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

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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.