Magnesium-doped Nanostructured Titanium Surface Modulates Macrophage-mediated Inflammatory Response for Ameliorative Osseointegration

Abstract

Background: Next generation of coating materials on the surface of implants is designed with a paradigm shift from an inert material to an osteoimmunomodulatory material. Regulating immune response to biomedical implants through influencing the polarization of macrophage has been proven to be an effective strategy.

Methods: Through anodization and hydrothermal treatment, magnesium ion incorporated TiO₂ nanotube array (MgN) coating was fabricated on the surface of titanium and it is hypothesized that it has osteoimmunomodulatory properties. To verify this assumption, systematic studies were carried out by in vitro and in vivo experiments.

Results: Mg ion release behavior results showed that MgN coating was successfully fabricated on the surface of titanium using anodization and hydrothermal technology. Scanning electron microscopy (SEM) images showed the morphology of the MgN coating on the titanium. The expression of inflammation-related genes (IL-6, IL-1β, TNF-α) was downregulated in MgN group compared with TiO₂ nanotube (NT) and blank Ti groups, but anti-inflammatory genes (IL-10 and IL-1ra) were remarkably upregulated in the MgN group. The in vitro and in vivo results demonstrated that MgN coating influenced macrophage polarization toward the M2 phenotype compared with NT and blank-Ti groups, which enhanced osteogenic differentiation of rat bone mesenchymal stem cells rBMSCs in conditioned media (CM) generated by macrophages.

Conclusion: MgN coating on the titanium endowed the surface with immune-regulatory features and exerted an advantageous effect on osteogenesis, thereby providing excellent strategies for the surface modification of biomedical implants.

Keywords: TiO2 nanotube; macrophage; magnesium ions; osteoimmunomodulation.

Figure 1

Surface characteristics. Notes: (A) the process of fabricating MgN coating on the titanium. (B) SEM images of (i, ii) NT and (iii, iv) MgN coating on the titanium. (C) Release kinetics of Mg ion from MgN sample at day 3, 5, 7, 14, 21, and 28. (D) Contact angle of SBF on the surface of blank-Ti, NT and MgN samples. *p <0.05. Abbreviations: MgN, magnesium ion incorporated TiO₂ nanotube arrays; NT, TiO₂ nanotube; SEM, scanning electron microscopy; SBF, simulated body fluid.

Figure 2

FACS results of RAW 264.7 cells cultured on different sample surfaces. Notes: The mean fluorescence intensity of M2 marker (CD206) was increased after stimulated by MgN group compared with that by NT and blank-Ti groups; however, the mean fluorescence intensity of M1 marker (CCR7) declined under the same treatment. Abbreviation: FACS, fluorescence activated cell sorting.

Figure 3

In vitro responses of RAW 264.7 cells cultured on different samples for four days. (A) The expression of inflammation-related genes TNF-α, IL-6, IL-1β, IL-10 and IL-1ra were detected by RT-PCR (blank-Ti group has been standardized as 1). (B) Western blotting analyses of VEGF and BMP2 respectively and their corresponding gray values. (C, D) ELISA determination of cytokines: (C) BMP2 (D) VEGF. Notes: **denotes highly significant difference between groups; *denotes the statistically significant difference between groups; (*p <0.05, **p <0.01). Abbreviations: TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-1ra, interleukin-1ra; IL-10, interleukin–10; VEGF, vascular endothelial growth factor; BMP2, bone morphogenetic protein 2; ELISA, enzyme-linked immunosorbent assay.

Figure 4

(A) Cell proliferation of rBMSCs cultured for one, three, five, and seven days in CM on various samples. (B) ALP activity of rBMSCs cultured for three and seven days in conditioned medium on various samples. (C) mRNA expression of osteogenic genes (ALP, RUNX2, OCN, COL1) of rBMSCs cultured on various samples in standard culture medium vs in CM after osteogenic induction for seven days. Notes: *Statistically significant difference between groups (p <0.05); **highly significant difference between groups (p <0.01); #no significant difference between groups (p >0.05). Abbreviations: rBMSCs, rat bone mesenchymal stem cells; RUNX2, runt-related transcription factor 2; COL1, type I collagen; OCN, osteocalcin; CM, conditioned medium.

Figure 5

Morphology of rBMSCs on different surfaces of Ti, NT and MgN in CM by fluorescence microscopy. Notes: The rBMSCs attached well and kept a normal shape on the three surfaces. Compared with NT and blank Ti groups, rBMSCs stretching on MgN surfaces were facilitated to different degrees. Abbreviations: rBMSCs, rat bone mesenchymal stem cells; CM, conditioned medium.

Figure 6

Detection of M2 marker (CD206) in tissue sections from rat bone by immunolabeling and confocal immunofluorescence imaging. Notes: The area of CD206-positive sites (M2; red) was the smallest in blank Ti and NT groups and the largest in the MgN group. Abbreviation: CD206, the surface markers M2 of macrophages.

Figure 7

Detection of M1 marker (CCR7) in tissue sections from rat bone by immunolabeling and confocal immunofluorescence imaging. Notes: The area of CCR7-positive sites (M1; red) was the smallest in MgN group and the largest in NT and Ti group. Abbreviation: CCR7, the surface markers M1 of macrophages.

Figure 8

H&E staining and Masson’s trichrome staining evaluation of the effects of MgN, NT and blank-Ti on bone regeneration. Notes: Four weeks after implantation, cubic and short pre-osteoblasts were lined surrounding the trabecular bone and MgN implants. On the same day, newly generated small vessels were scattered in trabecular bone. Angiogenesis and osteogenesis were intricately modulated in the formation of bone tissues. The MgN group showed robust new formation of bone tissues, which delimited the defected area with the implantation of MgN implant. The defected area filled by the blank Ti and NT groups showed less bone area than that of MgN group, indicating that MgN could enhance the formation of bone tissues compared with the NT and blank-Ti groups. Abbreviation: H&E staining, hematoxylin and eosin staining.