Nanotopographical Coatings Induce an Early Phenotype-Specific Response of Primary Material-Resident M1 and M2 Macrophages

Abstract

Implants elicit an immunological response after implantation that results in the worst case in a complete implant rejection. This biomaterial-induced inflammation is modulated by macrophages and can be influenced by nanotopographical surface structures such as titania nanotubes or fractal titanium nitride (TiN) surfaces. However, their specific impact on a distinct macrophage phenotype has not been identified. By using two different levels of nanostructures and smooth samples as controls, the influence of tubular TiO₂ and fractal TiN nanostructures on primary human macrophages with M1 or M2-phenotype was investigated. Therefore, nanotopographical coatings were either, directly generated by physical vapor deposition (PVD) or by electrochemical anodization of titanium PVD coatings. The cellular response of macrophages was quantitatively assessed to demonstrate a difference in biocompatibility of nanotubes in respect to human M1 and M2-macrophages. Depending on the tube diameter of the nanotubular surfaces, low cell numbers and impaired cellular activity, was detected for M2-macrophages, whereas the impact of nanotubes on M1-polarized macrophages was negligible. Importantly, we could confirm this phenotypic response on the fractal TiN surfaces. The results indicate that the investigated topographies specifically impact the macrophage M2-subtype that modulates the formation of the fibrotic capsule and the long-term response to an implant.

Keywords: combination of physical vapor deposition and electrochemical etching; defined humanized test system; inflammatory response; nanotopographical surfaces.

Figure 1

Scheme of the study design. Study design for the testing of two different types of nanostructured surfaces, with glass and suitable smooth coatings as control surfaces.

Figure 2

Surface characterization. (a) Scanning electron microscopy (SEM) picture displaying the smooth, mirror-like Ti film, deposited on a glass substrate at a substrate temperature of 200 °C; (b,c) TiO₂ NTs generated by 2h-anodization of the Ti coatings at 10 and 20 V in an H₃PO₄/HF electrolyte. AFM scans of 5 µm × 5 µm areas in 3D view; (d) untreated Ti film deposited on a pre-heated glass substrate; (e,f) TiO₂ NT arrays generated by anodization with voltages of 10 and 20 V in H₃PO₄/HF electrolyte—Figure 2 (a–f) reprinted with permission from [36]; (g) Smooth TiN coating produced with the substrates heated to 200 °C; (h,i) Nanorough TiN coatings deposited on unheated substrates with deposition times of 15 (TiN 15), or 60 (TiN 60) minutes, respectively. Small inserts depict higher magnification images with areas of 1 µm × 1 µm; (j) Atomic force microscopy scans of 5 µm × 5 µm areas in three-dimensional (3D) view of smooth TiN coating and (k,l) nanorough TiN coatings.

Figure 3

X-ray diffraction (XRD) analysis. X-ray diffraction patterns of an untreated Ti layer, smooth and nanorough TiN coatings on glass slides, as well as the TiO₂ nanotube arrays after annealing at 450 °C for 3 h. The different phases are marked as follows: A: anatase, R: rutile, Ti: Ti coating, TiN: TiN coating. Ti and TiN lattice planes are marked with the corresponding Miller indices.

Figure 4

Morphological analysis of biomaterial-resident macrophages after 48 h of culture. Following 48 h of culture, cell morphology of M1 macrophages was visualized by (a) confocal microscopy and (b) SEM imaging. Images are exemplarily shown for one of five donors. The small inserts in the SEM-images show a higher magnification image with an area of 2 µm × 2 µm. (c) Confocal images of M2 macrophages and the (d) respective SEM analysis facilitate the comparison of both subtypes. Glass, Ti coating, and Ti nanotubes (NT) produced by anodization of the physical vapor deposition (PVD) coatings at 10 and 20 V in an H₃PO₄/HF electrolyte for 2 h. The cell numbers counted for each material and phenotype in relation to the glass control samples are inserted in the respective images.

Figure 5

Statistical analysis of cell morphology. Probability density of (a,b) cell area and (c,d) aspect ratio for the M1- and M2-polarized macrophages on glass, Ti and the Ti nanotubes (NT) surfaces obtained from fluorescence microscopy images. Data were merged for all five donors. Images of cells typically found at a size indicated by the dashed lines are inserted into the graphs to facilitate conceivability. The darkened parts in graph (a,b) indicate the range of cell size where no cells could be detected.

Figure 6

Morphological analysis of biomaterial-resident macrophages. Following 48 h of culture, cell morphology of the two phenotypes (a–c) M1 and (d–f) M2 was visualized by SEM imaging. Images are exemplarily shown for one of three donors. The small inserts in the SEM-images show a higher magnification image with an area of 2 µm × 2 µm with focus on the filopodia of the cells.

Figure 7

Statistical analysis of cell morphology. Probability density of (a,b) cell area and (c,d) aspect ratio for the M1- and M2-polarized macrophages on smooth and nanorough TiN surfaces obtained from fluorescence microscopy images. Data were merged for all three donors. Images of cells typically found at a size indicated by the dashed lines are inserted into the graphs to facilitate conceivability. The darkened parts in graph A and B indicate the range of cell size where no cells could be detected.