Langmuir–Schaefer films of fibronectin as designed biointerfaces for culturing stem cells

Abstract

Glycoproteins adsorbing on an implant upon contact with body fluids can affect the biological response in vitro and in vivo, depending on the type and conformation of the adsorbed biomacromolecules. However, this process is poorly characterized and so far not controllable. Here, protein monolayers of high molecular cohesion with defined density are transferred onto polymeric substrates by the Langmuir–Schaefer (LS) technique and were compared with solution deposition (SO) method. It is hypothesized that on polydimethylsiloxane (PDMS), a substrate with poor cell adhesion capacity, the fibronectin (FN) layers generated by the LS and SO methods will differ in their organization, subsequently facilitating differential stem cell adhesion behavior. Indeed, atomic force microscopy visualization and immunofluorescence images indicated that organization of the FN layer immobilized on PDMS was uniform and homogeneous. In contrast, FN deposited by SO method was rather heterogeneous with appearance of structures resembling protein aggregates. Human mesenchymal stem cells showed reduced absolute numbers of adherent cells, and the vinculin expression seemed to be higher and more homogenously distributed after seeding on PDMS equipped with FN by LS in comparison with PDMS equipped with FN by SO. These divergent responses could be attributed to differences in the availability of adhesion molecule ligands such as the Arg-Gly-Asp (RGD) peptide sequence presented at the interface. The LS method allows to control the protein layer characteristics, including the thickness and the protein orientation or conformation, which can be harnessed to direct stem cell responses to defined outcomes, including migration and differentiation.