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 its 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.

三维（3D）打印技术是制造钛（Ti）及其合金用于骨组织工程的一种有前途的方法。然而，钛材料的生物惰性性质限制了其直接与骨组织结合的能力。本文旨在通过在表面上构建分层的微观/纳米形貌来增强3D打印的Ti-6Al-4V植入物的生物活性和成骨作用。通过电子束熔化（EBM）技术制备了Ti-6Al-4V植入物。在本研究中，提出了一种将超声酸蚀刻与阳极氧化相结合的方法，以对EBM Ti-6Al-4V植入物进行表面改性。酸蚀刻步骤是去除植入物表面上任何存在的残留粉末，并在EBM微粗糙表面上构建微坑和-沟槽。通过阳极氧化将直径为40-50nm的纳米管阵列叠加在微结构化的衬底上。结果体外实验表明，酸蚀和阳极氧化（AN）后，Ti-6Al-4V上的分层微/纳米结构表面通过增强表面亲水性和表面活性来促进成骨细胞（MC3T3-E1）的增殖和成骨分化。与抛光的钛表面（P）相比具有更高的生物活性。显微CT和组织学分析用于评估体内成骨特性增强。手术后8周的结果显示，AN植入物的骨体积与总体积之比（BV / TV）为43.4％，是未经任何后处理的印刷植入物（AM）的1.5倍。与AM植入物和酸蚀刻（AE）EBM植入物相比，还可以从micro-CT重建的3D模型中检测到骨骼与植入物接触面积的显着增加。总之，在EBM自然表面上生成的分层微观/纳米形貌显示出生物活性和成骨特性的改善，这有望加速3D打印整形外科和牙科植入物在临床中的应用。

重要声明

传统的钛植入物具有生物惰性的性质，这限制了其直接与骨组织结合的能力。植入几年后植入物的失败将给患者带来巨大的痛苦。在这项工作中，开发了一种用于3D打印植入物的表面改性方法，以构建分层的微/纳米结构。通过体外和体内实验，我们证明了这种分层的微/纳米结构与未经处理的表面或抛光的表面相比，对成骨细胞的增殖和分化具有更好的促进作用，并且还能够促进新的骨形成，表明改善3D打印钛植入物生物学特性的有效策略。