To stick or not to stick: Investigating cell adhesion dynamics and cell function using nanometric topography.

在人体的器官和组织中，细胞附着在一种名为细胞外基质(ECM)的复杂的支架状蛋白质网上。细胞外基质为细胞提供了许多刺激和信号，细胞需要这些刺激和信号才能正常运作，从而使身体组织发挥作用。然而，由于组织损伤和生物材料的使用，人类细胞越来越多地接触人造材料。许多生物工程应用的核心是将人类细胞附着到人造材料上，但成功的细胞-材料相互作用的重要因素仍未被很好地了解，这限制了新功能生物材料的设计。利用微电子工业的技术，有可能制造出具有精确定义的表面的结构，用于细胞生物学研究，从而能够观察到细胞对定义的地形特征的反应。我们之前在提案中描述的研究已经确定，可以改变材料的形貌来增强细胞附着、运动和组织发育。当细胞附着在表面时，它会激活细胞生存和发挥功能所需的重要信号分子。细胞通过称为焦点粘连(FA)的特殊位置附着到表面，研究强调这些粘连在细胞中是非常重要的位置。Fas不仅负责将附着事件传递到细胞内的其他区域，而且它们还成熟以形成纤维粘连，这些粘连是细胞沉积和排列支架样蛋白质网(细胞外基质)的位置。现在已经知道，并不是所有的细胞都具有相同的附着能力，在该方案中，我们将比较两种类型的细胞的反应：真皮成纤维细胞(皮肤)和牙龈成纤维细胞(牙床组织)。尤其相关的是，在这些组织的愈合过程中，牙龈组织可以愈合而不会形成疤痕，但皮肤不会。尽管数十年的研究，这些观察到的机制仍然很不清楚，但粘连改变被认为是重要的。我们在这个方案中的目的是通过在纳米和微米尺度上改变培养底物的形貌来探索每种细胞类型的黏附能力的差异。底物形态的变化是细胞黏附的一个非常强大的调节器。具体地说，我们将：1)量化纳米级地形对人牙龈和真皮成纤维细胞的附着、黏附组装和整合素募集的影响，以及2)评估纳米级地形对局部和纤维黏附形成的变化如何改变真皮和牙龈成纤维细胞的细胞内信号、增殖和基因表达。我们预计，真皮和牙龈成纤维细胞在纳米结构上的黏附水平将显示出显著的差异，这将导致细胞内信号模式的改变。总体而言，我们预计增加牙龈成纤维细胞黏附的地形将导致与瘢痕形成相关的信号模式和基因表达，反之，限制真皮成纤维细胞黏附形成的地形将诱导组织重塑表型。这项研究的结果将直接解决细胞黏附如何将外部刺激转化为调节细胞行为的信号，这对细胞生物学的所有领域都将是重要的。此外，我们预计，从长远来看，我们的计划产生的信息可以用于细胞和组织工程应用，在这些应用中，我们对细胞-材料相互作用的缺乏正在限制功能工程组织替代品的开发和生物材料的寿命。

In organs and tissues of the human body, cells adhere to a complex scaffold-like protein mesh called the extracellular matrix (ECM). The ECM provides cells with many stimuli and signals that cells need to behave normally allowing tissues of the body to function. However, increasingly human cells contact artificial materials due to tissue damage and the use of biomaterials. Central in many bioengineering applications is the attachment of human cells to artificial materials, but the factors important in successful cell-material interactions are still not well understood, which limits the design of new functional biomaterials. Using techniques harnessed from the microelectronic industry, it became possible to produce structures with precisely defined surfaces for cell-biological studies, thus allowing the reaction of cells to defined topographic features to be observed. Our previous research described in the proposal has identified that the topography of a material can be altered to enhance cell attachment, movement and tissue development. When a cell attaches to a surface, it results in activation of important signaling molecules required by the cells to survive and function. Cells attach to surfaces through specialized sites called focal adhesions (FAs), and research is highlighting that these adhesions are very important sites in cells. FAs are not only responsible for relaying the attachment event to other areas within the cell, but they also mature to form fibrillar adhesions, which are sites where cells deposit and arrange the scaffold-like protein mesh (extracellular matrix). It is now known that not all cells possess the same ability to attach, and in the proposal we will compare two the response of two such cell types: dermal fibroblasts (skin) and gingival fibroblasts (gum tissue). Of particular relevance is that in healing of these tissues, gingival tissue heals without scar formation, but skin doesn’t. Despite decades of research, the mechanisms underlying these observations are still largely unknown, but altered adhesion is postulated to be important. Our aim in this proposal is to probe the differences in adhesion capacity of each cell type by altering the topography of the culture substrata at the nano- and micro-metric scale. Changes in substratum topography are a very powerful modulator of cell adhesion. Specifically we will: 1) quantify the influence of nanometric scale topographies on attachment, adhesion assembly and integrin recruitment in human gingival and dermal fibroblasts, and 2) assess how changes in focal and fibrillar adhesion formation by nanometric scale topographies alter intracellular signaling, proliferation, and gene expression in dermal and gingival fibroblasts. We anticipate that dermal and gingival fibroblasts will show significant differences in levels of adhesion to nanotopographies, which will result in altered patterns of signaling within the cells. Overall, we expect that topographies that increase gingival fibroblast adhesion will result in signaling patterns and gene expression associated with scarring and conversely, topographies which limit dermal fibroblast adhesion formation will induce a tissue remodeling phenotype. The results of the study will directly address how and cell adhesion turns external stimuli into signals that regulate cell behaviour, which will be important for all areas of cell biology. Furthermore, we anticipate that the information produced by our program could in the long term be used in cell and tissue engineering applications, where our lack of understanding of cell-material interactions are limiting the development of functional engineered tissue substitutes and the longevity of biomaterials.