Lateral Chain Length in Polyalkyl Acrylates Determines the Mobility of Fibronectin at the Cell/Material Interface

Abstract

Cells, by interacting with surfaces indirectly through a layer of extracellular matrix proteins, can respond to a variety of physical properties, such as topography or stiffness. Polymer surface mobility is another physical property that is less well understood but has been indicated to hold the potential to modulate cell behavior. Polymer mobility is related to the glass-transition temperature (Tg) of the system, the point at which a polymer transitions from an amorphous solid to a more liquid-like state. This work shows that changes in polymer mobility translate to interfacial mobility of extracellular matrix proteins adsorbed on the material surface. This study has utilized a family of polyalkyl acrylates with similar chemistry but different degrees of mobility, obtained through increasing length of the side chain. These materials are used, in conjunction with fluorescent fibronectin, to determine the mobility of this interfacial layer of protein that constitutes the initial cell-material interface. Furthermore, the extent of fibronectin domain availability (III9, III10, - the integrin binding site), cell-mediated reorganization, and cell differentiation was also determined. A nonmonotonic dependence of fibronectin mobility on polymer surface mobility was observed, with a similar trend noted in cell-mediated reorganization of the protein layer by L929 fibroblasts. The availability of the integrin-binding site was higher on the more mobile surfaces, where a similar organization of the protein into networks at the material interface was observed. Finally, differentiation of C2C12 myoblasts was seen to be highly sensitive to surface mobility upon inhibition of cell contractility. Altogether, these findings show that polymer mobility is a subtle influence that translates to the cell/material interface through the protein layer to alter the biological activity of the surface.

Figure 1

Fibronectin and material systems. Conceptual figure of how polymer side-chain length affects the mobility by increasing free volume (represented by the white area) and its effect on the fibronectin layer (monomer shown above). PMA(x = 1) leads to adsorption of globular fibronectin, with PEA(x = 2), PBA(x = 4), and PHA(x = 6) all leading to the formation of fibronectin nanonetworks. The fact that the same organization of the protein occurs on these three polymers allows discussion of the effect of surface mobility on protein layers with the same initial conformation/distribution and assessment of the effect of mobility, disregarding major conformational effects of the proteins induced by surface chemistry.

Figure 2

Nanoindentation of polymer films. (A) Sketch of nanoindentation showing cantilever indenting a polymer surface. (B) Force curve from measurements, showing an example of the initial 50 nm indentation of a polymer surface (δ), on which the Hertz model was applied to calculate the Young’s moduli. (C) Young’s moduli of polymer films; each point on the graph is an average derived from 64 measurements (per sample).

Figure 3

Surface hydrophilicity. Dynamic water contact angles were measured on the polymer surfaces before and after coating with a FN solution of concentration 20 μg/mL. (A) Contact-angle hysteresis. (B) Advancing (ACA) and receding (RCA) angles of the uncoated polymer surface and (C) of the surfaces after coating with FN and labeled FN.

Figure 4

Atomic force microscopy. Fibronectin distribution on the polymer surfaces by AFM (AC mode) after adsorption from a solution of concentration 20 μg/mL. FN is organized into nanonetworks on PEA (x = 2), PBA (x = 4), and PHA (x = 6). These 1 μm × 1 μm phase images demonstrate the similarities in protein distribution in the labeled and unlabeled forms. The values shown underneath the network forming polymers relate to the fractal dimension (D), a descriptor of the complexity of a pattern that accounts for the network connectivity.

Figure 5

Fibronectin conformation. Exposure of epitopes of FN when adsorbed on polymer surfaces from a solution of concentration 20 μg/mL. (A) FNIII_7–10 model with arrows pointing to binding sites for mAb1937 and HFN 7.1. Image from the RCSB PDB (www.rcsb.org) of PDB ID 1FNF. (B) Overall availability of FN using a polyclonal antibody. (C) RGD cell-binding site exposure (HFN 7.1 Ab). (D) Exposure of the synergy site (mAb 1937 Ab). Panels C and D were normalized using panel B.

Figure 6

Interfacial mobility. Measuring the rate of change in fluorescence intensity profile as an indicator of protein mobility. (A) Bleach border region, with the manually selected area highlighted. (B) Linear gradient value of the bleach border used to determine any changes of the intensity profile at each time point. (C) Comparison of the gradient value at each time point (N = 10) for each of the polymer surface. This is then used to determine the rate constant (k value) of the exponential growth equation. (D) Gradient of the linear region of the bleach border changes with respect to time for each of the polymers. (E) Ascertained k values compared with the glass-transition temperature, T_g, of the polymers.

Figure 7

FN reorganization. Cell-mediated FN reorganization on differently mobile polymer surfaces. (A) Relative differences in the fluorescence intensity within the cell area (as defined by the stained actin) compared with outside the cell area. (B) Cell (actin, red) with the corresponding fibronectin layer (green) below for glass, PMA (x = 1), PEA(x = 2), PBA(x = 4), and PHA(x = 6), respectively (scale bar = 25 μm).

Figure 8

Cell differentiation. Differentiation of C2C12 cells cultured on FN coated Collagen I, PMA, PEA, PBA, and PHA (A) without and (B) with a contractility inhibitor (blebbistatin). COLI is the differentiation control, and the percentage of differentiation is measured as the ratio of sarcomeric myosin-positive cells. (C) Representative pictures: nuclei (blue) and sarcomeric myosin (red) for each of the surfaces. In panel B there was significant differences between COLI and polymer surfaces with 1,2 (p = ****) and 3 (p = *) carbons in the side chain.