Immune response of macrophages on super-hydrophilic TiO 2 nanotube arrays

Abstract

Macrophages are attracting increasing attention in promoting implant-mediated osteogenesis by modulating the microenvironment of the implant site. Biomaterial surface properties including topography and wettability regulate macrophage responses to influence tissue repair. The objective of our present study was to investigate the effects of hydrogenated titanium dioxide nanotube surfaces on the immune response of macrophages in vitro. Hydrogenated titanium dioxide nanotubes (TNTs) were synthesized on Ti surfaces by anodic oxidation and hydrogenation to form super-hydrophilic nanotubular surfaces. Macrophages were seeded directly onto three substrates (hydrogenated TNTs (H₂-TNTs), air-annealed TNTs, and commercially pure Ti substrates) and grown under standard or lipopolysaccharide-stimulated culture conditions. Cell proliferation and viability were evaluated by Cell Counting Kit-8 and live/dead staining at 24 and 48 h. Secretion and expression of pro- and anti-inflammatory cytokines were evaluated at 24 and 48 h to determine whether the surfaces elicited differential macrophage behaviors. Gene expression of the M1/M2 surface markers of macrophages was analyzed to assess the effect of the H₂-TNT surface on macrophage polarization. The results showed that hydrogenation of the TNT surface resulted in a super-hydrophilic substrate, which exhibited markedly improved wettability attributable to the formation of oxygen vacancies in the nanotubes. The H₂-TNT group induced a significantly lower macrophage proliferation rate and up-regulated anti-inflammatory cytokines (interleukin-10, bone morphogenetic protein-2, and transforming growth factor-β1) irrespective of lipopolysaccharide stimulation, while alleviating the secretion of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, nitric oxide, and macrophage chemotactic protein-1) induced by lipopolysaccharide. Moreover, the H₂-TNT surface elicited up-regulated gene expression of M2 surface markers and down-regulation of M1 surface markers. We concluded that the hydrogenated TNT surface modulated macrophage immune responses, which could be useful in accelerating inflammation resolution and facilitating tissue repair.