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物或人工材料中时，成纤维细胞呈现高度收缩的肌成纤维细胞表型，其特征是a-平滑肌肌动蛋白（a- sma）上调。假设这种a- sma收缩表型，增加基质或底物硬度是成纤维细胞的已知驱动因素，但我们最近表明，通过对培养底物进行地形修饰，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，并且与培养底物硬度无关。这两种表型之间的触发因素似乎与细胞在响应地形特征变化时形成的粘附的大小、稳定性和组成有关。局灶粘连（FA）的稳定性对于纤维粘连的发展至关重要，我们最近表明，f -肌动蛋白结合蛋白的接触蛋白被募集到平滑地形上的FA位点，促进成纤维细胞的肌成纤维细胞分化。在粗糙的地形上，接触不被FAs吸收，这一观察结果对应于基质重塑基因表达的显著增加。在2008年由NSERC资助的项目的延续中，我们将评估纳米和微观形貌的变化如何调节牙龈成纤维细胞粘附和成纤维细胞向肌成纤维细胞的转变。在这个应用中，我们假设通过地形改变限制细胞表面接触改变粘附位点的分子组成，激活细胞内信号并抑制牙龈成纤维细胞的肌成纤维细胞分化。我们将在此应用程序中：

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.