Introduction: Cells are sensitive to the chemistry, the topography and the mechanical properties of their substrate surface. Chemical composition and topography of surfaces used for interaction with biological matter become more and more controlled, at the supra- or the subcellular level. However, control of surface mechanical properties at the subcellular scale remains challenging. The aim of this study is to prepare surfaces with mechanical cues at the nanometer scale, and to evaluate cell behavior in contact with these surfaces. The research strategy consists in coating a substrate showing rigid topographical nanostructures with a thin layer of an elastomer (Fig 1). This layer ensures the homogeneous chemistry and topography of the surface, while the rigid structures underneath provide mechanical contrast.
Materials and Methods: Substrates with mechanical nanoheterogeneities: The combination of colloidal lithography and layer-by-layer assembly allows a defined topography to be created. Glass surfaces were covered with positively-charged polyallylamine, and then with negatively-charged 500 nm silica colloids. Particle distribution was examined using scanning electron microscopy (SEM). To obtain a homogenous chemistry and topography, thin films of poly(dimethylsiloxane) (PDMS) were spin-coated on top of the silica colloids. The stiffness of PDMS was adjusted using different cross-linking agent concentrations. Atomic force microscopy (AFM) was used to investigate the topography and the mechanical properties of the obtained substrates. Surface chemical composition and wettability were checked using X-ray photoelectron spectroscopy and water contact angle measurements. Cell culture experiments: MC3T3 preosteoblasts, gingival fibroblasts and C2C12 skeletal myoblasts were examined after 4h and 72h in culture on the elaborated substrates, and the observed behavior was compared to the one found on PDMS layers in absence of silica colloids. Cell motility was investigated using time-lapse imaging, and gene expression was determined by qPCR.
Results: A regular distribution of the silica colloids was obtained (Fig 1, Left). Spin-coating parameters were adjusted to produce surfaces with a very limited topography and a homogeneous surface chemistry. The thickness of the PDMS layer was of ~50 nm on top of the 500 nm colloids (Fig 1, Center). AFM force maps evidenced the mechanical contrast produced by the embedded rigid particles. Cell response is shown to be affected by the mechanical nanoheterogeneities, in a cell type-dependent manner. Global trends may however be extracted. Cell adhesion increases on heterogeneous substrates. Moreover, cell proliferation is maximized on heterogeneous substrates, although the different cell types proliferate better on homogeneous substrates of different stiffness. This suggests that on mechanically nanoheterogeneous substrates, cells can make use of areas of the most favorable stiffness, which will control their response. Finally, cell motility was enhanced in presence of rigid mechanical nanocues (Fig 1, Right). This is attributed to increased formation of focal adhesions on more rigid domains, but the limited size of these domains prevents extensive maturation of these focal adhesions.
Conclusion: The results demonstrate that cells are sensitive to mechanical heterogeneities at the subcellular level, which opens the way to novel strategies for a better control of cell-material interactions.
SD thanks the FNRS for funding