RESUMO
The nanoscale geometry and topography of the extra-cellular matrix (ECM) is an important parameter controlling cell adhesion and phenotype. Similarly, integrin expression and the geometrical maturation of adhesions they regulate have been correlated with important changes in cell spreading and phenotype. However, how integrin expression controls the nanoscale sensing of the ECM geometry is not clearly understood. Here we develop a new nanopatterning technique, electrospun nanofiber lithography (ENL), which allows the production of a quasi-2D fibrous nanopattern with controlled dimensions (250-1000nm) and densities. ENL relies on electrospun fibres to act as a mask for the controlled growth of protein-resistant polymer brushes. SEM, AFM and immunofluorescence imaging were used to characterise the resulting patterns and the adsorption of the extra-cellular matrix protein fibronectin to the patterned fibres. The control of adhesion formation was studied, as well as the remodelling and deposition of novel matrix. Cell spreading was found to be regulated by the size of fibres, similarly to previous observations made on circular nanopatterns. However, cell shape and polarity were more significantly affected. These changes correlated with important cytoskeleton reorganisation, with a gradual decrease in stress fibre formation as the pattern dimensions decrease. Finally, the differential expression of αvß3 and α5ß1 integrins in engineered cell lines was found to be an important mediator of cell sensing of the nanoscale geometry of the ECM. STATEMENT OF SIGNIFICANCE: The novel nanofiber patterns developed in this study, via ENL, mimic the geometry and continuity of natural matrices found in the stroma of tissues, whilst preserving a quasi-2D character (to facilitate imaging and for comparison with other 2D systems such as micropatterned monolayers and circular nanopatches generated by colloidal lithography). These results demonstrate that the nanoscale geometry of the ECM plays an important role in regulating cell adhesion and that this is modulated by integrin expression. This is an important finding as it implies that the knowledge of the biochemical context underlying the integrin-mediated adhesive machinery of specific cell types should allow better design of biomaterials and biointerfaces. Indeed, changes in integrin expression are often associated with the control of cell proliferation and differentiation.
