Accelerated Formation of Organized Hydroxyapatite Microstructures by Amelogenin Assemblies

Objectives: It has been suggested that cooperative assembly and interactions between forming crystals and assembling protein/peptide is pivotal to enamel biomineralization. To understand the formation mechanisms of enamel microstructures mediated by matrix protein, amelogenin, we use a newly developed constant composition (CC) method under precisely defined thermodynamic driving forces and near-physiological conditions (óHAP=15.1, ionic strength=0.15 M, 37 °C, and pH 7.40). Self-organized microstructures, compositionally and morphologically similar to natural enamel, are achieved without the use of extreme hydrothermal conditions. Methods: Amel proteins were added to supersaturated reaction solutions to achieve concentrations ranging from 0.5 to 5.0 ug mL^-1. Nucleation of hydroxyapatite (HAP) was monitored using a pH electrode coupled with a single-junction Ag/AgCl reference electrode, and nucleated HAP crystallites were characterized by HRTEM. Results: The delay (induction) time for nucleation in pure supersaturated solutions was 778 ± 30 min (n=3). However in the presence of 0.5, 1.25 or 5.0 ug mL^-1. Amel, the induction times decreased to 705 ± 25 (n=3), 398 ± 20 (n=3), and 255 ± 20 (n=3) min, respectively, suggesting that Amel kinetically promotes HAP nucleation. Elongated ribbon-like apatite crystals were grown in the presence of 5.0 ug mL^-1; whereas in the absence of Amel, HAP crystallites were randomly aggregated. Intermediate nanorod structures were successfully captured following the formation of the critical nuclei at the earliest nucleation stages. These intermediate structures further assemble by a self-epitaxial nucleation mechanism to form the final hierarchically organized microstructures. Conclusion: Under cooperative kinetic control, self-assembly of nucleated HAP nanoparticle-Amel nanosphere mixtures plays an explicit role in the formation of organized microstructures from two dissimilar organic and inorganic nanophases during a slow crystallization process. Supported by NIDCR (DE03223).