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Ricardo Gouveia, Valeria Castelletto, Ian Hamley, Che Connon, Stem Cells and Nanomaterials Group; Peptide Amphiphiles as Versatile Substrates for Oriented Cell Adhesion and Proliferation of Human Cornea Stromal Keratocytes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4556.
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© ARVO (1962-2015); The Authors (2016-present)
To develop novel bioactive surfaces able to support adhesion and proliferation of human corneal stroma keratocytes (HCSK) in vitro whilst emulating the cell’s in vivo phenotype. This includes maintaining proper cell morphology, the expression of specific markers such as keratocan, lumican, and decorin, as well as the aligned deposition of collagen type-I. To this purpose, we developed peptide amphiphile (PA) molecules that self-assemble into bioactive, stable nanostructures. PAs composed by a hexadecyl lipid chain attached to a peptide headgroup comprising the integrin-binding motifs RGD or RGDS, or the negatively charged ETTES sequence were designed and tested for their nanostructure, biocompatibility, and cell adhesion properties.
The bulk properties of RGD, RGDS, ETTES, and of binary PA systems consisting of RGD(S) mixed with diluent ETTES were studied by transmission electron microscopy, small-angle X-ray scattering, and X-ray diffraction. In addition, fluorescence spectroscopy was used to determine the critical concentration for PA self-assembly (c.a.c.) in water. PA solutions at 1×10-2-10-4 M and at various RGD(S):ETTES molar ratios were dried and used as coatings to enhance HCSK adhesion, viability, and proliferation. Furthermore, the effect of these PA substrates on the expression of HCSK markers was evaluated by QPCR.
Above their c.a.c. (>0.01% w/v), PAs formed well defined tapes, with bilayer structures and β-sheet features. When used as coating substrates, PAs containing the RGD(S) motifs specifically promoted integrin-dependent adhesion and proliferation of HCSKs without significantly altering the expression patterns of analysed keratocyte markers. However, no adhesion was observed with ETTES coating alone. Optimal adhesion and maximal cell proliferation was achieved with 1.25×10-3 M RGDS:ETTES at 13:87 (mol/mol) ratio. This binary system enhanced adhesion 1.4-fold relatively to substrates composed of only the RGD or RGDS molecules, suggesting that spacing between RGD(S) motifs promotes cell adhesion, whilst epitope crowding impairs it, possibly due to a charge effect.
Self-assembling nanostructures formed by co-assembly of RGD(S)-displaying PAs may constitute a versatile tool for corneal tissue engineering through modulation of HCSK adhesion and proliferation.
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