Cell adhesion and polarisation on molecularly defined spacing gradient surfaces of cyclic RGDfK peptide patches

Abstract

In vivo cell migration and location are orchestrally guided by soluble and bound chemical gradients. Here, gradients of extracellular matrix molecules are formed synthetically by the combination of a surface nanopatterning technique called block copolymer nanolithography (BCN) and a biofunctionalisation technique. A modified substrate dip-coating process of BCN allows for the formation of precise molecular gradients of cyclic RGDfK peptide patches at interfaces, which are presented to cells for testing cell adhesion and polarisation. Surfaces formed by BCN consist of hexagonally ordered gold dot patterns with a gradient in particle spacing. Each dot serves as a chemical anchor for the binding of cyclic RGDfK peptides, which are specifically recognised by alpha(v)beta(3) integrins. Due to steric hindrance only up to one integrin binds to one functionalised gold dot which forms a peptide patch spacing. We demonstrate how cell morphology, adhesion area, actin and vinculin distribution as well as cell body polarisation are influenced by the peptide patch spacing gradient. As a consequence, these gradients of adhesive ligands induce cell orientation towards smaller particle spacing when the gradient strength is 15nm/mm at least. This implicates that an adherent cell's sensitivity to differentiate between ligand patch spacing is approximately 1nm across the cell body.

Fig. 1

Nanoparticle spacing gradient surfaces. (A) Gradient sample with a 2 mm length from substrate position 3 mm to 5 mm which covers nanodot spacings ranging from ca. 55 nm to 85 nm. Each gold particle has a diameter of approximately 6 nm. Origin of the graph indicates the dipping edge as explained in (B). Error bars represent standard deviations (standard errors ~1 %). (B) Schematic drawing of the dip-coated substrates illustrating the differently patterned areas. (I) 3 mm homogenously nanopatterned area, small constant particle spacing; (II) 2 mm particle spacing gradient; (III) homogenously nanopatterned area, large constant particle spacing, including the dripping edge which displays an area of uncontrolled nanoparticle aggregation forming upon solvent drying. (C) Scanning electron microscopy pictures taken at different positions of the substrate: area (I) at position 2 mm, area (II) at position 4.3 mm and area (III) at position 6 mm.

Fig. 2

(Upper panel) Scheme of biofunctionalised nanopatterns to control integrin clustering (Arnold et al., 2004): gold dots are functionalised by c(-RGDfK-) thiols; glass areas between cell-adhesive gold dots are covalently bound to polyethyleneglycol to prevent unspecific protein binding. Therefore, cell adhesion is only mediated via c(-RGDfK-)-covered gold nanodots. (Bottom panels) Mc3t3 osteoblast in contact with a biofunctionalised 80-nm pattern and exhibiting cell protrusions sensing the pattern. Bars: 20 µm (left); 200 nm (right).

Fig. 3

Projected cell area as a function of substrate position. Projected cell area along a 2-mm c(-RGDfK-) patch spacing gradient on a sample covering a spacing from 50–80 nm after 23 h cell culture. Insets: Mc3t3 osteoblasts after 23 h adherence on a homogeneously nanopatterned area with 50 nm c(-RGDfK-) patch spacing and along the spacing gradient, respectively. The latter one displays a section of the gradient which represents approximately 70 nm c(-RGDfK-) patch spacing. Cells were immunostained for vinculin (green), and actin was visualised using TRITC-phalloidin (red). Bars: 20 µm.

Fig. 4

Cell polarisation along c(-RGDfK-) patch spacing gradients. Angles of the main cell body axis with the gradient direction after 23 h on 55 nm homogenously nanopatterned samples (A) and on the c(-RGDfK-) patch spacing gradient ranging from 50 to 80 nm spacing along a 2 mm substrate length (B). Data were acquired from three separate experiments. For graph (A) 347 cells and graph (B) 428 cells were evaluated, error bars present standard error, p<0.01. (C) Describes the analysis of the main cell body angle with the direction of the gradient and the gradient polarisation ratio (GPR). The GPR is given by the ratio of the maximum x-width (length parallel to the gradient axis) with the maximum y-width (length perpendicular to the gradient axis) of a cell body. GPR of a cell adhering on homogeneously nanopatterned areas (50 nm, 80 nm) and on the c(-RGDfK-) patch spacing gradient area with spacing ranging from 50 to 80 nm along 2 mm substrate length after 23 h are given in (D).