Biomaterials 25 (2004) 2731–2739 Promotion of fibroblast activity by coating with hydrophobins in the b-sheet end state M.I. Janssen a,c , M.B.M. van Leeuwen b , T.G. van Kooten b , J. de Vries b , L. Dijkhuizen a , H.A.B. W . osten d, * a Groningen Biotechnology and Biomolecular Sciences Institute, Kerklaan 30, 9751 NN Haren, The Netherlands b Department of Biomedical Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands c BioMaDe, Nijenborgh 4, 9747 AG Groningen, The Netherlands d Microbiology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Received 19 June 2003; accepted 17 September 2003 Abstract Hydrophobins such as SC3 and SC4 of Schizophyllum commune self-assemble into an amphipathic film at hydrophilic/ hydrophobic interfaces. These proteins can thus change the nature of surfaces, which makes them attractive candidates to improve physio– and physico–chemical properties of implant surfaces. At a hydrophobic solid, assembly of the hydrophobin is arrested in an intermediate state, called the a-helical state. The conversion to the stable b-sheet end state can be induced by treating the solid at elevated temperatures in the presence of detergent. We here show that SC3 and SC4 in the a-helical state homogeneously cover Teflon sheets when coating was performed at 20  C. However, when the protein was adsorbed at 80  C aggregates were shown to bind tightly to the adsorbed hydrophobin film. The transition to the b-sheet state created pores of about 50 nm in the SC3 and SC4 coatings when coating was performed at 20  C. Cell growth and morphology on SC4 coatings was better than on SC3. In case of both hydrophobins, fibroblast growth and morphology was not influenced by the coating temperature or the conformation of the protein. However, in contrast to the a-helical state, the b-sheet state of both SC3 and SC4 hardly, if at all, affected mitochondrial activity. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydrophobin; Protein coating; Biocompatibility; Fibroblast growth; Surface modification 1. Introduction By modifying the surface properties of a biomaterial, its interaction with cells can be improved. Surface properties can be modified by plasma modification [1–4] or by coating with e.g. block copolymers [5], sugars [6] or proteins [6–11]. Proteins that seem very attractive to improve the physio– and physical–chemical properties of surfaces are the class I hydrophobins [12,13]. Class I hydrophobins are secreted fungal proteins of about 100 amino acids that are characterized by eight conserved cysteine residues and a typical spacing of hydrophilic and hydrophobic regions [13]. Class I hydrophobins fulfil a broad spectrum of functions in fungal growth and development [14,15]. For instance, they coat reproductive structures (including edible mushrooms), thus making them hydrophobic, and mediate attach- ment of fungi to hydrophobic surfaces. Class I hydro- phobins self-assemble at any hydrophilic/hydrophobic interface into a highly insoluble 10nm thin amphipathic film [16–18]. The film attaches strongly to hydrophobic solids and may also do so on hydrophilic surfaces [19]. The wettability of the hydrophilic side of the film is highly variable and depends on the nature of the hydrophobin. In contrast, the wettability of the hydro- phobic side is extremely low in all cases [19]. By self- assembly, hydrophobins change the wettability of a surface. Hydrophobic surfaces become hydrophilic and vice versa [16–18, 20–22]. The strong attachment of the hydrophobin film to a solid and the fact that wettability can be varied by using different hydrophobins make these proteins of interest for coating biomaterials. The SC3 and SC4 hydrophobins of Schizophyllum commune have been purified [16,22] and characterized. ARTICLE IN PRESS *Corresponding author. Tel.: +31-30-2533448; fax: +31-30- 2513655. E-mail address: h.a.b.wosten@bio.uu.nl (H.A.B. W. osten). 0142-9612/$-see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.09.060