We report on the covalent immobilization of bone tissue morphogenetic proteins 6 (BMP-6) and its own co-presentation with integrin ligands on the nanopatterned platform to review cell adhesion and signaling responses which regulate the transdifferentiation of myoblasts into osteogenic cells. ImageJ was utilized to procedure images. Traditional western blot bands and myotubes area were quantified using ImageJ . All plotted data show mean values with standard deviations calculated from at least three impartial experiments (samples in duplicates or triplicates). 3. Results 3.1. BMP-6 Influences Cell Spreading and Focal Adhesion Assembly To evaluate the influence of BMP-6 on cell adhesion and spreading, C2C12 cells were cultured for 4 h on nanopatterned surfaces presenting RGD ligands at 50 nm spacing in absence of BMP-6 (No BMP-6) or in presence of the growth factor added to the media (sBMP-6) (Physique 1a). The 50 nm spacing has been reported to promote cell spreading and focal adhesion assembly . As expected, RGD-ligands allowed integrin mediated cell adhesion and spreading of C2C12 AR-A 014418 cells (Physique 1b). Comparing the formation of focal adhesions (FAs) and total AR-A 014418 cell area of samples treated or not with BMP-6 we found that FAs were larger and increased in presence of BMP-6. Interestingly, cells in AR-A 014418 presence of both RGD and sBMP-6 showed two-fold reduction in cell spreading (Physique 1c). This suggests that BMP-6 acts on cell cytoskeletal tension, which is usually Mouse monoclonal to Influenza A virus Nucleoprotein tightly couple with BMP-induced osteogenic signaling . 3.2. Surface Immobilized BMP-6 is Not Internalized by Cells and Triggers Smad Signaling To study the effect of binding proximity of integrins and BMP receptors on C2C12 adhesion and signaling which regulate cell fate, we applied a selective chemistry approach, using a self-assembled monolayer of the heterobifunctional linker mercaptoundecanoic-< 0.05, < 0.01, * < 0.001. Students < 0.05, < 0.01, * < 0.001. Students < 0.05, < 0.01, * < 0.001. Students t-test was used for single comparison. To determine the long-term effects of RGD-iBMP-6 surfaces, we cultured C2C12 cells for 4 days and performed immunofluorescence microscopy studies of myotube formation by detecting myosin heavy chain (MHC) (Physique 5b). In the control group, cells were left to adhere and further cultured on nanopatterned surfaces with clicked RGD, but in absence of BMP-6 (Physique 5b left). For the iBMP-6 and sBMP-6 groups, cells were additionally exposed to 19, 6 or 1 ng of the growth factor, either covalently immobilized on the surface (Physique 5b middle) or added to the culture media (Physique 5b right). C2C12 cells form myotubes in the control group as evidenced by the green staining for MHC; in presence of iBMP-6 and sBMP-6, MHC positive myotubes formation is significantly prevented when the factor is usually added at an amount of 19 ng. This inhibitory effect on myotubes formation decreases with the reduction of BMP-6 concentration used (Physique 5b). In particular, for 1 ng of sBMP-6, corresponding to the amount offered at 100 nm spacing, iBMP-6 is more effective than sBMP-6, suggesting that the sustained presentation of the growth factor, activates signaling pathways even after several days in culture. As such, surface copresentation of low amounts of BMP-6 together with integrin ligands guides cell fate through adhesion and regulation of signaling. 4. Conversation The objective of this study was to develop surfaces containing controlled density of immobilized BMP-6 and co-present it with RGD ligands to direct cell adhesion and signaling. Cell adhesion and distributing were key elements to.