Anodized 3D–printed titanium implants with dual micro- and nano-scale topography promote interaction with human osteoblasts and osteocyte-like cells Karan Gulati 1† , Matthew Prideaux 2† , Masakazu Kogawa 2 , Luis Lima-Marques 3 , Gerald J. Atkins 2‡ , David M. Findlay 2‡ and Dusan Losic 1 * ‡ 1 School of Chemical Engineering, University of Adelaide, SA 5005, Australia 2 Discipline of Orthopaedics & Trauma, University of Adelaide, SA 5005, Australia 3 The Institute for Photonics and Advanced Sensing, University of Adelaide, SA 5005, Australia Abstract The success of implantation of materials into bone is governed by effective osseointegration, requiring biocompatibility of the material and the attachment and differentiation of osteoblastic cells. To enhance cellular function in response to the implant surface, micro- and nano-scale topography have been suggested as essential. In this study, we present bone implants based on 3D–printed titanium alloy (Ti6Al4V), with a unique dual topography composed of micron-sized spherical particles and vertically aligned titania nanotubes. The implants were prepared by combination of 3D–printing and anodization processes, which are scalable, simple and cost-effective. The osseointegration properties of fabricated implants, examined using human osteoblasts, showed enhanced adhesion of osteoblasts compared with titanium materials commonly used as orthopaedic implants. Gene expression studies at early (day 7) and late (day 21) stages of culture were consistent with the Ti substrates inducing an osteoblast phenotype conducive to effective osseointegration. These implants with the unique combination of micro- and nano-scale topography are proposed as the new generation of multi-functional bone implants, suitable for addressing many orthopaedic challenges, including implant rejection, poor osseointegration, inflammation, drug delivery and bone healing. Copyright © 2016 John Wiley & Sons, Ltd. Received 8 January 2016; Revised 11 April 2016; Accepted 16 June 2016 Keywords 3D–printing; titanium; titania nanotubes; bone implants; osteoblast phenotype 1. Introduction Titanium (Ti) and its alloys have been used for many decades as bone implants, mainly due to their corrosion resistance and appropriate biomechanical properties (Popat et al., 2007a). Besides providing mechanical support and function, a bone implant must also serve as a substrate for various protein and cellular interactions that determine the extent of bone to implant bonding (osseointegration) and the rate of peri-implant bone healing. As a result, the implant surface, being the first site of contact with the surrounding tissue, plays an important role in determining the fate of the implant. The porosity and pore size of a biomaterial intended for bone implant applications are important determinants of its osteogenic properties in vitro and in vivo (Karageorgiou and Kaplan, 2005). The surface chemistry or energy of the implant material also influences the nature of the interac- tion with bone cells, in particular by the extent to which the material binds extracellular matrix (ECM) proteins, such as vitronectin and fibronectin, either present in the serum or synthesized by the bone cells themselves (Anselme, 2000). In addition, the surface topography of implant materials influences osteoblast attachment and influences subsequent osteogenesis, thought due in part to bone being a material with natural micro- and nano- scale topographical features. As a result, various surface modification strategies have been utilized to enhance the surface roughness of implant materials, including sand- blasting, acid-etching, plasma reaction and electrochemi- cal anodization (Losic et al., 2015). These approaches render the implant surface micro- to nano-rough, with electrochemical anodization offering good control over the structural characteristics. In fact, in vitro and in vivo investigations, together with mathematical modelling, have established that micrometre roughness, particularly hemispherical pits (1.5 μm deep and 4 μm wide), pro- vides the optimal surface features to enhance integration with the surrounding tissue (Bauer et al., 2013). Other studies conclude that implants having roughened surfaces with irregular morphologies promote high levels of cellular attachment at the bone–implant contact region (Bowers et al., 1992). A number of reports have shown that nano-scale rough- ness and topography further improves bone cell interac- tion compared with micro-scale roughness (Webster and *Correspondence to: Dusan Losic, School of Chemical Engineering, The University of Adelaide, SA 5005, Australia. E-mail: dusan.losic@adelaide.edu.au † K.G. and M.P. have equal first-author status. ‡ G.J.A., D.M.F. and D.L. have equal senior-author status. Copyright © 2016 John Wiley & Sons, Ltd. JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2016. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2239