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Self-assembling Peptide Scaffolds Promote Enamel Remineralization

J. Kirkham^1,*, A. Firth^1,2, D. Vernals², N. Boden², C. Robinson¹, R.C. Shore¹, S.J. Brookes¹, and A. Aggeli^2,

¹ Department of Oral Biology, Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK; and
² Centre for Self Organising Molecular Systems, School of Chemistry, University of Leeds, UK

View larger version (25K): [in this window] [in a new window]   Figure 1. Schematic representation of a self-assembled fibrillar network and its relationship with individual self-assembled fibrils and peptide primary structure. (a) Fibrillar network of assembled peptide. (b) Each fibril is comprised of 4 ribbons. The thin lines on the ribbons represent individual oligopeptides, each in ß-strand conformation. (c) Dimeric antiparallel ß-sheet tape of P11-4, the self-assembling peptide used in the present study. The labels correspond to the side-chains of the peptide at the back only.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of P11-4 on remineralization and demineralization behavior of caries-like lesions in human enamel under simulated intra-oral conditions. (a) Effect of treatment with P11-4 on mineral loss/gain by caries-like lesions of human enamel following 5 days of cycling in an oscillating pH model (5 x 24-hour periods, each comprised of 3 x 20-minute acid challenges [pH 4.8], intervening time at neutral pH; solutions supersaturated with respect to hydroxyapatite at pH 7.4, undersaturated at pH 4.8). Histograms show net mineral gain by lesions treated with the self-assembling peptides after 5 days of cycling, compared with untreated controls, and mineral loss/gain during periods at low pH compared with intervening periods at neutral pH. Results show mean values of 8 separate experiments ± SD. (b) Effect of treatment with a self-assembling peptide on mineral loss/gain by caries-like lesions of human enamel during each 24-hour period of 5 days’ cycling in an oscillating pH model (5 x 24-hour periods, each comprised of 3 x 20-minute acid challenges [pH 4.8], intervening time at neutral pH; solutions supersaturated with respect to hydroxyapatite at pH 7.4, undersaturated at pH 4.8). Histograms show net mineral gain by lesions treated with the self-assembling peptides compared with untreated controls. Results show mean values of 8 separate experiments ± SD.

View larger version (156K): [in this window] [in a new window]   Figure 3. TEM of P11-4 gels and fibrils. TEM micrographs show: (a) section through an unstained, undiluted P11-4 gel following 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4). Electron-dense deposits, some apparently aligned, may be seen (arrows). Scale bar corresponds to 250 nm. (b) P11-4 fibrils from gels that were not incubated in "mineralizing solution", stained with uranyl acetate and prepared by dilution of the gels immediately before application to the TEM grid. Individual fibrils with clear helical twist can be seen. Scale bar corresponds to 100 nm. (c) Section through an undiluted P11-4 gel stained with uranyl acetate after 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4, supersaturated with respect to hydroxyapatite). Fibrils appear to be arranged in twisted bundles with a width of hundreds of nm to µm and lengths of several µm, reminiscent of collagen fibers. Electron-dense deposits associated with the bundles following in vitro mineralization may reflect sites where hydroxyapatite nucleation has taken place. Arrows show bundles of fibrils in cross-section (white arrow) and running longitudinally (black arrow). Scale bar corresponds to 1 µm.

View larger version (43K): [in this window] [in a new window]   Figure 4. De novo nucleation of hydroxyapatite by P11-4 in vitro. (a) Transmission electron micrographs (unstained) showing needle-like electron-dense deposits in the body of a self-assembled P11-4 gel following 7 days’ incubation at 35°C in "mineralizing" solution (22 mM NaHCO3, 3.75 mM CaCl2, 1.67 mM Na2HPO4, pH 7.4). (b) Electron diffraction patterns of needle-like deposits. Lattice planes 110 (A); 002 (B); 211 (C), camera focal length 400 mm; 100 (D); 200 (E); and 102 (F), camera focal length 575 mm, are indicated. (c) EDX spectrum of needle-like deposits.