The gradient localization of biological cues is of paramount importance to guide directional migration of cells. In this study, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate)-block- poly(2-hydroxyethyl methacrylate) (P(HEMA-co-GMA)-b-PHEMA) brushes with a uniform underneath P(HEMA-co-GMA) layer and a gradient thickness of PHEMA blocks were prepared by using surface-initiated atom-transfer radical polymerization and a dynamically controlled polymerization process. The polymer chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) peptides by reaction with the glycidyl groups, and their structures and properties were characterized by X-ray photoelectron spectrometry (XPS), quartz crystal microbalance with dissipation (QCM-D) and air contact angle. Adhesion and migration processes of smooth muscle cells (SMCs) were then studied. Compared with those on the sufficiently exposed RGD surface, the cell adhesion and mobility were well maintained when the RGD peptides were localized at 18.9 nm depth, whereas the adhesion, spreading and migration rate of SMCs were significantly impaired when the RGD peptides were localized at a depth of 38.4 nm. On the RGD depth gradient surface, the SMCs exhibited preferential orientation and enhanced directional migration toward the direction of reduced thickness of the second PHEMA brushes. Half of the cells were oriented within ± 30° to the x-axis direction, and 72% of the cells moved directionally at the optimal conditions. Cell adhesion strength, arrangement of cytoskeleton, and gene and protein expression levels of adhesion-related proteins were studied to corroborate the mechanisms, demonstrating that the cell mobility is regulated by the complex and synergetic intracellular signals resulted from the difference in surface properties.

Statement of Significance

The cell migration is of paramount importance for the processes of tissue repair and regeneration. So far, the gradient localization of biological cues perpendicular to the substrate, which is the usual case for the biological signaling molecules to locate in ECM in vivo, has been scarcely studied, and has not been used to guide the directional migration of cells. In this study, we prepare a depth gradient of RGD peptides along the polymer chains, which is used to guide the directional migration of SMCs after a second hydrophilic bock is prepared in a gradient manner. For the first time the directional migration of SMCs is achieved under the guidance of a depth gradient of RGD ligands. The mechanisms of different cell migration abilities are further discussed based on the results of cell adhesion, cell adhesion force, cytoskeleton alignment and expression of relative proteins and genes.

This work paves a new strategy by fabricating a gradient polymer brushes with immobilized bioactive molecules to dominate the directional cell migration, and elucidates the mechanisms underlining the biased migration along RGD depth localization gradients, shedding a light for the design of novel biomaterials to control and guide cell migration and invasion.

中文翻译：

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通过在与RGD肽共价固定的聚合物刷上垂直分布的聚（甲基丙烯酸2-羟乙酯）梯度来调节平滑肌细胞的迁移

生物学线索的梯度定位对于指导细胞的定向迁移至关重要。在这项研究中，聚（2-羟乙基丙烯酸甲酯-共-缩水甘油基甲基丙烯酸酯） -嵌段-聚（2-羟乙基甲基丙烯酸酯）（P（HEMA-共-GMA） - b -PHEMA）以P下方的均匀刷（HEMA-共-GMA）层和PHEMA嵌段的梯度厚度是通过使用表面引发的原子转移自由基聚合和动态控制的聚合过程制备的。随后通过细胞粘附的精氨酸-甘氨酸-天冬氨酸（RGD）肽与缩水甘油基反应，将聚合物链官能化，并通过X射线光电子能谱（XPS），具有耗散作用的石英晶体微天平对它们的结构和性质进行了表征。 （QCM-D）和空气接触角。然后研究了平滑肌细胞（SMC）的粘附和迁移过程。与在足够暴露的RGD表面上的那些相比，当RGD肽定位在18.9 nm深度时，细胞的粘附力和迁移率得到了很好的维持，而粘附力 当RGD肽定位在38.4 nm的深度时，SMC的扩散和迁移速率会受到严重损害。在RGD深度梯度表面上，SMC朝着第二PHEMA刷厚度减小的方向表现出优先的定向和增强的方向迁移。一半的单元格在x轴方向的±30°内定向，并且有72％的单元格在最佳条件下定向移动。研究了细胞粘附强度，细胞骨架排列以及粘附相关蛋白的基因和蛋白表达水平，以证实其机制，表明细胞迁移率受表面性质差异产生的复杂且协同的细胞内信号调节。SMC表现出优先的取向，并朝着第二PHEMA刷厚度减小的方向增强了方向迁移。一半的单元格在x轴方向的±30°内定向，并且72％的单元格在最佳条件下定向移动。研究了细胞粘附强度，细胞骨架排列以及粘附相关蛋白的基因和蛋白表达水平，以证实其机制，表明细胞迁移率受表面性质差异产生的复杂且协同的细胞内信号调节。SMC表现出优先的取向，并朝着第二PHEMA刷厚度减小的方向增强了方向迁移。一半的单元格在x轴方向的±30°内定向，并且有72％的单元格在最佳条件下定向移动。研究了细胞粘附强度，细胞骨架排列以及粘附相关蛋白的基因和蛋白表达水平，以证实其机制，表明细胞迁移率受表面性质差异产生的复杂且协同的细胞内信号调节。在最佳条件下，有72％的细胞会定向移动。研究了细胞粘附强度，细胞骨架排列以及粘附相关蛋白的基因和蛋白表达水平，以证实其机制，表明细胞迁移率受表面性质差异产生的复杂且协同的细胞内信号调节。在最佳条件下，有72％的细胞会定向移动。研究了细胞粘附强度，细胞骨架排列以及粘附相关蛋白的基因和蛋白表达水平，以证实其机制，表明细胞迁移率受表面性质差异产生的复杂且协同的细胞内信号调节。

重要声明

细胞迁移对于组织修复和再生过程至关重要。到目前为止，垂直于底物的生物学线索的梯度定位，这是生物学信号分子在体内定位于ECM中的常见情况，很少被研究，并且还没有被用来指导细胞的定向迁移。在这项研究中，我们准备了沿聚合物链的RGD肽的深度梯度，该梯度用于指导以梯度方式制备第二个亲水块后SMC的定向迁移。首次在RGD配体的深度梯度的指导下实现SMC的定向迁移。基于细胞粘附，细胞粘附力，细胞骨架排列以及相关蛋白质和基因表达的结果，进一步讨论了不同细胞迁移能力的机制。

这项工作通过用固定化的生物活性分子制造梯度聚合物刷来主导细胞的定向迁移，为新的策略铺平了道路，阐明了沿RGD深度定位梯度偏向迁移的机制，为新型生物材料的设计提供了控制和指导的思路。细胞迁移和侵袭。