Introducing chemical functionality in Fmoc-peptide gels for cell culture.
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-F(2)) 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 NH(2), COOH or OH, enhances compatibility with different cell types. A series of hydrogel compositions consisting of combinations of Fmoc-F(2) and n-protected Fmoc amino acids, lysine (K, with side chain R=(CH(2))(4)NH(2)), glutamic acid (D, with side chain R=CH(2)COOH), and serine (S, with side chain R=CH(2)OH) 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 beta-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-F(2)/D) to 21.2 KPa (Fmoc-F(2)). All gels supported the viability of bovine chondrocytes as assessed by a live-dead staining assay. Fmoc-F(2)/S and Fmoc-F(2)/D hydrogels in addition supported viability for human dermal fibroblasts (HDF) while Fmoc-F(2)/S hydrogel was the only gel type that supported viability for all three cell types tested. Fmoc-F(2)/S was therefore investigated further by studying cell proliferation, cytoskeletal organization and histological analysis in 2D culture. In addition, the Fmoc-F(2)/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.
Jayawarna
Vineetha
V
School of Materials & Manchester Interdisciplinary Biocentre, Materials Science Centre, The University of Manchester, Grosvenor Street, Manchester M1 7HS, UK.
Richardson
Stephen M
SM
Hirst
Andrew R
AR
Hodson
Nigel W
NW
Saiani
Alberto
A
Gough
Julie E
JE
Ulijn
Rein V
RV
D016475:3T3 Cells; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002417:Cattle; D002448:Cell Adhesion; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020405:Dermis / Q000166:cytology; D055119:Elastic Modulus; D004548:Elasticity; D005347:Fibroblasts; D005449:Fluorenes / Q000737:chemistry; D018014:Gene Transfer Techniques; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D045022:Lactate Dehydrogenases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D049329:Nanostructures / Q000737:chemistry / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D055672:Static Electricity; D054457:Tissue Scaffolds; D014783:Viscosity
C496789:9-fluorenylmethoxycarbonyl; D001672:Biocompatible Materials; D005449:Fluorenes; D020100:Hydrogels; D010455:Peptides; D018698:Glutamic Acid; D012694:Serine; D004247:DNA; D045022:Lactate Dehydrogenases; D008239:Lysine