基于扫描式电填充的空间变刚度微柱阵列生物模板成形机理研究

The stiffness of extracellular matrix has a significant influence on cell’s growth and differentiation. To mimic the nonuniform stiffness environment in vivo, it is necessary to fabricate the extracellular matrix with spatially varying stiffness. Such a matrix may found plenty applications in cell culture in vitro and human disease studying. However it is difficult to precisely control the stiffness for the current techniques, we thus proposed a novel method, scanning electrical filling technique, to fabricate the micro- pillar array with spatially varying stiffness as the extracellular matrix. This technique is based on the fact that liquid fluid can be well controlled by the electric field. The strategy is as follows, first imposing a sequential electric field between the conductive nozzle and template to drive the polymer for spatially varying ‘partial filling’, and then filling another material via the second scanning for a ‘full filling’. After cured and demoulded, the hybrid-material pillar array can be obtained which features a plain top surface but spatially varying stiffness. For such a technique, we intend to investigate some fundamental scientific problems, such as the driving mechanism of the sequential electric field on the functional polymer and its control on the height, the formation of hybrid-material micro-pillar by the second scanning and the influence factors on the stiffness, and also the regulation effect of extracellular matrix’s stiffness on cell behavior. The major aim is to develop a well controlled and highly efficient fabrication method and its associated forming theory for micro-pillar array with spatially varying stiffness which may provide a platform for in vitro cell culture. This project is opening a new door in engineering the extracellular matrix with spatially varying stiffness and will serve as a toolbox to solve issues in cell culture.

细胞外基质的刚度特性对细胞的生长分化有重要的调控作用，构建空间变刚度的生物模板来模拟人体内非均一的刚度环境是开展细胞体外培养、研究人体疾病的关键。针对目前细胞外基质生物模板的制造方法难以定点调控刚度的难题，本项目提出扫描式电填充的新方法制备空间变刚度微柱阵列。该技术利用液体材料在外电场作用下精确可控的流动特性，采用电场力辅助压印方法实现聚合物在模板空腔中的变高度填充；并利用扫描方式结合时序变化的电压，实现大幅面空间变高度微柱阵列的制备。此外，项目提出了二次扫描填充的方法在变高度微柱阵列上垒加异种材料，成形的异质材料微柱顶面平整且刚度精确可调。项目拟通过研究时序变化电场对聚合物的变高度填充机理、二次扫描填充对异质材料微柱成形及刚度影响、微柱结构生物模板刚度特性对细胞行为的调控机理等科学问题，建立一套空间变刚度微柱阵列的精确可控的制造方法及成形理论，为细胞体外培养提供技术平台。

细胞的体外培养是研究人体疾病、探寻治疗方案的重要手段，其中细胞周围的材料称为细胞外基质，科研工作者通常制备生物模板作为细胞外基质进行细胞的体外培养。研究表明细胞外基质生物模板的刚度特性对细胞的生长分化有重要的调控作用，并且人体组织和器官的刚度并不均匀，而是存在一定的空间梯度，如肌肉和骨骼的界面处存在刚度突变，其梯度的数量级可达MPa/mm。但目前的制备方法难以精准调控生物模板的刚度，为此，本项目开发了扫描式电填充的新方法来成形具有刚度梯度的微柱阵列，作为生物模板进行细胞培养。该方法采用导电微孔阵列模板进行模塑成形，利用导电喷头挤出材料，通过在喷头和模板间施加电场利用电场力实现材料在模板中的填充，且填充高度可通过电压值来调控。导电喷头在模板上扫描，即可实现大幅面的微柱阵列结构的制备，且每根微柱的高度及等效刚度精确可控。此外，项目还开发了二次扫描填充的方法在高度变化的微柱阵列上垒加异种材料，成形的异质材料微柱顶面平整且刚度精确可调。项目搭建了三维运动实验平台，完成了扫描式电填充制造工艺的研发；建立了电场驱动材料填充的三维相场模型，模拟了聚合物在模板中填充的动态过程。项目制备了高度精准可控的微柱阵列，微柱直径为数十微米，高度可达数百微米。采用制备的细胞外基质生物模板，开展了MDPC-23成牙本质细胞、3T3成纤维细胞、β-TC-6胰岛细胞的体外培养实验。