The regulatory effect of TiO2 nanotubes loaded with graphene oxide on macrophage polarization in an inflammatory environment

Abstract

Background: Excessive inflammation is a major cause of implant failure. The surface morphology, hydrophilicity, and loading of biomaterials are major properties modulating anti-inflammatory macrophage activation. This paper investigates the regulatory effects of modifying the surface of Titanium dioxide nanotubes (TNTs) with graphene oxide (GO) on the polarization of mouse monocyte macrophages (RAW264.7).

Methods: TNT was produced by the anodic oxidation of titanium. GO was subsequently electrodeposited on the TNT to obtain a TNT-GO composite. The samples were characterised through scanning electron microscopy (SEM), Raman spectroscopy, and X-ray diffraction. RAW264.7 cells were separately seeded onto the surface of three groups of samples: pure Ti, TNT, and TNT-GO. Under the condition of lipopolysaccharide stimulation, the influence of the sample surfaces on the gene expression profiles was investigated through RNA sequence analysis. In addition, cell spreading was observed through SEM, cell adhesion and proliferation were analysed using the CCK8 assay, and the expression of inflammation-related factors was investigated by ELISA and cellular immunofluorescence staining. The production of reactive oxygen species (ROS) in the RAW264.7 cells on the surface of the three groups was detected via immunofluorescence staining.

Results: The CCK8 results indicated that the adhesion and proliferation of the RAW264.7 cells were reduced on the TNT and TNT-GO surfaces. ELISA results revealed significant differences in the pro-inflammatory factors tumour necrosis factor-α and interleukin-6 secretion among the three groups at 24 h (p < 0.05). The secretion of pro-inflammatory factors significantly reduced and the expression of anti-inflammatory factor IL-10 increased on the TNT and TNT-GO surfaces. The RNA sequencing, ELISA, and cell immunofluorescence staining test results suggested that the inflammatory response of M1 polarization was reduced and the M2 polarization of macrophages was induced on the TNT-GO surface, which may be attributed to the reduction in ROS production.

Conclusions: Under lipopolysaccharide stimulation, the inflammatory response of the RAW264.7 cells was reduced and the M2 polarization of macrophages was promoted on the TNT-GO surface, which may be caused by the reduced ROS production. Consequently, the designed TNT-GO material is promising for implants owing to its excellent inflammation regulation ability.

Keywords: Biomaterials; Graphene oxide; Immune regulation; Macrophage polarization; Nanotubes; Reactive oxygen species.

Fig. 1

Surface characterisation of the Ti, TNT, and TNT–GO samples. A Photographs, B SEM images (magnification: 50,000×), and C Raman spectra of the samples

Fig. 2

Surface analysis of the Ti, TNT, and TNT–GO samples. A Reconstructed AFM topographical images of the samples, B water contact angles, C surface roughness, D water contact angle, and E XRD patterns. *p < 0.05

Fig. 3

Results of RNA-Seq analysis of the RAW 264.7 cells on the Ti, TNT, and TNT–GO samples under LPS stimulation. A Volcano map showing DEGs in pairwise comparisons. The upregulated, downregulated, and unchanged genes are shown as red, blue, and grey dots, respectively; B gene ontology enrichment; and C KEGG enrichment of Ti, TNT, and TNT–GO

Fig. 4

A SEM images of the RAW 264.7 cells on the different surfaces under LPS stimulation (magnification: 2000×). B Live/dead viability of the RAW 264.7 cells at 24 and 48 h under LPS stimulation (magnification 100×). C Cell adhesion and proliferation of the RAW 264.7 cells on the samples after 4, 24, and 48 h under LPS stimulation. *p < 0.05

Fig. 5

Pro- and anti-inflammatory cytokine levels under LPS stimulation at 24 and 48 h. A TNF-α, B IL-6, and C IL-10. *p < 0.05

Fig. 6

Immunofluorescence staining of the macrophages under LPS stimulation. A iNOS was labelled green, and nuclei were stained with DAPI. B ARG was labelled green, and nuclei were stained with DAPI. ‘Merge’ represents the merged images of iNOS or ARG and nuclei

Fig. 7

Immunofluorescence staining of the macrophages under LPS stimulation. A CD80 was labelled green, and nuclei were stained with DAPI. B CD163 was labelled green, and nuclei were stained with DAPI. ‘Merge’ represents the merged images of CD80 or CD163 and nuclei

Fig. 8

Results of quantitative analysis of fluorescent staining. A iNOS, B ARG, C CD80, and D CD163. *p < 0.05

Fig. 9

ROS production under LPS stimulation at 24 and 48 h

Fig. 10

Results of quantitative analysis of ROS production under LPS stimulation at 24 and 48 h. *p < 0.05