Abstract
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.
Highlights
Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β superfamily.They exert pleiotropic effects and BMP-2 and -7 have been approved in clinical use for their potent induction of bone formation [1]
To evaluate the influence of bone morphogenetic protein 6 (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)
Comparing the formation of focal adhesions (FAs) and total cell area of samples treated or not with BMP-6 we found that FAs were larger and increased in presence of BMP-6
Summary
Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β superfamily. They exert pleiotropic effects and BMP-2 and -7 have been approved in clinical use for their potent induction of bone formation [1]. Showing efficacy in in vivo studies at low amounts, e.g., 50 μg in whole blood containing devices. These doses are much lower when compared to the ones (e.g., 3.5 mg in a bovine collagen carrier). Cells 2019, 8, 1646 used for BMP-2 and -7 [4,5]. First validated in C2C12 mouse myoblasts [6], and further investigated in a variety of primary human cells [7,8], the binding profile of BMP-6 to the type I (ALK-2 strongly and weakly to ALK-6)
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