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-N
-hydroxysuccinimide ester (MU-NHS) to immobilize BMP-6 on gold surfaces at controlled surface density of 80 ng/cm2
as previously shown with BMP-2 [23
]. We used quartz crystal microbalance with dissipation monitoring (QCM-D) to demonstrate the validity of our platform by monitoring the binding kinetics of BMP-6 to the MU-NHS linker and its binding specificity (Figure A1
To visualize the growth factor on surfaces, we detected the surface immobilized BMP-6 (iBMP-6) by fluorescence microscopy (Figure 2
a). We used four different glass coverslips (Figure 2
a); in brief, a (i) glass coated with a 50-nm homogeneous gold layer by metal sputtering (indicated as Au), (ii) glass uncoated (indicated as Glass), (iii) glass coated with PEG (indicated as PEG), (iv) glass decorated with 8-nm size gold nanoparticles having a spacing of 30 nm arranged in hexagonal lattice, with the interparticle surface covered by PEG to prevent unspecific binding of protein (indicated as PEG/AuNP) (Figure 2
a). The sample was first incubated with the MU-NHS linker and then with BMP-6, followed by detection with antibodies. Indirect immunofluorescence staining shows signal on Au, glass and PEG/AuNP whereas on PEG coated surfaces no binding takes place, proving that PEG completely prevents the unspecific deposition of the growth factor and antibodies on the surface, which otherwise takes place on glass. Additionally, on PEG/AuNP, the staining for BMP-6 is rather homogeneous in comparison to Au coated, further indicating the binding specificity of BMP-6 and the antibodies. Furthermore, the bond between the NHS group of the linker and the amine residues of the growth factor is stable, even after washing and further treatment with detection reagents.
We next determined the stability of the chemical bond of BMP-6 to the surface in presence of cells (Figure 2
b). C2C12 were cultured in tissue culture dishes and approached from the top [23
] with (i) glass coverslips without growth factor (control, No BMP-6), (ii) glass coverslips functionalized with iBMP-6, (iii) glass coverslips in presence of BMP-6 added to the culture media (sBMP-6). The surfaces were in contact to cells for different time points (30, 60 and 120 min) and detection of remaining BMP-6 on the samples was done by chemiluminescence (Figure 2
b). BMP-6 was labeled with primary anti-BMP-6 antibody and secondary antibody conjugated with HRP, then detected by chemiluminescence. In samples where the growth factor is absent or added to the culture media, BMP-6 is not detected neither before nor after incubation with cells. When BMP-6 is covalently immobilized on the surface (iBMP-6), it is detected after presentation to cells, indicating that cells could not remove it.
To determine if iBMP-6 retains its biological activity, we next performed Western blotting to detect Smad1/5/8 phosphorylation levels (Figure 2
c). The activation of receptor-regulated Smads by BMP-6 converts the myogenic differentiation of C2C12 cells into osteogenic [6
]. Exposing cells to control surfaces without BMP-6 does not lead to BMP-mediated signaling. In cells exposed for 30, 60 or 120 min to 80 ng/cm2
of BMP-6 (calculated according to molecular size and weight to obtain complete surface coverage [23
]) either added to the media (sBMP-6) or immobilized on the surface (iBMP-6), SMAD 1/5/8 is phosphorylated and no statistically significant differences can be observed between the two groups. The peak of short term signaling, i.e., Smad 1/5/8 phosphorylation, has been reported to be at 120 min when the growth factor is immobilized on the surfaces and at 60 min after addition of sBMP-6 to cell cultures.
3.3. Surface Copresentation of Immobilized BMP-6 and RGD Ligands Promotes Adhesion and Induces BMP-Mediated Cell Responses
Although our system based on gold coating and self-assembled monolayer of MU-NHS linkers allows long term studies [23
], cell adhesion to the surfaces is rather unspecific and guided by deposition of cell own matrix, which in turn mediates further signaling modulation of Smad pathways, as also reported for other systems [29
]. Thus, to create an adhesive background, while preserving the setup for the presentation of iBMP-6 covalently bound to the surface, we further developed the nanopatterned surfaces (PEG/AuNP) shown in Figure 3
a by functionalizing the PEG layer with c(RGDfE)K(N3
), an integrin binding peptide. This setup is adapted from Schenk et al., where the dual functionalization of cRGD on the gold nanoparticles and synergy peptide PHSRN on the PEG layer was developed to promote cell adhesion [21
]. Following the coating with a PEG layer consisting in a mixture of PEG2000 and PEG3000-alkyne at a 99:1 ratio, the adhesive ligand carrying an azido functional group is introduced by copper(I)-catalyzed- azide-alkyne cycloaddition. Cells adhering to this surface were used as control (No BMP-6). Then, the gold nanoparticles (AuNPs) were functionalized with the MU-NHS linker so that the covalent immobilization of single BMP-6 homodimers (iBMP-6) or BMP-6 can be added to the media in soluble form (sBMP-6) (Figure 3
a). The amount of cRGD immobilized on the surface is tuned by varying the number of functional groups in PEG [21
] and the amount of BMPs on the surface by varying the AuNPs spacing [24
], to achieve a controlled spatial distribution of the peptide and the protein, and monitor the specific response of cells to the co-presentation of biomolecule in proximity to each other.
In absence of the adhesive ligand, cells do not adhere and spread on the surfaces containing gold nanodots with an interparticle distance of 30, 50 or 100 nm, further demonstrating the passivating properties of the PEG layer. The functionalization with RGD peptides on the PEG3000-alkyne end groups promotes adhesion, as indicated by the high number of spread cells on the surfaces 3 h after seeding (Figure 3
b). Regardless of the presence of BMP-6 (either added to the media or immobilized on the surface) and the amount of immobilized BMP-6, cell adhesion and spreading was comparable in presence of integrin ligands. This indicates that the immobilization of RGD on the PEG layer is fundamental for early adhesion and suggests that BMP-6-mediated cell surface interactions are not required for this process.
To study the impact of surface co-presentation of RGD and BMP-6 on cell signaling, we seeded C2C12 cells on nanopatterned surfaces and, following lysis to extract their protein content, we monitored Smad 1/5/8 phosphorylation by Western blotting 3 h after seeding (Figure 4
C2C12 cells not treated with BMP-6 were used as control (No BMP-6), treated cells were exposed to sBMP-6 (19, 6 or 1 ng) or to the corresponding amount of the immobilized growth factor (iBMP-6, at 30, 50 or 100 nm interparticle spacing). Adhesion of cells to RGD functionalized surfaces in absence of BMP-6 does not result in Smad 1/5/8 phosphorylation, confirming the independence of Smad activation from integrin mediated signaling. When BMP-6 is either added to the media or immobilized on the surface, phosphorylation of Smad1/5/8 is observed. The phosphorylation kinetics and levels in cells adhering to RGD surfaces and exposed to different concentrations of sBMP-6 indicate that signaling takes place, but the amount of growth factor presented in the soluble form is not sufficient to trigger a strong response on 50 and 100 nm distance surfaces. Combining RGD with iBMP-6 on the surfaces enhances Smad phosphorylation levels with all the different nanoparticles surfaces and the highest pSMAD 1/5/8 expression is observable with the 30 nm surfaces. It should be noted that with our platforms we deliver here to cells 1 ng of the growth factor (when the 100 nm distance is used), which is extremely lower than the amount typically used in standard in vitro assays (20 nM, corresponding to 520 ng in 1 mL solution).
We next investigated long-term responses of cells to iBMP-6 by imaging and quantifying myotube formation in C2C12 cell cultures on the different surfaces detecting myosin heavy chain (MHC) (Figure 5
a,b). In cells cultured for 6 days on Au homogeneous surfaces (Figure 5
a), staining for myosin heavy chain reveals the presence of myotubes (Figure 5
a, No BMP-6). When BMP-6 is either covalently immobilized on the Au surface (iBMP-6) or added to the culture media (sBMP-6), it suppresses myotube formation. Thus, iBMP-6 maintains its short- and long-term biological activity. The effective immobilization of BMP-6 on material surfaces, showing that the growth factor is still active even after a longer period of time, has not been reported thus far.
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) (Figure 5
b). In the control group, cells were left to adhere and further cultured on nanopatterned surfaces with clicked RGD, but in absence of BMP-6 (Figure 5
b 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 (Figure 5
b middle) or added to the culture media (Figure 5
b 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 added at an amount of 19 ng. This inhibitory effect on myotubes formation decreases with the reduction of BMP-6 concentration used (Figure 5
b). In particular, for 1 ng of sBMP-6, corresponding to the amount presented 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.