Investigating adhesion-based regulation of cell phenotype with nano- and micro-metric topography

假设和目的：当将成纤维细胞置于细胞培养或人工材料上时，成纤维细胞呈现高度收缩的肌成纤维细胞表型，其特征是α-平滑肌肌动蛋白(a-SMA)表达上调。基质或基质硬度的增加是假定这种a-SMA收缩表型的成纤维细胞的已知驱动因素，但我们最近表明，通过改变培养底物的形态可以减弱人牙龈成纤维细胞的肌成纤维细胞分化。此外，这些抑制肌成纤维细胞分化的特征促进了成纤维细胞的基质重塑表型，并且与培养底物的硬度无关。这两种表型之间的触发似乎与细胞对地形特征的变化所形成的粘连的大小、稳定性和组成有关。局灶性粘连(FA)的稳定性对纤维粘连的发展至关重要，我们最近发现，F-肌动蛋白结合蛋白Cortactin被募集到平滑地形上的FA位置，促进成纤维细胞的肌成纤维细胞分化。在粗糙的地形上，皮质素没有被吸收到FA，这一观察结果对应于基质重塑基因表达的显著增加。在我们自2008年以来由NSERC资助的项目的继续中，我们将评估纳米和微米地形的变化如何调节牙龈成纤维细胞的黏附以及成纤维细胞向肌成纤维细胞的转变。在这一应用中，我们假设通过局部修饰来限制细胞与表面的接触，改变了黏附位置的分子组成，激活了细胞内的信号，并抑制了牙龈成纤维细胞的肌成纤维细胞分化。在此应用程序中，我们将： 1)研究光滑、纳米和微米形态对牙周成纤维细胞黏附成分、细胞内信号转导、α-平滑肌肌动蛋白表达和细胞硬度的影响。 2)研究在光滑的纳米和微米地形图上，牙龈成纤维细胞向肌成纤维细胞分化是否需要皮质肌动蛋白的磷酸化和向局灶性粘连(FA)的募集。 3)确定地形对HGFs基质重塑基因表达的影响。 意义：我们预计，从长远来看，我们的计划产生的信息将提供对细胞-材料相互作用的更好理解。对于软组织工程应用，抑制肌成纤维细胞表型而促进基质分泌和重塑表型的能力将是有利的。此外，抑制肌成纤维细胞分化的拓扑学可以用来“设计”新一代细胞培养底物，以防止成纤维细胞在常规培养中采用这种表型。

HYPOTHESIS AND OBJECTIVES: When placed in cell culture or on artificial materials, fibroblasts assume a highly contractile myofibroblast phenotype characterized by upregulation of a-smooth muscle actin (a-SMA). Increased matrix or substrate stiffness is a known driver of fibroblasts assuming this a-SMA contractile phenotype, but we have recently shown it is possible to attenuate myofibroblast differentiation of human gingival fibroblasts using topographical modification of the culture substrate. Moreover, such features that inhibit myofibroblast differentiation promoted a matrix remodeling phenotype in fibroblasts and were independent of culture substrate stiffness. It appears the trigger between the two phenotypes relates to the size, stability, and composition of the adhesions formed by the cells in response to the changes in topographical features. Stability of focal adhesions (FA) is pivotal to allow development of fibrillar adhesions and we have recently shown that the F-actin binding protein cortactin is recruited to FA sites on smooth topographies that promote myofibroblast differentiation of fibroblasts. On rough topographies, cortactin is not recruited to FAs, an observation that corresponds to a significant increase in matrix-remodeling gene expression. In a continuation of our program funded by NSERC since 2008, we will assess how changes in nano- and micro-metric topography regulate gingival fibroblast adhesion and the transition of fibroblasts to myofibroblasts. In this application, we hypothesize that limiting cell to surface contact through topographical modifications alters the molecular composition of adhesion sites, activation of intracellular signaling and suppresses myofibroblast differentiation in gingival fibroblasts. We will in this application: 1) Investigate how smooth, nano- and micro- metric topographies influences gingival fibroblast adhesion composition, intracellular signaling, a-smooth muscle actin expression and cell stiffness. 2) Investigate if cortactin phosphorylation and recruitment to focal adhesions (FA) is required for gingival fibroblast to myofibroblast differentiation on smooth, nano- and micro-metric topographies. 3) Determine the role of topography on the expression of matrix-remodeling genes in HGFs. SIGNIFICANCE: We anticipate that the information produced by our program will in the long term provide a better understanding of cell-material interactions. For soft tissue engineering applications, the ability to suppress a myofibroblast phenotype while promoting a matrix secreting and remodeling phenotype would be advantageous. Furthermore, topographies that suppress myofibroblast differentiation could be applied to “engineer” new generation cell culture substrates to prevent fibroblasts adopting this phenotype during routine culture.