Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.

中文翻译：

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成骨细胞与激光烧结钛合金结构微粗糙表面界面的成像分析

先前使用聚焦离子束 (FIB) 分析光滑和微粗糙 Ti 表面上的成骨细胞的工作表明，粗糙表面上的平均细胞纵横比和距表面的距离更大。为了使用多种成像方式更好地研究单个细胞与其基质之间的关系，我们开发了一种方法，通过共聚焦激光扫描显微镜 (CLSM) 跟踪同一细胞，以将表面微观粗糙度与细胞形态和细胞骨架相关联；扫描电子显微镜 (SEM) 为通过能量色散 X 射线光谱观察纳米粗糙度和化学映射提供更高的分辨率；和用于高分辨率成像的透射电子显微镜（TEM）。FIB 用于制备 TEM 细胞-材料界面的薄片，或用于三维电子断层扫描。细胞在具有抛光或蚀刻表面的激光烧结 Ti-6Al-4V 基底上培养。跨表面观察到细胞与表面的直接附着，尽管在粗糙的基材上发生跨宏观表面特征的桥接。我们的结果表明，表面粗糙度、细胞骨架和大体形态可以通过多种高分辨率成像方式与单细胞水平的细胞-材料横截面界面相关联。这项工作为进一步研究细胞-材料界面的机制提供了一种平台方法。尽管跨越宏观表面特征的桥接发生在粗糙的基材上。我们的结果表明，表面粗糙度、细胞骨架和大体形态可以通过多种高分辨率成像方式与单细胞水平的细胞-材料横截面界面相关联。这项工作为进一步研究细胞-材料界面的机制提供了一种平台方法。尽管跨越宏观表面特征的桥接发生在粗糙的基材上。我们的结果表明，表面粗糙度、细胞骨架和大体形态可以通过多种高分辨率成像方式与单细胞水平的细胞-材料横截面界面相关联。这项工作为进一步研究细胞-材料界面的机制提供了一种平台方法。