Cell migration on material-driven fibronectin microenvironments

Abstract

Cell migration is a fundamental process involved in a wide range of biological phenomena. However, how the underlying mechanisms that control migration are orchestrated is not fully understood. In this work, we explore the migratory characteristics of human fibroblasts using different organisations of fibronectin (FN) triggered by two chemically similar surfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA); cell migration is mediated via an intermediate layer of fibronectin (FN). FN is organised into nanonetworks upon simple adsorption on PEA whereas a globular conformation is observed on PMA. We studied cell speed over the course of 24 h and the morphology of focal adhesions in terms of area and length. Additionally, we analysed the amount of cell-secreted FN as well as FN remodelling. Velocity of human fibroblasts was found to exhibit a biphasic behaviour on PEA, whereas it remained fairly constant on PMA. FA analysis revealed more mature focal adhesions on PEA over time contrary to smaller FAs found on PMA. Finally, human fibroblasts seemed to remodel adsorbed FN more on PMA than on PEA. Overall, these results indicate that the cell-protein-material interface affects cell migratory behaviour. Analysis of FAs together with FN secretion and remodelling were associated with differences in cell velocity providing insights into the factors that can modulate cell motility.

Fig. 1. Chemical structure of PEA and PMA (a). Fibronectin distribution on PEA and PMA as observed with the height magnitude in the AFM. The protein was adsorbed for 10 min from a solution of concentration 20 μg ml^–1 (b). Schematic representation of cell migration on PEA and PMA coated with fibronectin (c).

Fig. 2. Characterization of cell migration on fibrillar (on PEA) and globular (on PMA) FN over the course of 24 h. Phase contrast pictures of human fibroblasts (a) on fibrillar FN and globular FN 0 h, 12 h, 24 h after attachment. Red arrows indicate the migration of a single cell over time. Velocity (μm h^–1) of human fibroblasts (b). Videos of migratory cells were quantified to build the migration graphs (ESI Video 1†).

Fig. 3. Characterisation of focal adhesions of human fibroblasts. Representative inverted binary images of focal adhesions on fibrillary (on PEA) and globular (on PMA) FN (a). Area and length distribution of focal adhesions of human fibroblasts on fibrillary (PEA) and globular (PMA) FN 6 h (b) and 22 h (c) after seeding. Scale bar: 50 μm.

Fig. 4. Fluorescent images of human fibroblasts (a) on fibrillar (PEA) and globular (PMA) FN 6 h and 22 h after seeding. Staining of cellular FN (red), actin (green) and nuclei (blue). Scale bar is 100 μm. Quantification of FN secreted by human fibroblasts (b) on fibrillar and globular FN (white and black bars respectively) 6 h and 22 h after seeding.

Fig. 5. FN reorganisation. Fluorescent pictures of FITC-labelled FN on PEA (fibrillar) and PMA (globular) after reorganisation by human fibroblasts for 6 h and 22 h. Scale bar is 30 μm (a). Normalised fluorescence intensity of FN within the cell compared with the intensity outside the cell area (b). Schematic representation of cell-mediated reorganisation of FN. 2 h.