Amelogenin Peptide-Chitosan Hydrogel for Biomimetic Enamel Regrowth

Abstract

We designed synthetic peptides that have demonstrated an effective remineralization potential to restore incipient enamel decay. In order to develop a clinically viable approach we incorporated the amelogenin-derived peptides P26 and P32 into chitosan hydrogel and examined their efficacy in the remineralization of enamel. Peptides in chitosan exhibited increased stability in vitro as compared to peptides in solution at room temperature and at 37°C. Tooth models for enamel erosion (sections) and white spot lesions (blocks) were subject to periods of demineralization. Treatment groups were subjected to remineralization in artificial saliva in the presence of P26 and P32 in solution and in chitosan hydrogel (P26-CS and P32-CS). Quantitative light-induced fluorescence (QLF) was employed to analyze mineral density following demineralization and remineralization across all the treatment groups. Scanning electron microscopy and nanoindentation were used to characterize the surface structure and mechanical strength of regrown enamel. Control enamel sections treated in artificial saliva demonstrated randomly distributed, tiny, needle-shaped crystals with a low packing density and porosities displaying mineralization defects. In samples treated with P26-CS or P32-CS a denser coating of organized hydroxyapatite (HAP) crystals was formed covering the entire surfaces of demineralized enamel window. The hardness and modulus of enamel surfaces were increased after treatment with P26-CS and P32-CS with no significant difference in the mechanical properties between the two peptide hydrogels. Analysis of mineral density by QLF showed that in enamel sections P26 peptide alone or P26-CS significantly enhanced the remineralization. In enamel blocks P26 in solution had a better efficacy than P26-CS.

Keywords: QLF; bioactive peptide; caries; enamel; hydroxyapatite; remineralization.

FIGURE 1 ∣

SEM images of demineralized enamel surface showing clear outlines of enamel prisms/rods with remnants of inter-rods (A). HAP crystals grown on demineralized enamel after 3 days of incubation at pH 7.0, 37 °C in artificial saliva (control) (B), with P32 (C), P32-CS (D), P26 (E), P26-CS (F) (scale = 1 um).

FIGURE 2 ∣

Nanoindentation data showing the modulus (A) and hardness (B) for healthy enamel, demineralized enamel, and samples treated in control (0.2% NaF), P26-CS (0.2 mg/ml) and P32-CS (0.2 mg/ml) hydrogel for 7 days in artificial saliva. The hydrogel applications were made daily. Demin: demineralization (2 h). The error bars represent standard deviation (n = 5–8 per group). Tukey’s multiple comparisons test was applied to identify differences in the hardness and elastic modulus between demineralized and repaired enamel. * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001.

FIGURE 3 ∣

Representative white-light images of the visual comparisons made between the three experimental groups tested. Demineralized samples (A, C, E) and remineralized samples (B, D, F). The images clearly show a minimal visual decrease in the gray areas in the control remineralized in artificial saliva only (B), whereas there is visual decrease in the gray patches (mineral gain) of the enamel sections post remineralization with P26-CS (D) and P32-CS (F).

FIGURE 4 ∣

Evaluation of Remineralized Enamel Mineral Density and stability of amelogenin peptides. Comparative quantitative analysis of ΔΔF_remin between the three experimental groups before and after remineralization: control, P26-CS and P26. In enamel sections (A) there was no significant difference between the P26 solution and P26-CS treatment group (p > 0.05). In enamel blocks (B) there was a highly significant difference between P26 solution and control group (p < 0.007). Percentage of peptide remaining after 1, 7, and 28 days in P26 and P26-CS solutions (n = 3 per group) incubated at room temperature (C) and 37°C (D). While P26 and P26-CS were comparably stable after 1 day, P26-CS is remarkably more stable after 7 and 28 days at RT and 37°C, as indicated by a greater % area. * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001.