Cell biological responses of osteoblasts on anodized nanotubular surface of a titanium-zirconium alloy Subhash Sista, 1,2 Alireza Nouri, 2 Yuncang Li, 2 Cuie Wen, 3 Peter D. Hodgson, 2 Gopal Pande 1 1 CSIR – Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India 2 Institute for Frontier Materials, Deakin University, Pigdons Road, Waurn Ponds, Geelong, Victoria 3217, Australia 3 Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia Received 31 October 2012; revised 29 January 2013; accepted 31 January 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34638 Abstract: Anodization of titanium and its alloys, under con- trolled conditions, generates a nanotubular architecture on the material surface. The biological consequences of such changes are poorly understood, and therefore, we have ana- lyzed the cellular and molecular responses of osteoblasts that were plated on nanotubular anodized surface of a titanium-zirconium (TiZr) alloy. Upon comparing these results with those obtained on acid etched and polished surfaces of the same alloy, we observed a significant increase in adhesion and proliferation of cells on anodized surfaces as compared to acid etched or polished surface. The expression of genes related to cell adhesion was high only on anodized TiZr, but that of genes related to osteo- blast differentiation and osteocalcin protein and extracellular matrix secretion were higher on both anodized and acid etched surfaces. Examination of surface morphology, topog- raphy, roughness, surface area and wettability using scan- ning electron microscopy, atomic force microscopy, and contact angle goniometry, showed that higher surface area, hydrophilicity, and nanoscale roughness of nanotubular TiZr surfaces, which were generated specifically by the anodiza- tion process, could strongly enhance the adhesion and pro- liferation of osteoblasts. We propose that biological properties of known bioactive titanium alloys can be further enhanced by generating nanotubular surfaces using anodiza- tion. V C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2013. Key Words: anodization, titanium alloy, osteoblast, gene expression, nanotubes How to cite this article: Sista S, Nouri A, Li Y, Wen C, Hodgson PD, Pande G. 2013. Cell biological responses of osteoblasts on anodized nanotubular surface of a titanium-zirconium alloy. J Biomed Mater Res Part A 2013:00A:000–000. INTRODUCTION Osseointegration of implant materials is necessary for their successful application in bone tissue engineering. 1 A direct and strong bonding of the material with the bone is essen- tial for the bone healing process. The surface properties of implant materials play an important role in determining the bonding between the implant and the newly formed bone tissue and they are critical in controlling the cell–material interactions that occur at the surface upon implantation. 1,2 Thus, physicochemical properties of the material surface have a significant role in determining the cellular and molecular response of osteoblasts and other cells that come in their contact after the implant is placed in the body or during the implant testing process in vitro. 3–6 Titanium (Ti) and some of its alloys, due to their supe- rior mechanical properties and good biocompatibility, have been used in the fields of orthopedic biomaterials and bone tissue engineering for a long time. 7–9 Recently, with the advent of newer alloys and derivatives of Ti, it has been shown that some Ti based biomaterials are more cytocom- patible than others for bone tissue engineering. 10,11 In this comparative analysis, titanium-zirconium (TiZr) alloys have emerged as strong candidates and suitable as bone implant materials, 12–15 but this analysis has also suggested that fur- ther improvements in the surface properties of these alloys, including TiZr, are necessary to enhance the formation of new bone tissue in the peri-implant region and to accelerate the process of osseointegration. 16–18 Thus, many strategies for surface modification and ren- dering the alloy surfaces more bioactive are being devel- oped. 19–24 These approaches also give us the opportunity to better analyze the molecular responses of cells towards ma- terial surfaces and to do basic studies on how specific sur- face modifications of osteogenic biomaterials affect signaling pathways that regulate adhesion, proliferation and differen- tiation of osteoblasts. 25–30 Since surface properties of biomaterials are interdepend- ent, it is difficult to pinpoint the role of one particular sur- face property that is responsible for a specific response of osteoblast cells. The comparative analysis of in vitro cellular Correspondence to: G. Pande; e-mail: gpande@ccmb.res.in or C. Wen; e-mail: cwen@swin.edu.au Contract grant sponsor: Department of Science and Technology and Department of Biotechnology, Government of India; contract grant number: GAP 220 and GAP 311 V C 2013 WILEY PERIODICALS, INC. 1