Laser Direct Writing of Dual-Scale 3D Structures for Cell Repelling at High Cellular Density

Abstract

The fabrication of complex, reproducible, and accurate micro-and nanostructured interfaces that impede the interaction between material's surface and different cell types represents an important objective in the development of medical devices. This can be achieved by topographical means such as dual-scale structures, mainly represented by microstructures with surface nanopatterning. Fabrication via laser irradiation of materials seems promising. However, laser-assisted fabrication of dual-scale structures, i.e., ripples relies on stochastic processes deriving from laser-matter interaction, limiting the control over the structures' topography. In this paper, we report on laser fabrication of cell-repellent dual-scale 3D structures with fully reproducible and high spatial accuracy topographies. Structures were designed as micrometric "mushrooms" decorated with fingerprint-like nanometric features with heights and periodicities close to those of the calamistrum, i.e., 200-300 nm. They were fabricated by Laser Direct Writing via Two-Photon Polymerization of IP-Dip photoresist. Design and laser writing parameters were optimized for conferring cell-repellent properties to the structures, even for high cellular densities in the culture medium. The structures were most efficient in repelling the cells when the fingerprint-like features had periodicities and heights of ≅200 nm, fairly close to the repellent surfaces of the calamistrum. Laser power was the most important parameter for the optimization protocol.

Keywords: cell repellent; dual-scale structure; laser direct writing; two-photon polymerization.

Figure 4

(Upper panel) Atomic force microscopy images (AFM) (enhanced colors) of dual-scale structures in the shape of nanostructured mushroom-like pillars (NMPs) fabricated by LDW via TPP of IP-Dip photopolymer, having the same design and fabricated using different laser powers; (Middle panel) 2D profiles along the radial segments marked in red in the upper panel. For all samples, the scanning speed was 100 μm/s; (Lower panel) Nanostructures’ heights as a function of incident laser power step (the error bars are the statistical deviations). The greeen oval from the right part of the middle panel points out the best result in terms of laser power optimization.

Figure 5

(Upper panel) Atomic force microscopy images (AFM) (enhanced colors) of dual-scale structures in the shape of nanostructured mushroom-like pillars (NMPs) fabricated by LDW via TPP of IP-Dip photopolymer using the same writing parameters and different designs/geometries; (Middle panel) 2D profiles along radial segments across the structures imaged in the upper panel; (Middle panel) Simulation of the laser path with different scaling of the spiral step in the z-direction (side view of the pillar lid). For all structures, the laser power was 13.25 mW and the scanning speed was 100 μm/s. (Lower panel) Nanostructures’ heights as a function of spiral step (the error bars are the statistical deviations).

Figure 6

(a) Scanning electron micrograph (SEM) of a nanostructured mushroom-like pillar (NMP) inclined at 30 degrees; (b) Atomic force microscopy (AFMJ) image of NMP from (a); (c) Cross-section of the indentation surface through the center of a mushroom-like pillar (star-like points: experimental data; continuous lines: parabolic fit).

Figure 7

Fluorescent images of cells on: (a) glass (control), (b) nanostructured mushroom-like pillars with optimized laser power (NMPLP), (c) nanostructured mushroom-like pillars with optimized design (NMPD); green: structures’ autofluorescence; blue: cells nuclei, Hoechst; red: cells cytoskeleton, Phalloidin; (d–f) image processing by Image J showing only the cells’ outlines; (g–i) cells area, cells circularity and reduction of adhering cells for flat, NMP_LP and NMP_D structures, respectively. The scale (40 µm) from (f) is valid for all figures, i.e., (a–f). The sign “*” from (g–i) indicate that the data are statistically significant.

Figure 1

Optical images of arrays of nanostructured mushroom-like pillars (NMPs) arranged in hexagonal lattices, fabricated by LDW via TPP of IP-Dip photopolymer: (a) using the same geometry and writing parameters; (b) using different laser powers (upper panel) and scanning speeds (lower panel). Writing parameters (laser power, scanning speed) are indicated on each image. On both (a,b) the red stars mark the structures that lost their stability on the glass substrate.

Figure 2

Scanning electron micrographs (SEM) of nanostructured mushroom-like pillars (NMPs), fabricated by LDW via TPP of IP-Dip photopolymer, imprinted using the same design and different laser powers, as indicated above each figure: (Upper panel) Tilted top views of NMPs arrays (tilted at 30 degrees); (Middle panel) Close top views of single NMP; (Lower panel) Tilted top views of single NMP (tilted at 30 degrees). For all samples, the scan speed was kept constant at 100 μm/s.

Figure 3

Scanning electron micrographs (SEM) of nanostructured mushroom-like pillars (NMPs), fabricated by LDW via TPP of IP-Dip photopolymer. The original spiral step (h) was multiplied with coefficients indicated above each figure: (Upper panel) Tilted top views of NMPs arrays (tilted at 30 degrees); (Middle panel) Close top views of single NMP; (Lower panel) Tilted top views of single NMP (tilted at 30 degrees). Writing parameters: 12.5 mW laser power and 100 μm/s scanning speed. The spiral step, h, represents the height difference of the laser focus after completing a 360-degree trip while writing the structure.