Evaluation of single-cell force spectroscopy and fluorescence microscopy to determine cell interactions with femtosecond-laser microstructured titanium surfaces Pooyan Aliuos, 1 Elena Fadeeva, 2 Muhammad Badar, 3 Andreas Winkel, 4 Peter P. Mueller, 3 Athanasia Warnecke, 1 Boris Chichkov, 2 Thomas Lenarz, 1 Uta Reich, 1† * Guenter Reuter 1 * 1 Department of Otorhinolaryngology, Head and Neck Surgery, Carl-Neuberg-Str. 1, Hannover Medical School, 30625 Hannover, Germany 2 Laser Zentrum, Hannover e.V., Hannover, Germany 3 Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany 4 Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Hannover, Germany Received 25 May 2012; accepted 24 July 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34401 Abstract: One goal in biomaterials research is to limit the for- mation of connective tissue around the implant. Antiwetting surfaces are known to reduce ability of cells to adhere. Such surfaces can be achieved by special surface structures (lotus effect). Aim of the study was to investigate the feasibility for creating antiwetting surface structures on titanium and to characterize their effect on initial cell adhesion and prolifera- tion. Titanium microstructures were generated using femto- second- (fs-) laser pulses. Murine fibroblasts served as a model for connective tissue cells. Quantitative investigation of initial cell adhesion was performed using atomic force mi- croscopy. Fluorescence microscopy was used for the charac- terization of cell-adhesion pattern, cell morphology, and proliferation. Water contact angle (WCA) measurements evinced antiwetting properties of laser-structured surfaces. However, the WCA was decreased in serum-containing me- dium. Initial cell adhesion to microstructured titanium was significantly promoted when compared with polished tita- nium. Microstructures did not influence cell proliferation on titanium surfaces. However, on titanium microstructures, cells showed a flattened morphology, and the cell orientation was biased according to the surface topography. In conclu- sion, antiwetting properties of surfaces were absent in the presence of serum and did not hinder adhesion and prolifera- tion of NIH 3T3 fibroblasts. V C 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2012. Key Words: cell-substrate adhesion, atomic force microscopy, femtosecond-laser, titanium microstructure, connective tissue growth How to cite this article: Aliuos P, Fadeeva E, Badar M, Winkel A, Mueller PP, Warnecke A, Chichkov B, Lenarz T, Reich U, Reuter G. 2012. Evaluation of single-cell force spectroscopy and fluorescence microscopy to determine cell interactions with femtosecond- laser microstructured titanium surfaces. J Biomed Mater Res Part A 2012:00A:000–000. INTRODUCTION Biomaterials are routinely used for organ replacement 1 and also for diagnostic 2 and pharmaceutical 3 purposes. However, despite the technological progress, there are still challenges to overcome in order to ensure an optimized integration into the human organism. Foreign body response (FBR), as the natural reaction of the organism to foreign objects, is based on nonspecific protein adsorption and activation of immune and inflammatory cells. 4 Because of an initial inter- action between blood and the material, formation of a fibrin scaffold occurs. Macrophages and fibroblasts migrate toward the injury site and build a fibrous tissue capsule around permanent implants in order to shield them from the non- specific immune system response. 5,6 This is associated with implant failure. For example, the growth of connective tissue in dental implants precludes the osseointegration and proper mechanical fixation of implants. 7 In neural implants for direct stimulation of nerve fibers and neuronal cells, the encapsulation of the electrodes with connective tissue nega- tively affects the electrode-tissue impedance. 8,9 Adhesion of cells to their environment (cell-substrate adhesion) plays a crucial role in the regulation of diverse cellular processes such as cell division, migration, assembly of tissues, inflammation, wound healing, 10,11 and conse- quently in the formation of connective tissue. Cell adhesion is a highly complex and dynamic process. 12,13 After cells *These authors contributed equally to this work. † Present address: Department of Otorhinolaringology, Charite-Universit€ atsmedizin, Berlin, Germany. Correspondence to: P. Aliuos; e-mail: aliuos.pooyan@mh-hannover.de Contract grant sponsor: Deutsche Forschungsgemeinschaft; contract grant number: SFB599 (D2 and D1) Contract grant sponsor: DAAD and HEC, Pakistan V C 2012 WILEY PERIODICALS, INC. 1