Combining substratum compliance and topography to investigate cell adhesion and contraction

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OBJECTIVES: When a mammalian cell attaches to an artificial surface, eg, tissue cell culture plastic (TCP), fibroblasts assume an activated state (myofibroblast) characterized by incorporation of alpha-smooth muscle actin (SMA) into stressfibres. This phenotypic change in fibroblasts is largely attributed to the elastic moduli of polystyrene tissue culture plastic (0.01 GPa), but we have shown that 1) alterations in substratum topography can inhibit myofibroblast differentiation of fibroblasts even on stiff surfaces (110 GPa), and 2) fibroblasts isolated from different tissues show inherent differences in the amount of SMA incorporated into stressfibres even under the same culture conditions. In particular, gingival fibroblasts seem particularly resistant to transitioning to the myofibroblast phenotype which is in stark contrast to dermal fibroblasts which differentiate into myofibroblasts on TCP alone. Our research and that of others, suggests the initiator of fibroblast to myofibroblast differentiation relates to the size, stability, and composition of the adhesion sites formed. Our preliminary data has identified an alteration in the integrin subunits recruited to focal adhesion sites in fibroblasts susceptible to myofibroblast differentiation. In most mammalian cells, alphaVbeta3 is the integrin present in focal adhesion sites, but we have characterized that in highly contractile cells, alpha5beta1 is associated with vinculin containing focal adhesions. In a continuation of our program funded by NSERC, in this application, we hypothesize that topographies that increase cell to surface contact induce beta1 integrin recruitment to focal adhesion sites, inducing myofibroblast differentiation. Specifically, we will in this application: 1) Define how nanometric topographical features and substratum elastic moduli influence integrin recruitment to adhesion sites and the relationship to ?-smooth muscle actin and fibronectin expression in fibroblasts. 2). Quantify how nanometric topographical features and substratum elastic moduli modify adhesion mediated signaling upstream of ?-smooth muscle actin and fibronectin synthesis. 3). Assess whether specific combinations of nanometric topographical features fabricated in materials of varying elastic moduli suppress cellular contraction while promoting extracellular matrix synthesis. SIGNIFICANCE: We anticipate that the information produced by our program will in the long term provide a better understanding of how mammalian cells interact with extracellular matrix and artificial materials. Identification of surface compliances and topographies that suppress myofibroblast differentiation could be applied to "engineer" new generation cell culture substrates to prevent fibroblasts adopting this phenotype during routine culture. For soft tissue engineering applications, the ability to suppress a myofibroblast phenotype while promoting a matrix secreting and remodeling phenotype would be advantageous.