Biomimetic organic–inorganic nanocomposite coatings for titanium implants. In vitro and in vivo biological testing R. Schade, 1 M. Dutour Sikiric ´, 2 S. Lamolle, 3 H. J. Ronold, 3 S. P. Lyngstadass, 3 K. Liefeith, 1 F. Cuisinier, 4 H. Fu ¨ redi-Milhofer 5 1 Department of Biomaterials, Institute for Bioprocessing and Analytical Measurement Techniques (iba), Heilbad Heiligenstadt, Germany 2 Department of Physical Chemistry, Laboratory for Organic Molecules Synthesis and Selfassembly Processes, Rudjer Bos ˇ kovic ´ Institute, Zagreb, Croatia 3 Department of Biomaterials, Faculty of Dentistry, University of Oslo, Oslo, Norway 4 BioNano, EA 4203, UFR Odontologie, Universite’ Montpellier I, Montpellier Cedex 5, France 5 Casali Institute of Applied Chemistry, Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel Received 23 October 2009; revised 4 February 2010; accepted 16 March 2010 Published online 19 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32888 Abstract: Recently described organic–inorganic nanocompo- site coatings of the chemical composition: (PLL/PGA) 10 - CaP[(PLL/PGA) 5 CaP] 4 (coating A) and (PLL/PGA) 10 CaP[(PLL/ PGA) 5 CaP] 4 (PLL/PGA) 5 (coating B), applied to chemically etched titanium plates, have been tested by extensive cell culture tests and in vivo biological experiments, with uncoated titanium plates serving as controls. Before testing, coated samples were stored for extended periods of time (from 2 weeks to 8 months) under dry, sterile conditions. Cells of the cell-lines MC3T3-E1 and/or SAOS-2 were used for the following cell culture tests: initial adhesion (4 h) and proliferation (up to 21 days), cell activity (XTT test), morphol- ogy, synthesis of collagen type I and alkaline phosphatase activity (all incubation up to 21 days). In addition, coating B was tested against uncoated control in a validated in vivo pull-out model in rabbit tibia. The results of both in vitro and in vivo experiments show excellent biological properties of chemically etched titanium which are even surpassed by surfaces covered with coating B. Thus, after 8 weeks of heal- ing the implants coated with B were significantly better attached to the cortical bone of rabbit thibiae than uncoated titanium controls with more than twice the force needed to detach coated implants. However, coating A (top crystal layer) had an adverse effect on both cell proliferation and activity, which is explained by morphological observations, showing inhibited spreading of the cells on its rough surfa- ces. The results also show the remarkable stability of the coatings when shelved under dry and sterile condi- tions. V C 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A: 691–700, 2010. Key Words: biomimetic composite coatings, calcium deficient apatite, polyelectrolyte multilayers, cell culture testing, in vivo biological testing INTRODUCTION Many of the materials used for replacement and/or repair of bones and teeth, such as certain types of metals, glass ceramics, and polymers, meet biomechanical requirements but poorly integrate with the surrounding bone and, there- fore, benefit from surface modifications. For this purpose, surface coatings consisting of calcium phosphates have been prepared by different methods, including biomimetic coating methods. 1–4 Recently, novel biomimetic organic–inorganic nanocomposite coatings, consisting of polyelectrolyte multi- layers and different calcium phosphates, have been pre- pared and characterized. 5–7 The method of preparation of the coatings closely mimics some forms of biomineralization, that is, calcium phosphate crystals are grown in situ within an organic matrix. It involves a three-step procedure: (1) depositing an organic matrix in the form of polyelectrolyte multilayers (PL MLs), (2) coad- sorbing amorphous calcium phosphate (ACP) as seed mate- rial, and (3) immersing the material into a metastable calcify- ing solution (MCS) to sustain crystallization induced by the ACP particles. Such coatings have numerous advantages, such as satisfactory mechanical stability, flexibility with regard to the substrate, and a simple, energy saving, and environmen- tally friendly building procedure. Preliminary in vitro biologi- cal testing 6,7 showed that coatings topped by a polyelectrolyte multilayer rather than a crystalline layer satisfactorily pro- mote cell adhesion and proliferation. Here, we report the results of further in vitro and in vivo biological testing, dem- onstrating excellent biological properties and remarkable long term stability (shelf life) of our coatings. Correspondence to: M. Dutour Sikiric ´ ; e-mail: sikiric@irb.hr Contract grant sponsor: The German-Israeli Foundation for Scientific Research and Development, G.I.F. (cell culture studies); contract grant number: I-907-39.3/2006 Contract grant sponsor: Commission of the European Union (animal studies, SIMI project); contract grant number: GRDI-2000-26823 Contract grant sponsor: Croatian Ministry of Science, Education and Sports; contract grant number: 098-0982915-2949 V C 2010 WILEY PERIODICALS, INC. 691