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We report on the covalent immobilization of bone morphogenetic protein 6 (BMP-6) and its co-presentation with integrin ligands on a nanopatterned platform to study cell adhesion and signaling responses which regulate the transdifferentiation of myoblasts into osteogenic cells. To immobilize BMP-6, the heterobifunctional linker MU-NHS is coupled to amine residues of the growth factor; this prevents its internalization while ensuring that its biological activity is maintained. Additionally, to allow cells to adhere to such platform and study signaling events arising from the contact to the surface, we used click-chemistry to immobilize cyclic-RGD carrying an azido group reacting with PEG-alkyne spacers via copper-catalyzed 1,3-dipolar cycloaddition. We show that the copresentation of BMP-6 and RGD favors focal adhesion formation and promotes Smad 1/5/8 phosphorylation. When presented in low amounts, BMP-6 added to culture media of cells adhering to the RGD ligands is less effective than BMP-6 immobilized on the surfaces in inducing Smad complex activation and in inhibiting myotube formation. Our results suggest that a local control of ligand density and cell signaling is crucial for modulating cell response.

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

We tested the hypothesis that RGDS peptides regulate osteoblast survival in culture. Osteoblast-like MC3T3-E1 cells were allowed to attach to RGDS peptides that had been tethered to a silicone surface utilizing a previously described grafting technique. The RGDS-modified surface caused up-regulation of alpha(v)beta(3) integrin. We noted that there was an increase in expression of activated focal adhesion kinase and activated Akt. There was no change in the expression level of the anti-apoptotic protein Bcl-2, the pro-apoptotic protein Bad, or the inactivated form of Bad, pBad. Attachment to the RGDS-treated membrane completely abolished apoptosis induced by staurosporine, the Ca(2+).P(i) ion pair, and sodium nitroprusside. However, the surface modification did not interfere with apoptosis mediated by the free RGDS peptide or serum-free medium. When the activity of the phosphatidylinositol 3-kinase pathway was inhibited, RGDS-dependent resistance to apoptosis was eliminated. These results indicated that the binding of cells to RGDS abrogated apoptosis via the mitochondrial pathway and that the suppression of apoptosis was dependent on the activity of phosphatidylinositol 3-kinase.

To study the function behind the molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell-adhesive gold nanodots coated with cyclic RGDfK peptide by using block-copolymer micelle nanolithography. The diameter of the adhesive dots is < 8 nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73, and 85 nm spacing at interfaces. A separation of > or = 73 nm between the adhesive dots results in limited cell attachment and spreading, and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in the cell-matrix interface since "micro-nanopatterned" substrates consisting of alternating fields with dense and no nanodots do support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.