Improved osseointegration of 3D printed Ti-6Al-4V implant with a hierarchical micro/nano surface topography: An in vitro and in vivo study

Abstract

Three-dimensional (3D) printing technology is serving as a promising approach of fabricating titanium (Ti) and its alloys used for bone tissue engineering. However, the biological inertness nature of Ti material limits its capability to bind directly with the bone tissue. This paper aims to enhance the bioactivity and osteogenesis of 3D printed Ti-6Al-4V implants by constructing a hierarchical micro/nano-topography on the surface. Ti-6Al-4V implants were prepared by the electron beam melting (EBM) technique. A method combining ultrasonic acid etching with anodic oxidation is proposed for surface modification of EBM Ti-6Al-4V implants in this study. The acid etching step was to remove any existent residual powders on the implant's surface and construct micro-pits and -grooves on the EBM microrough surface. Nanotube arrays with a diameter of 40-50 nm were superimposed on the micro-structured substrate via anodic oxidation. The results of in vitro experiments showed that the hierarchical micro/nano-structured surface on Ti-6Al-4V after acid etching and anodic oxidation (AN) promoted the proliferation and osteogenic differentiation of pre-osteoblast cells (MC3T3-E1) via enhancing the surface hydrophilicity and bioactivity compared with the polished Ti surface (P). Micro-CT and histological analysis were used to assess the in vivo osteogenic properties enhancement. The results 8 weeks after the surgery showed the ratio of bone volume to total volume (BV/TV) of AN implant was 43.4%, which represented 1.5 times that of as-printed implants (AM) without any post-treatment. Considerable increment of bone-to-implant contact area was also detected from the micro-CT reconstructed 3D models in comparison with AM implants and acid etched (AE) EBM implants. In conclusion, the hierarchical micro/nano topography generated on the EBM native surface showed an improvement of bioactivity and osteogenic properties, which is expected to accelerate the application of 3D printed orthopedic and dental implants in clinics. STATEMENT OF SIGNIFICANCE: Traditional titanium implants have the nature of biological inertness, which limits their capability to bind directly with the bone tissue. The failure of implants after couple of years of implantation will cause huge pain to the patients. In this work, a surface modification method for 3D printed implants was developed to construct a hierarchical micro/nano-structure. Through the in vitro and in vivo experiments, we proved that this hierarchical micro/nano-structure induced a better promotion effect on osteoblast proliferation and differentiation comparing with untreated surface or polished surface, and was also capable of bolstering the new bone formation, suggesting a potent strategy to improve the biological properties of 3D printed titanium implants. The work is expected to accelerate the application of 3D printed orthopedic and dental implants in clinics.

Keywords: Anodic oxidation; Micro/nano-structure; Osseointegration; Surface modification; Titanium implants; Ultrasonic acid etching.