Protein adsorption as a key mediator in the nanotopographical control of cell behavior

Surface nanotopography is widely employed to control cell behavior and in particular controlled disorder has been shown to be important in cell differentiation/maturation. However, extracellular matrix proteins, such as fibronectin (FN), initially adsorbed on a biomaterial surface are known to mediate the interaction of synthetic materials with cells. In this work, we examine the effect of nanotopography on cell behavior through this adsorbed layer of adhesive proteins using a nanostructured polycarbonate surface comprising 150 nm-diameter pits originally defined using electron beam lithography.

We address the effect of this nanopitted surface on FN adsorption and subsequently on cell morphology and behavior using C2C12 myoblasts. Wettability measurements and atomic force microscopy imaging showed that protein is adsorbed both within the interpits spaces and inside the nanopits. Cells responded to this coated nanotopography with the formation of fewer but larger focal adhesions and by mimicking the pit patterns within their cytoskeleton, nanoimprinting, ultimately achieving higher levels of myogenic differentiation compared to a flat control. Both focal adhesion assembly and nanoimprinting were found to be dependent on cell contractility and are adversely affected by the use of blebbistatin. Our results demonstrate the central role of the nanoscale protein interface in mediating cell-nanotopographical interactions and implicate this interface as helping control the mechanotransductive cascade.

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表面纳米形貌（Surface nanotopography）被广泛用于调控细胞行为，尤其是受控无序已被证明在细胞分化/成熟中具有重要作用。然而，初始吸附于生物材料表面的细胞外基质蛋白（extracellular matrix proteins）（如纤连蛋白（FN））已知可介导合成材料与细胞之间的相互作用。本研究中，我们利用由电子束光刻（electron beam lithography）定义的直径150 nm凹坑组成的纳米结构化聚碳酸酯表面（nanostructured polycarbonate surface），通过该吸附的黏附蛋白（adhesive proteins）层探究纳米形貌对细胞行为的影响。

我们研究了这种纳米凹坑表面对FN吸附的影响，并随后探究其对C2C12成肌细胞（C2C12 myoblasts）形态和行为的作用。润湿性测量和原子力显微镜（atomic force microscopy）成像显示，蛋白质既吸附于凹坑间隙，也吸附于纳米凹坑内部。与平坦对照组相比，细胞对这种涂层纳米形貌的响应表现为形成更少但更大的黏着斑（focal adhesions），并通过其细胞骨架（cytoskeleton）模拟凹坑图案（即纳米印记），最终实现更高水平的肌源性分化。研究发现，黏着斑组装和纳米印记均依赖于细胞收缩性（cell contractility），且受blebbistatin的使用负面影响。我们的结果表明，纳米级蛋白界面（nanoscale protein interface）在介导细胞-纳米形貌相互作用中发挥核心作用，并暗示该界面有助于调控机械转导级联（mechanotransductive cascade）。

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