A micron-scale surface topography design reducing cell adhesion to implanted materials

Abstract

The micron-scale surface topography of implanted materials represents a complementary pathway, independent of the material biochemical properties, regulating the process of biological recognition by cells which mediate the inflammatory response to foreign bodies. Here we explore a rational design of surface modifications in micron range to optimize a topography comprised of a symmetrical array of hexagonal pits interfering with focal adhesion establishment and maturation. When implemented on silicones and hydrogels in vitro, the anti-adhesive topography significantly reduces the adhesion of macrophages and fibroblasts and their activation toward effectors of fibrosis. In addition, long-term interaction of the cells with anti-adhesive topographies markedly hampers cell proliferation, correlating the physical inhibition of adhesion and complete spreading with the natural progress of the cell cycle. This solution for reduction in cell adhesion can be directly integrated on the outer surface of silicone implants, as well as an additive protective conformal microstructured biocellulose layer for materials that cannot be directly microstructured. Moreover, the original geometry imposed during manufacturing of the microstructured biocellulose membranes are fully retained upon in vivo exposure, suggesting a long lasting performance of these topographical features after implantation.

Figure 1

Replica molding and micro-pattern characterization. (a) SEM micrograph of elastomeric microstructured substrate manufactured using soft-lithography and coated with fibronectin. Scale bar: 10 µm. Inset, scale bar: 5 µm. (b) Layout of the patterns on the elastomeric microstructured substrate for cell adhesion experiments. (c) Elemental cells of the investigated patterns for cell adhesion reduction. Parametric design space for elastomeric microstructured substrate in the (d) hexagonal and (e) squared patterns considering manufacturing constraints and rationale mechanobiological design principles.

Figure 2

Cell morphology on surface-structured PDMS membranes. HDFs (a) density, (b) circularity and (c) area on different patterns normalized to the HDFs values measured on unstructured flat PDMS surfaces. (d) Representative fluorescence image of HDF on flat elastomeric substrate revealing f-Actin (red) and Vinculin (green). Scale bar: 50 µm. (e) HDF on Hexa_d20,i23 PMDS sample. Scale bar: 50 µm. Detail: focal adhesions (green) were preferentially established on the top surface of the walls separating the wells, with short bridging actin fibers (red). The semi-transparent hexagonal structures are artificially overlaid for illustration purposes. Scale bar: 10 µm. (f) Representative fluorescence images of HDFs on different patterns revealing F-Actin (red) and nuclei (blue). Scale bar: 80 µm.

Figure 3

Cell morphology on surface-structured biocellulose. (a) First row: hexagonal patterns with well-diameter ranging from 3 µm to 10 µm. Second row: squared patterns with microwells diameter ranging from 3 µm to 10 µm. Scale bar: 10 µm. (b) HDFs density on different patterns on biocellulose normalized to the HDFs density measured on unstructured biocellulose substrates. (c) HDFs circularity on different patterns. Representative scanning electron microscopy images of HDFs on (d) gratings and on (e) Hexa10 (d = 10 µm, i = 20 µm) micropatterns. Scale bar: 10 µm. Cell surface is colored for visualization purposes.

Figure 4

Macrophages adhesion onto different materials. (a) THP-1 cells density on different materials normalized to the THP-1 cells density measured on Tissue Culture Plastic. (b) THP-1 cells density on different patterns on biocellulose normalized to the THP-1 cells density measured on unstructured biocellulose substrates. Representative widefield images of THP-1 macrophages on (c) microstructured biocellulose and on (d) MED 6015. Scale bar: 100 µm.

Figure 5

Expression levels of selected pro- and anti-inflammatory genes. The expression levels of selected target genes were calculated in fold change (2-ddCt) and plotted in the graphs (antilog scale). n = 5 were used for the analysis. (a,b) The additional x axis at y = 0 indicates the expression levels of each specific marker in THP-1 cells cultured onto silicone coated plates. CCL17 is significantly up-regulated by cells seeded on the flat surface (p < 0.001) and on the microstructured (Hexa5, d = 5 µm, i = 10 µm) cellulose (p = 0.02) with comparison to the expression levels of the same markers onto silicone. PTGS1 appears to be significantly down regulated (p = 0.03) in THP-1 cells cultured onto flat surfaces. (c) The x axis at y = 0 indicates the expression levels of each marker in THP-1 cells cultured onto flat surfaces. CCL17 appears down-regulated (p < 0.001) microstructured (Hexa5, d = 5 µm, i = 10 µm) cellulose with comparison to unstructured cellulose.