Histological and three-dimensional evaluation of osseointegration to nanostructured calcium phosphate-coated implants Ryo Jimbo a,b,⇑ , Paulo G. Coelho c , Stefan Vandeweghe a , Humberto Osvaldo Schwartz-Filho a,d , Mariko Hayashi a , Daisuke Ono e , Martin Andersson f , Ann Wennerberg a,b a Surface Biology Group, Department of Prosthodontics, Faculty of Odontology, Malmö University, Sweden b Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Sweden c Department of Biomaterials and Biomimetics, New York University, NY, USA d Division of Periodontology, Department of Oral Diagnosis and Surgery, School of Dentistry, UNESP, São Paulo State University, Araraquara, SP, Brazil e Department of Applied Prosthodontics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan f Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg, Sweden article info Article history: Received 29 May 2011 Received in revised form 8 July 2011 Accepted 13 July 2011 Available online 21 July 2011 Keywords: Nano structures Micro-CT Surface chemistry Bone implant interactions abstract Nanostructures on implant surfaces have been shown to enhance osseointegration; however, commonly used evaluation techniques are probably not sufficiently sensitive to fully determine the effects of this process. This study aimed to observe the osseointegration properties of nanostructured calcium phosphate (CaP)-coated implants, by using a combination of three-dimensional imaging and conventional histology. Titanium implants were coated with stable CaP nanoparticles using an immersion technique followed by heat treatment. Uncoated implants were used as the control. After topographical and chemical characterizations, implants were inserted into the rabbit femur. After 2 and 4 weeks, the samples were retrieved for micro-computed tomography and histomorphometric evaluation. Scanning electron microscopy evaluation indicated that the implant surface was modified at the nanoscale by CaP to obtain surface textured with rod-shaped structures. Relative to the control, the bone-to-implant contact for the CaP-coated implant was significantly higher at 4 weeks after the implant surgery. Further, corresponding 3-D images showed active bone formation surrounding the implant. 3-D quantification and 2-D histology demonstrated statistical correlation; moreover, 3-D quantification indicated a statistical decrease in bone density in the non-coated control implant group between 2 and 4 weeks after the surgery. The application of 3-D evaluation further clarified the temporal characteristics and biological reaction of implants in bone. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Recent developments in the surface properties of osseointegrated implants have significantly enhanced both the quality and rate of osseointegration. Current advances enable modifications of implants at the macro, micro, as well as nano levels, and several commercially available implants possess modifications on all these levels [1]. Recent studies have reported that the application of nanostructures to implant surfaces seems to further increase bone apposition [2–4]. Possible explanations for this phenomenon are widely sought, since it has been suggested that it may be due to the cumulative effect of various factors. From a topographical perspective, both in vitro and animal studies have suggested that the enhanced surface area seems to create an optimal basis for bone responses. Lamers et al. [5] reported that osteoblasts sensitively reacted to nanogrooves (width: 50 nm, diameter: 17 nm), resulting in osteogenic gene expression, and suggested that nanogrooves on implant surfaces enhanced the bone response. In another study, Puckett et al. [6] reported reduced bacterial attachment on nano-scale rough surfaces than on other surfaces; furthermore, the same nano-scale surface showed higher affinity to fibronectin, which is essential for the initial osseointegration process [7]. Meirelles et al. [8] conducted an animal study with implants containing nanostructures and polished implants deliberately lacking them, and found that the former had higher bone-to-implant contact than the latter implant surfaces. These reports suggest that cells, particularly osteoblasts, respond to topographical alterations at the nanometer scale. In addition to topography, the physical and chemical properties of the deposited nano-size materials could enhance the osseointegration cascade. Studies have reported the beneficial 1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.07.017 ⇑ Corresponding author at: Department of Prosthodontics, Faculty of Odontology, Malmö University, 205 06 Malmö, Sweden. Tel.: +46 40 665 8679; fax: +46 40 665 8503. E-mail address: ryo.jimbo@mah.se (R. Jimbo). Acta Biomaterialia 7 (2011) 4229–4234 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat