Reduced adhesion of macrophages on anodized titanium with select nanotube surface features

Abstract

One of the important prerequisites for a successful orthopedic implant apart from being osteoconductive is the elicitation of a favorable immune response that does not lead to the rejection of the implant by the host tissue. Anodization is one of the simplest surface modification processes used to create nanotextured and nanotubular features on metal oxides which has been shown to improve bone formation. Anodization of titanium (Ti) leads to the formation of TiO(2) nanotubes on the surface, and the presence of these nanotubes mimics the natural nanoscale features of bone, which in turn contributes to improved bone cell attachment, migration, and proliferation. However, inflammatory cell responses on anodized Ti remains to be tested. It is hypothesized that surface roughness and surface feature size on anodized Ti can be carefully manipulated to control immune cell (specifically, macrophages) responses. Here, when Ti samples were anodized at 10 V in the presence of 1% hydrofluoric acid (HF) for 1 minute, nanotextured (nonnanotube) surfaces were created. When anodization of Ti samples was carried out with 1% HF for 10 minutes at 15 V, nanotubes with 40-50 nm diameters were formed, whereas at 20 V with 1% HF for 10 minutes, nanotubes with 60-70 nm diameters were formed. In this study, a reduced density of macrophages was observed after 24 hours of culture on nanotextured and nanotubular Ti samples which were anodized at 10, 15, and 20 V, compared with conventional unmodified Ti samples. This in vitro study thus demonstrated a reduced density of macrophages on anodized Ti, thereby providing further evidence of the greater efficacy of anodized Ti for orthopedic applications.

Keywords: TiO2 nanotube; anodization; titanium implants.

Figure 1

Scanning electron microscopy images. (A) Unanodized Ti sample showing a nearly smooth surface at the nanometer level. (B) Ti sample anodized in 1% HF for 1 minute at 10 V showing a nanotextured non-nanotubular surface; the black arrows show the ridge-like nanotextures interspersed between the tiny holes. (C) Ti sample anodized with 1% HF for 10 minutes at 15 V showing nanotubes of 40–50 nm diameters. (D) Ti sample anodized with 1% HF for 10 minutes at 20 V showing nanotubes of 60–70 nm diameters. (E) Enlarged portion of nanotubes of 40–50 nm diameter. (F) Enlarged portion shows nanotubes 60–70 nm diameter (scale bar = 200 nm). Abbreviations: HF, hydrofluoric acid; Ti, titanium.

Figure 2

Fluorescent microscopy images. (A) Adherent macrophages on unanodized Ti; round blue-green shape encircled by a red line shows a well spread macrophage. (B) Nanotextured, non-nanotubular Ti. (C) Nanotubular Ti with 40–50 nm diameters, after 24 hours of culture. (D) Nanotubular Ti with 60–70 nm diameters, after 24 hours of culture, showing macrophages (encircled in red lines) with little spreading. Abbreviation: Ti, titanium.

Figure 3

Macrophage density on conventional and anodized Ti samples. Macrophage density on conventional unmodified Ti, nanotextured non-nanotubular Ti and nanotubular Ti (tubes with diameters of 40–50 nm and 60–70 nm, respectively), n = 6, data are mean ± standard error of the mean. Notes: **P < 0.0001, *P < 0.0002 compared with conventional unanodized Ti. Abbreviation: Ti, titanium.