Enamel Remineralization By A Novel Biomimetic Mineralization Method

Objective To biomimetic the process of enamel mineralization, we designed a novel amelogenin-like oligopeptide as organic matrix templates for simulating the control of amelogenin in the nucleation and growth of HA crystals and synthesized Calcium-and phosphate-loaded temperature sensitive liposomes supplying calcium and phosphate ion which simulate the secreting calcium and phosphorus ions of ameloblast. This induce regeneration of the enamel microstructure and promote enamel remineralization.Methods The preparation of temperature-sensitive liposomes. The calcium-and phosphate-loaded temperature-sensitive liposomes were prepared using the interdigitation-fusion method. It composed of1,2-bis(palmitoyl)-sn-glycero-3-phosphor-choline which the lipid chain melting transition temperature(Tm) at24℃and1,2-bis(myristoyl)-sn-glycero-3-phosphocholine which the Tm at41℃. By adjusting the ratio of DPPC and DMPC(9:1), we got the liposome which Tm at37℃. Above37℃, the liposomes melt transition, increase permeability and release encapsulated Ca2+and PO43-. Hydrated with CaCl2or Na2HPO4solution, we gained Calcium-loaded temperature-sensitive liposomes.The preparation of oligropeptide. We designed a novel polyanion amelogenin-inspired oligopeptide (Gln-Pro-Ala)4-Thr-Lys-Arg-Glu-Glu-Val-Asp) by reference to the domain of amelogenin self-assembly and its hydrophilic tail with charged. The oligopeptide was synthesized by standard solid-phase, purified and characterized by high performance liquid chromatography and mass spectrometry. Peptide self-assembly was induced with CaCl2solution, tested its size by particle size analyzer and characterized with scanning electron microscope (SEM) and transmission electron microscope (TEM).The biomimetic minerialization of the enamel. Firstly, the surface of dental films about1.5mm were etched by37%phosphoric acid in lmin. Secondly, we mixed the liposomes and oligopeptide solution at the ratio (oligopeptide accounted for20%of the total mass of the suspension). Finally, we immersed the dental films in the suspension at37℃for2weeks. The new depositions on samples were characterized by FTIR, XRD, SEM and TEM.Results We prepared the temperature-sensitive liposomes by the interdigitation-fusion method (IF) method, which average diameters of161.3nm and the encapsulation efficiency of Ca2+and PO43-loaded vesicles was ranged from60-85%. It were stable at room temperature, but entrapped Ca2+and PO43-rapidly diffused out of the liposomes at37℃. The amelogenin-like oligopeptide of (Gln-Pro-Ala)4-Thr-Lys-Arg-Glu-Glu-Val-A-sp was synthesized by synthesized by standard solid-phase peptide synthesis. Peptide molecule weight (MV) and purity were2061.26and99.46%accordingly, identified with mass spectrometry and high performance liquid chromatography respectively. The oligopeptide could self-assemble by Ca2+induction. The average size of oligopeptide which were self-assemble are1415nm by particle size analyzer. SEM and TEM showed that the oligopeptide could self-assemble to form some nanospheres. We applied the mixture of the liposomes and oligopeptide to the experiment of remineralization. It was found that the enamel surface only treated with liposomes suspension had a few amount of HA crystals deposition, and the precipitate distributed randomly. In contrast, the experimental groups treated with mixture solution of liposomes and polyanion peptide showed that the enamel-like crystals almost fully covered the etched enamel surface, and distributed evenly to make the surface smoothly. The FTIR and the XRD spectra showed that the deposition corresponded well to HA.Conclusions We could prepared calcium-and phosphate-loaded temperature-sensitive liposomes. When heated to body temperature, calcium and phosphate ions were released. It provided inorganic ions in biomimetic mineralization. The amelogenin-like oligopeptide of (Gln-Pro-Ala)4-Thr-Lys-Arg-Glu-Glu-Val-Asp was synthesized by standard solid-phase peptide synthesis. The oligopeptide could self-assemble by Ca2+induction to form some nanospheres. The Ca2+and PO43-loaded temperature-sensitive liposomes and amelogenin-like oligopeptide could induce the enamel microstructure regeneration in the specific mineral system.