Washburn Newell R, Yamada Kenneth M, Simon Carl G, Kennedy Scott B, Amis Eric J
Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Biomaterials. 2004 Mar-Apr;25(7-8):1215-24. doi: 10.1016/j.biomaterials.2003.08.043.
A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic acid) using a gradient in annealing temperature. The resultant morphologies were characterized using an atomic force microscope. Root-mean-square (rms) roughness values ranging from 0.5 to 13 nm were created on a single sample. MC3T3-E1 osteoblastic cells were cultured for 1, 3 and 5 d, and the number of cells was measured using automated fluorescence microscopy. It is shown that the rate of proliferation on the smooth regions of the films is much greater than that on the rough regions, and a monotonic variation in rate is observed as a function of roughness. The critical rms roughness, above which a statistically significant reduction in rate of proliferation occurs, was approximately 1.1 nm. Fluorescence microscopy measurements on immunostained cells indicate there is no significant change in cell area, the number or type of adhesions formed, or the degree of actin polymerization. Results from enzyme-linked immunofluorescence assays indicated that there was no detectable change in adhesion protein accessibility, suggesting the cells directly respond to substrate topography. The use of the gradient library approach yielded the functional dependence of cell proliferation on nanometer-scale roughness and gave a sensitive estimate of the critical roughness for which a decrease in proliferation is observed.
本文介绍了一种用于分析细胞对聚合物材料结晶度反应的高通量方法。结晶度的变化会导致纳米尺度表面粗糙度的改变，研究表明细胞对这些变化极为敏感。利用退火温度梯度在聚（L-乳酸）薄膜上制备了聚合物结晶度梯度。使用原子力显微镜对所得形态进行了表征。在单个样品上产生了均方根（rms）粗糙度值范围为0.5至13nm的表面。将MC3T3-E1成骨细胞培养1、3和5天，并使用自动荧光显微镜测量细胞数量。结果表明，薄膜光滑区域的细胞增殖速率远高于粗糙区域，并且观察到增殖速率随粗糙度呈单调变化。增殖速率出现统计学显著降低时的临界均方根粗糙度约为1.1nm。对免疫染色细胞的荧光显微镜测量表明，细胞面积、形成的粘附数量或类型以及肌动蛋白聚合程度均无显著变化。酶联免疫荧光分析结果表明，粘附蛋白可及性没有可检测到的变化，这表明细胞直接对底物形貌做出反应。使用梯度库方法得出了细胞增殖对纳米尺度粗糙度的功能依赖性，并对观察到增殖下降的临界粗糙度进行了灵敏估计。
