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Introducing chemical functionality in fmoc-peptide gels for cell culture

Jayawarna, V. and Richardson, S.M. and Hirst, A.R. and Hodson, N.W. and Saiani, A. and Gough, J.E. and Ulijn, R.V. (2009) Introducing chemical functionality in fmoc-peptide gels for cell culture. Acta Biomaterialia, 5 (3). pp. 934-943. ISSN 1742-7061

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Abstract

Aromatic short peptide derivatives, i.e. peptides modified with aromatic groups such as 9-fluorenylmethoxycarbonyl (Fmoc), can self-assemble into self-supporting hydrogels. These hydrogels have some similarities to extracellular matrices due to their high hydration, relative stiffness and nanofibrous architecture. We previously demonstrated that Fmoc-diphenylalanine (Fmoc-F2) provides a suitable matrix for two-dimensional (2D) or three-dimensional (3D) culture of primary bovine chondrocytes. In this paper we investigate whether the introduction of chemical functionality, such as NH2, COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F2 and n-protected Fmoc amino acids, lysine (K, with side chain R = (CH2)4NH2), glutamic acid (D, with side chain R = CH2COOH), and serine (S, with side chain R = CH2OH) were studied. All compositions produced fibrous scaffolds with fibre diameters in the range of 32-65 nm as assessed by cryo-scanning electron microscopy and atomic force microscopy. Fourier transform infrared spectroscopy analysis suggested that peptide segments adopt a predominantly antiparallel β-sheet conformation. Oscillatory rheology results show that all four hydrogels have mechanical profiles of soft viscoelastic materials with elastic moduli dependent on the chemical composition, ranging from 502 Pa (Fmoc-F2/D) to 21.2 KPa (Fmoc-F2). All gels supported the viability of bovine chondrocytes as assessed by a live-dead staining assay. Fmoc-F2/S and Fmoc-F2/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F2/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F2/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F2/S gel was shown to support retention of cell morphology in 3D culture of bovine chondrocytes. These results demonstrate that introduction of chemical functionality into Fmoc-peptide scaffolds may provide gels with tunable chemical and mechanical properties for in vitro cell culture.