Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions Wei-Bor Tsai a, * , Rita Pei-Yeh Chen b , Kuang-Ling Wei a , Yi-Ru Chen c , Tai-Yan Liao b , Hsuan-Liang Liu c , Juin-Yih Lai d a Department of Chemical Engineering, National Taiwan University, No. 1, Roosevelt Road, Section 4, Taipei 106, Taiwan b Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan c Graduate Institute of Biotechnology and Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan d R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Chungli, Taoyuan, Taiwan Received 12 January 2009; received in revised form 24 May 2009; accepted 26 May 2009 Available online 2 June 2009 Abstract Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly (acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorpo- ration of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: RGD; Polyelectrolyte multilayer; Osteoblasts; Calcium deposition; Cell adhesion 1. Introduction Cell attachment to biomaterials is an important step in many biomedical applications, such as implants, tissue engineering and cell-based sensors [1–3]. Cells in native tis- sues adhere to the surrounding extracellular matrix (ECM) via cell membrane receptors (e.g. integrins [4]) that specifi- cally bind to ECM adhesion proteins such as fibronectin, vitronectin or laminin. Since synthetic biomaterials lack the natural mechanisms that mediate cell attachment, there is a broad need to couple bioactive molecules to artificial substrates that render artificial materials biologically functional. Among the strategies to improve cellular affinity of syn- thetic surfaces, the most popular one is adsorption or con- jugation of ECM adhesion proteins onto biomaterial surfaces [5–7]. Alternatively, conjugation of peptides con- taining the cell-binding sequences of ECM adhesion pro- teins also supports cell adhesion to biomaterials [8,9]. The advantages of peptides include cost effectiveness and less vulnerability to denaturation in comparison with intact adhesion proteins. The most commonly applied cell-bind- ing peptide is the tri-amino acid sequence arginine–gly- cine–aspartic acid (Arg–Gly–Asp or RGD), which is found in many ECM adhesion proteins [2,8,10]. Several types of integrins bind fibronectin and vitronectin via the RGD domain so as to mediate cell adhesion. Besides the integrin-binding domains, many adhesion proteins contain heparin-binding domains that support cell 1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.05.034 * Corresponding author. Tel.: +886 2 3366 3996; fax: +886 2 2362 3040. E-mail address: weibortsai@ntu.edu.tw (W.-B. Tsai). Available online at www.sciencedirect.com Acta Biomaterialia 5 (2009) 3467–3477 www.elsevier.com/locate/actabiomat