Recombinant human bone morphogenetic protein (BMP) -2 has been applied in clinical settings and BMP-6 will be clinically tested for the restoration and treatment of skeletal conditions. Engineering an appropriate delivery system for BMPs, which promotes bone formation while controlling its amount and minimizing side effects, still remains a challenge.
In this work we covalently immobilize BMP-2 and BMP-6 on surfaces to investigate its effect on BMP-dependent signaling pathways and osteogenic differentiation in responsive cells. A further aim is to determine the effect of BMPs density on Smad-dependent signaling by using nanostructured substrates functionalized with immobilized BMPs and substrates presenting integrin adhesive ligands and BMPs.
The substrates were produced by means of block copolymer micelle nanolithography. To vary the local density of immobilized BMPs, gold nanoparticles arranged in an hexagonal pattern, were deposited on glass surfaces at different spacing, ranging from 30 to 90 nm. To prevent unspecific binding of proteins to the glass surface, the substrate was coated with polyethylene glycol between the gold nanoparticles. Alternatively, to render the surface adhesive to cells, the polyethylene glycol was synthesized such that it carried azido or alkyne functional group to allow the covalent immobilization of adhesive ligands by click chemistry. Following an initial incubation with a heterobifunctional linker which binds to the gold nanoparticles, single BMP homodimers are then coupled covalently to the linker.
The amount of BMP immobilized on the nanoparticles is determined by atomic force microscopy (AFM), in situ colorimetric assay and quartz crystal microbalance with dissipation (QCMD). To determine the impact of the ligand density on BMP-dependent signal transduction, we prove that the phosphorylation of Smad 1/5/8 is modulated by the stimulation of cells with immobilized BMPs on nanoparticles at different spacing. Additionally, the immobilized BMP at low density (below 1 ng/cm2) proves to efficiently trigger signaling due to its sustained presentation to cells, whereas the correspoding amount added to the culture media does not induce Smad-dependent signaling response. The copresentation of BMPs with integrin adhesive ligands further promotes cell adhesion and specifically modulates spreading kinetics and focal adhesion assembly.
With this approach it is therefore possible to achieve a controlled local presentation of immobilized growth factors and adhesive molecules and to quantitatively investigate cellular responses.
|Category||Short file description||File description||File Size|
|Manuskript||File 1||Includes author list, affiliations, abstract and references||51 KB||Download|