Cell growth as a sheet on three-dimensional sharp-tip nanostructures Chang-Hwan Choi, 1 Sepideh Heydarkhan-Hagvall, 2,3 Benjamin M. Wu, 2 James C. Y. Dunn, 2,3 Ramin E. Beygui, 3 * Chang-Jin ‘‘CJ’’ Kim 1 1 Mechanical and Aerospace Engineering Department, University of California, Los Angeles, California 90095 2 Department of Bioengineering, University of California, Los Angeles, California 90095 3 Department of Surgery, University of California, Los Angeles, California 90095 Received 7 January 2008; revised 5 March 2008; accepted 26 March 2008 Published online 3 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32101 Abstract: Cells in vivo encounter with and react to the extracellular matrix materials on a nanometer scale. Recent advances in nanofabrication technologies allowing the pre- cise control of a nanostructure’s pattern, periodicity, shape, and height have enabled a systematic study of cell interac- tions with three-dimensional nanotopographies. In this report, we examined the behavior of human foreskin fibro- blasts on well-ordered dense arrays (post and grate pat- terns with a 230-nm pitch) of sharp-tip nanostructures with varying three-dimensionalities (from 50 to 600 nm in structural height) over time—until a cell sheet was formed. Although cells started out smaller and proliferated slower on tall nanostructures (both posts and grates) than on smooth surfaces, they became confluent to form a sheet in 3 weeks. On grate patterns, significant cell elongation in alignment with the underlying pattern was observed and maintained over time. On tall nanostructures, cells grew while raised on sharp tips, resulting in a weak total adher- ence to the solid surface. A sheet of cells was easily peeled off from such surfaces, suggesting that nanoscale topogra- phies can be used as the basis for cell-sheet tissue engi- neering. Ó 2008 Wiley Periodicals, Inc. J Biomed Mater Res 89A: 804–817, 2009 Key words: nanotopography; cell morphology; cell prolif- eration; cell attachment/detachment; cell sheet INTRODUCTION Within the extracellular matrix, cells interact with three-dimensional (3D) projections and depressions that vary in composition, size, and periodicity on a nanometer scale. 1–3 The matrix nanotopography is important for the formation of proper adhesions and the activation of desired intracellular pathways, affecting cell behaviors in several ways such as mor- phology, cytoskeletal arrangement, migration, prolif- eration, surface antigen display, and gene expres- sion. Thus, a systematic understanding of the com- plex effects of the 3D nanotopography on cell behaviors is necessary. One of the practical aims of such an understanding is to design novel biomateri- als for tissue engineering or implantable medical devices. Although the effects of surface topographies on the cell behaviors had been studied with various micro- and nanostructured surfaces, 4–6 the inability to independently control the dimension and period of the structures in the nanoscale range has pre- cluded a systematic study. For a systematic study of the nanotopographical 3D effects on cell behaviors, well-defined nanostructures with good regularity and controllability of their pattern, size, and shape over a relatively large sample area are necessary. A recent achievement in nanofabrication has made it possible to fabricate well-ordered, dense-array nanostructures (nanoperiodic post and grate struc- tures) over a large sample area (several cm 2 ) with in- dependent controllability for structural size (height up to 1 lm) and shape (sidewall profile and tip sharpness). 7 The well-regulated nanostructure surfa- ces have provided a unique opportunity to elucidate the 3D effect of the surface nanotopography on cell behaviors. 8 Previously in Ref. 8, we reported on the cell interactions of human foreskin fibroblasts with *Present address: Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA. Correspondence to: C.-H. Choi, Department of Mechanical Engineering, Stevens Institute of Technology, Hoboken, NJ 07030, USA; e-mail: cchoi@stevens.edu Contract grant sponsor: National Science Foundation (NSF) Nanoscale Interdisciplinary Research Teams (NIRT); contract grant number: 0103562 Contract grant sponsor: Fubon Foundation Contract grant sponsor: American Heart Association Ó 2008 Wiley Periodicals, Inc.