Nanopatterned Surfaces to Control Cell Fate

描述（由申请人提供）：细胞形态对一系列细胞事件有深远的影响，如增殖、分化、细胞骨架组织，可能还有基因表达。细胞形状信息转导到基因表达的一种被提出的机制是通过机械力通过细胞骨架与细胞核的直接联系传递，特别是通过核基质蛋白（NMP），如NMP 1和NMP 2。以前，我们已经开发了在微米长度尺度上控制细胞和核形状的方法，并测量原位mRNA表达，以帮助阐述控制细胞命运的关系。虽然以前的研究已经利用具有空间分辨化学的表面来研究细胞形状/功能关系及其对蛋白质合成、原位基因表达（mRNA）和蛋白质合成的影响，作为细胞投影面积和核形状的函数，但这些研究由于无法独立控制通过细胞表面接触进入细胞的输入而受到限制。这一限制阻碍了对配体类型、密度和纳米级分布在控制细胞命运中的作用的分析。为了缓解这个问题，有必要制造更精细的表面来设计实验，以检查控制细胞命运的表面特性。在本提案中，我们试图通过创建可以控制肽配体密度和配体纳米级分布的表面来绕过上述限制，以控制纳米（nm）长度尺度上焦点粘附的空间排列。通过控制细胞骨架，我们预计我们可以独立控制细胞的形状和细胞核的形状。我们将利用定制的NSOM激光器来“写入”肽配体的图案，这些图案随后与整合素受体结合，以精确控制纳米尺度上的焦点粘附位置。这些培养表面将在生物技术、牙科和医学领域有许多可以想象的应用。首先，因为它们将允许细胞和核形状作为一个独立变量进行控制，它们作为研究细胞形状/基因表达层次的实验平台是无价的。其次，由于这些表面将使细胞分化和增殖的基本控制，我们最终希望将纳米图案技术映射到生物材料的表面，如牙科种植体，以控制界面上的细胞行为。

DESCRIPTION (provided by applicant): Cell morphology has a profound effect on a range of cellular events, such as proliferation, differentiation, cytoskeletal organization, and presumably gene expression. One proposed mechanism for the transduction of cell shape information into gene expression is through mechanical forces transmitted via the direct link of the cytoskeleton to the nucleus, and in particular to nuclear matrix proteins (NMPs) such as NMP 1 and 2. Previously, we have developed methods for both controlling cell and nuclear shape on the micron length scale, and measuring in situ mRNA expression in an effort to help elaborate relationships that control cell fate. Although previous studies have employed surfaces with spatially resolved chemistry to study cell shape/function relationships and their effect on protein synthesis, in situ gene expression (mRNA) and protein synthesis as a function of the cell projected area and nuclear shape, these studies have been limited by the inability to independently control the input into the cell via cell surface contacts. This limitation has prevented the analysis of the effect of ligand type, density, and nanoscale distribution in controlling cell fate. To alleviate this problem, fabrication of more elaborate surfaces are necessary to design experiments that examine surface properties that control cell fate. In this proposal we attempt to circumvent the aforementioned limitation by creating surfaces that can control peptide ligand density and ligand nanoscale distribution to control the spatial arrangements of focal adhesion on a nanometer (nm) length scale. By controlling the cytoskeleton, we anticipate that we can independently control the cell's shape and the shape of its' nucleus. We will exploit a custom built NSOM laser to "write" patterns of peptide ligands that subsequently engage with integrin receptors for precise control of focal adhesion locations on the nm length scale. These culture surfaces will have a number of conceivable applications in the fields of biotechnology, dentistry and medicine. First, because they will allow cell and nuclear shape to be controlled as an independent variable, they are invaluable as experimental platforms for the study of cell shape/gene expression hierarchies. Second, since these surfaces will enable the fundamental control of cell differentiation and proliferation, we ultimately hope to map the nanopatterning technique to the surface of biomaterials, such as dental implants to control cell behavior at the interface.