基于电流体动力学（EHD）的增材制造（AM）工艺中，施加的电压将聚合物材料的圆柱形射流从针拉到收集器板。圆柱形射流形成获得特征电荷的沉积纤维。传统的EHD工艺能够以小规模直接打印材料，但受到沉积的带电纤维的有限精度和图案控制的阻碍。为了解决这个问题，本研究的重点是EHD过程中观察到的纤维电荷效应的基本理解。假设恢复中性纤维电荷将使纤维能够精确沉积。在没有净电荷的情况下，纤维可以对齐和分层，以产生用于细胞附着和功能的3D生物基质。具体地说，这些基础知识将产生一个生物基板与大鼠脑神经细胞和内皮细胞接种。在这种双细胞培养平台中，神经细胞的形态由沉积的纤维结构引导以与内皮细胞形成物理接触。这项工作的结果可以通过为药物筛选和基础细胞研究提供强大的3D细胞培养平台，对生命科学产生重大影响。此外，AM方法可适用于包括电子和医药在内的广泛行业的聚合物纤维结构的精确图案控制。研究成果将被整合到本科课程中，并向高中生介绍先进制造，扩大代表性不足的群体在研究中的参与。本研究的总体目标是基于EHD的放电纤维组成的生物基质的增材制造。这种纤维可以可靠地定向在规定的方向和位置，以空间引导细胞形态。目前，在连续沉积的EHD纤维内捕获的残余电荷在纤维之间产生静电力，这限制了印刷图案的分辨率。第一个研究目的是了解印刷表面温度对残余纤维电荷的影响。一个PID控制的热电打印床将被构造成允许关于打印纤维的热去极化到中性电荷状态的基本调查。一个皮安计将被集成，使过程中的光纤电荷测量。温度和时间分辨的电荷测量将被关联以量化电荷衰减现象。纤维放置精度将使用扫描电子显微镜测量为纤维间距离与纤维直径的比率。第二个研究目标是测试动态纤维放置精度对大鼠脑神经细胞和内皮细胞的双细胞培养模型中规定的细胞-细胞接触的影响。为了实现这一目的，排出的纤维表面将沿成层方向沿着用生长因子形成图案。这些生长因子将刺激神经细胞投射延伸，与最顶层的内皮细胞形成接触。将进行细胞表面标志物的免疫化学和屏障渗透性测量，以确认大鼠脑神经细胞和内皮细胞之间的细胞间通讯。

In an electrohydrodynamics (EHD)-based additive manufacturing (AM) process, an applied voltage pulls a cylindrical jet of polymer material from a needle to a collector plate. The cylindrical jet forms a deposited fiber that acquires a characteristic charge. Conventional EHD processes enable direct printing of materials at small scales, but are hampered by limited accuracy and pattern control of the deposited charged fibers. To address this limitation, this research focuses on the fundamental understanding of fiber charge effects observed in EHD processes. It is hypothesized that restoring a neutral fiber charge will enable fibers to be precisely deposited. In the absence of a net charge, the fibers can be aligned and layered to produce a 3D biological substrate for cells to attach and function. Specifically, this fundamental knowledge will yield a biological substrate seeded with rat brain neural cells and endothelial cells. In this two-cell culture platform, the morphology of the neural cells are directed by the deposited fiber architecture to form physical contacts with the endothelial cells. The results of this work can have a significant impact on the life sciences by furnishing robust 3D cell culture platforms for drug screening and fundamental cell studies. Moreover, the AM methodology can be adapted for precision pattern control of polymer fiber structures across a wide range of industries, including electronics and medicine. The research outcomes will be integrated into the undergraduate curriculum as well as introduce high school students to advanced manufacturing, and broaden participation of underrepresented groups in research.The overall goal of this research is EHD-based additive manufacturing of biological substrates composed of discharged fibers. Such fibers can be reliably oriented in prescribed directions and positions to spatially guide cell morphology. Currently, the residual charge entrapped within successively deposited EHD fibers yields electrostatic forces between fibers that constrain the printed pattern resolution. The first research objective is to understand the effects of print surface temperature on the residual fiber charge. A PID-controlled thermoelectric printbed will be constructed to allow fundamental investigations regarding thermal depolarization of the printed fibers to a neutral charge state. A picoammeter will be integrated to enable in-process fiber charge measurements. Temperature and time-resolved charge measurements will be correlated to quantify the charge decay phenomena. Fiber placement accuracy will be measured as a ratio of inter-fiber distance to fiber diameter using scanning electron microscopy. The second research objective is to test the effect of dynamic fiber placement accuracy on prescribed cell-cell contacts in a two-cell culture model of rat brain neural cells and endothelial cells. To accomplish this objective, the discharged fiber surface will be patterned with growth factors along the layering direction. These growth factors will stimulate the neural cells to project extensions that form contacts with endothelial cells on the topmost layer. Immunochemistry of cell surface markers and barrier permeability measurements will be conducted to confirm cell-cell communication between the rat brain neural and endothelial cells.