Nano-Roughness-Mediated Macrophage Polarization for Desired Host Immune Response

Abstract

Macrophage polarization is a significant event in the host immune response, which can be modulated by modifying the surface of a biomaterial. Previous studies have demonstrated the modulation of macrophage polarization using different surface features; however, none of these studies reflect the effect of surface properties on unstimulated macrophage polarization for a prolonged period. To better understand the impact of surface features, in this work differentiated THP-1 cells are employed to control macrophage polarization on nano-rough surfaces for a duration of 7 days. Model nano-rough substrates are fabricated by immobilizing gold nanoparticles (AuNPs) of predetermined sizes (16, 38, 68 nm) on a 2-methyl-2-oxazoline thin film, followed by tailoring the outermost surface chemistry. All modified surfaces support high levels of cell adhesion and proliferation. Over time, the expression of pro-inflammatory cytokines decreases, whereas the expression of anti-inflammatory cytokines increases on all modified surfaces. Similarly, pro-inflammatory interleukin (IL)-1β gene expression is downregulated, and anti-inflammatory IL-10-gene expression is upregulated, regardless of the surface roughness. Analysis of cell morphology reveals that the predominant cell type on the modified surfaces exhibits M2 anti-inflammatory phenotype. Herein, how surface features can modulate macrophage responses over an extended period is highlighted, offering insights for the development of future biomaterial implants.

Keywords: foreign body reaction; macrophage polarization; macrophage response; nanotopography; plasma polymerization.

Figure 1

a) Surface characterization of the nanotopography‐ and chemistry‐modified surfaces. Transmission electron microscopy (TEM) images of the nanoparticles used for surface immobilization. b) Particle size distribution obtained from TEM images. c) Scanning electron microscopy (SEM) images showing the distribution of nanoparticles of different sizes on the model surfaces; scale bar: 1 μm. d) 3D atomic force microscopy (3D‐AFM) images of the surfaces after overcoating. e) The number of nanoparticles per unit area and root‐mean‐square (RMS) roughness of different surfaces calculated from AFM images. f) X‐ray photoelectron spectroscopy (XPS) spectra after overcoating. g) Advancing water contact angle.

Figure 2

a) Representative bright‐field optical microscopy images of adhered macrophages on the modified surfaces at D1, D4, and D7. The red circles represent large cells and multinucleated giant cells. Significance difference: p < 0.05 (*), <0.0001(****), scale bar of images (black color line) = 0.1 mm. b) Number of adhered macrophages on modified surfaces (plasma‐polymerized 2‐methyl‐2‐oxazoline [pOX], 16 pOX, 38 pOX, and 68 pOX) at D1, D4, and D7.

Figure 3

SEM images of macrophages on nanotopographic surfaces at different time points—D1, D4, and D7. The red circles represent cells’ shapes, including round cells without cytoplasm expansion, bipolar spindle cells, pancake‐like cells, round cells with small cytoplasm, and foreign body giant cells (FBGCs). Scale bar = 10 μm.

Figure 4

Cytokines secretion profile from macrophages in D1, D4, and D7. a) Pro‐inflammatory TNF‐α. b) Pro‐inflammatory IL‐1β. c) Anti‐inflammatory arginase. d) Anti‐inflammatory IL‐4. Statistical difference = asterisk represents the significance among days, * <0.05, ** <0.01, *** <0.001, and **** <0.0001, simple letters represent the significance among surfaces each day compared to pOX surface; a >0.0001, b >0.0001, c >0.0001 for TNF α, d <0.0001 for IL‐1β, and e <0.5 for arginase.

Figure 5

a,b) Gene expression of IL‐1β (a) and IL‐10 (b) on the modified surface at different time points (Day [D]1, D4, and D7); the asterisks represent the significance among days, * <0.05, ** <0.01, *** <0.001, and **** <0.0001.