Electrochemical growth behavior, surface properties, and enhanced in vivo bone response of TiO2 nanotubes on microstructured surfaces of blasted, screw-shaped titanium implants

Abstract

TiO(2) nanotubes are fabricated on TiO(2) grit-blasted, screw-shaped rough titanium (ASTM grade 4) implants (3.75 x 7 mm) using potentiostatic anodization at 20 V in 1 M H(3)PO(4) + 0.4 wt.% HF. The growth behavior and surface properties of the nanotubes are investigated as a function of the reaction time. The results show that vertically aligned nanotubes of approximately 700 nm in length, with highly ordered structures of approximately 40 nm spacing and approximately 15 nm wall thickness may be grown independent of reaction time. The geometrical properties of nanotubes increase with reaction time (mean pore size, pore size distribution [PSD], and porosity approximately 90 nm, approximately 40-127 nm and 45%, respectively for 30 minutes; approximately 107 nm, approximately 63-140 nm and 56% for one hour; approximately 108 nm, approximately 58-150 nm and 60% for three hours). It is found that the fluorinated chemistry of the nanotubes of F-TiO(2), TiOF(2), and F-Ti-O with F ion incorporation of approximately 5 at.%, and their amorphous structure is the same regardless of the reaction time, while the average roughness (Sa) gradually decreases and the developed surface area (Sdr) slightly increases with reaction time. The results of studies on animals show that, despite their low roughness values, after six weeks the fluorinated TiO(2) nanotube implants in rabbit femurs demonstrate significantly increased osseointegration strengths (41 vs 29 Ncm; P = 0.008) and new bone formation (57.5% vs 65.5%; P = 0.008) (n = 8), and reveal more frequently direct bone/cell contact at the bone-implant interface by high-resolution scanning electron microscope observations as compared with the blasted, moderately rough implants that have hitherto been widely used for clinically favorable performance. The results of the animal studies constitute significant evidence that the presence of the nanotubes and the resulting fluorinated surface chemistry determine the nature of the bone responses to the implants. The present in vivo results point to potential applications of the TiO(2) nanotubes in the field of bone implants and bone tissue engineering.

Keywords: electrochemical fabrication; fluorinated TiO2 nanotubes; in vivo bone response; osseointegrated titanium implant; surface properties.

Figure 1

Macroscopic images of A) the TiO₂ nanotube-fabricated TEST implant B) the blasted CONTROL implant. The thread pitch is 600 μm. C) Schematic of the experimental setup used for the electrochemical nanofabrication. D and E) One nanotube implant and one blasted implant were inserted in the femur condyle close to the knee joint of each rabbit.

Figure 2

Characteristic current vs time curve showing the effects of A) the potential sweep mode and B) the potentiostatic mode performed on the blasted, screw-shaped implants using 100–150 μm particles of TiO₂.

Figure 3

FE-SEM images of top view (first column), cross-section (second column), and bottom view (inset) of the nanotube implants formed in 1M H₃PO₄ + 0.4 wt.% HF at 20 V for A) 30 minutes, B) one hour, C) three hours (scale bar = 500 nm) and D) the blasted implants formed using 100–150 μm particles of TiO₂ (scale bar = 5 μm).

Figure 4

Variation of pore size, pore size distribution (PSD), and porosity with the reaction time used in nanotube formation of 30 minutes, one hour, and three hours.

Figure 5

High-resolution XPS spectra at A) the Ti 2p, B) O1s, and C) F 1s core-level energy region of the nanotube implants formed after A) 30 minutes, B) one hour, C) three hours, and d) the blasted implant. In order to extract the peak contributions at the F 1s energy region, spectral deconvolutions were performed using a numerical fitting procedure. Individual line shapes were simulated using a combination of the Lorentzian and Gaussian functions. Abbreviation: XPS, X-ray photoelectron spectroscopy.

Figure 6

A) XRD spectra of the nanotube implants formed after A) 30 minutes, B) one hour, C) three hours, and D) the blasted implants. (B and C) The bar charts show how average roughness (Sa) decreases gradually and developed surface area (Sdr) increases slightly with reaction time. Abbreviation: XRD, X-ray diffraction.

Figure 7

Three-dimensional images of TiO₂ nanotubes electrochemically fabricated on blasted, screw-shaped titanium implants for A) 30 minutes, B) one hour, and C) three hours obtained using interferometry with a measuring volume of 230 × 230 × 5 μm³.

Figure 8

LM and BSE-SEM qualitative and quantitative observations of osseoconductivity on undecalcified cut and ground sections with the implants in bone. Basic fuchsin stained sections (A–E) show the following: well-developed trabecular architecture surrounding the implant placed in a rabbit femur A), pronounced new (immature woven) bone formation under the periosteum (dark pink staining) B), blood cells filling the space between the implant bottom and the new bone detached in the removal torque test C), newly formed bone (dark blue) inside the threads of the blasted implant D), and fluorinated TiO₂ implants at the endosteal reagions E), clearly distinguished from the demarcation lines between dark and pale stained bone tissue. Toluidine-blue stained sections show variations in the nature of the direct bone contact between the blasted F) and fluorinated TiO₂ implants G) (Scale bar = 500 μm). High-resolution BSE-SEM observations (3000×) show that the direct bone contact defined by LM observations turns out to be “real” direct bone/cell contact with the TiO₂ nanotube implant surface more frequently than in the blasted implant (Scale bar = 5 μm). Abbreviations: LM, light microscopy; BSE-SEM, back scattered electron scanning electron microscopy.

Figure 9

The fluorinated TiO₂ nanotube implants in a rabbit femur for six weeks demonstrate superior bone responses over the blasted implants: A) new bone formation (57.5% vs 65.5%; P = 0.008) and (B) osseointegration strength (41 vs 29 Ncm; P = 0.008) (n = 8). Notably, every single nanotube implant showed a stronger osseointegration than the equivalent blasted implant paired in the same animal (B).