Micro- and Nanopatterned Topographical Cues for Regulating Macrophage Cell Shape and Phenotype

Abstract

Controlling the interactions between macrophages and biomaterials is critical for modulating the response to implants. While it has long been thought that biomaterial surface chemistry regulates the immune response, recent studies have suggested that material geometry may in fact dominate. Our previous work demonstrated that elongation of macrophages regulates their polarization toward a pro-healing phenotype. In this work, we elucidate how surface topology might be leveraged to alter macrophage cell morphology and polarization state. Using a deep etch technique, we fabricated titanium surfaces containing micro- and nanopatterned grooves, which have been previously shown to promote cell elongation. Morphology, phenotypic markers, and cytokine secretion of murine bone marrow derived macrophages on different groove widths were analyzed. The results suggest that micro- and nanopatterned grooves influenced macrophage elongation, which peaked on substrates with 400-500 nm wide grooves. Surface grooves did not affect inflammatory activation but drove macrophages toward an anti-inflammatory, pro-healing phenotype. While secretion of TNF-alpha remained low in macrophages across all conditions, macrophages secreted significantly higher levels of anti-inflammatory cytokine, IL-10, on intermediate groove widths compared to cells on other Ti surfaces. Our findings highlight the potential of using surface topography to regulate macrophage function, and thus control the wound healing and tissue repair response to biomaterials.

Keywords: biocompatibility; groove; macrophage polarization; titanium; topography.

Figure 1. Deep-etched titanium surfaces with varied groove dimensions

(A) Schematic of the Ti substrate with micro- and nano-patterned surface topographies used in this study. D denotes groove width; P denotes pitch, which is twice the groove width. Groove depth is approximately 0.8 μm to 1.3 μm for all widths. (B) Scanning electron microscopy micrographs of BMDM after 36 h culture on Ti substrate with 200 nm, 450 nm, 5 μm, and 50 μm grating patterns and non-patterned control.

Figure 2. Grooved surfaces regulate macrophage elongation

(A) Fluorescence micrographs of BMDM after 24 h culture on bulk Ti substrate with 200 nm, 450 nm, 5 μm, and 50 μm grating patterns and non-patterned control. Cells were stained using CellTracker Green CMFDA dye for monitoring cell shape. Scale bar = 50 μm. (B) Quantification of BMDM elongation factor on Ti surfaces of varied groove widths. Mean ± SEM (*p < 0.05; ordinary one-way ANOVA with Dunnett's post test, in comparison to blank Ti substrate, n = 3). (C) Quantification of BMDM cell area on Ti surfaces of varied groove widths. Mean ± SEM, n = 3.

Figure 3. Adhesive and cytoskeletal structures of macrophages on grooved surfaces

Fluorescent images of vinculin (red; immunostaining) and actin filaments (green; phalloidin staining) in BMDMs cultured on various groove sizes.

Figure 4. Expression of macrophage polarization markers on grooved surfaces

(A) Representative Arg1 immunofluorescent images of BMDMs on control and grooved substrates. Scale bar = 50 μm (B) Quantification of Arg1 expression corresponding to the groove width. Mean ± SEM (*p < 0.05; ordinary one-way ANOVA with uncorrected Fisher's LDS, blank and grooved surface topography substrates compared to 150 nm grooved substrate, n = 3). (C) Representative iNOS immunofluorescent images of BMDMs on control and grooved substrates. Scale bar = 50 μm. (D) Quantification of iNOS expression corresponding to the groove width. Mean ± SEM, n = 3. No stim. denotes unstimulated, LPS/IFN-γ was stimulated with 10 ng/mL LPS and 10 ng/mL IFN-γ, LPS/IL4/IL13 was stimulated with 20 ng/mL IL4 and 20 ng/mL IL13, Blank denotes unstimulated cells on a flat Ti substrate.

Figure 5. Cytokine secretion from macrophages on grooved surfaces

(A) Quantified IL10 secretion from macrophages cultured on different groove widths. Mean ± SEM (*p < 0.05; ordinary one-way ANOVA with Dunnett's post test, in comparison to 200 nm grooved substrate, n = 3). (B) Quantified TNF-α secretion from macrophages cultured on different groove widths. Mean ± SEM, n = 3. No stim. denotes unstimulated, LPS/IFN-γ was stimulated with 0.5 ng/mL LPS and 10 ng/mL IFN-γ, LPS/IL4/IL13 was stimulated with 0.5 ng/mL LPS, 20 ng/mL IL4 and 20 ng/mL IL13, Blank denotes unstimulated cells on a flat Ti substrate.