Biomimetic reconstruction of tooth enamel is a significant topic of study in material science and dentistry as a novel approach for prevention, restoration, and treatment of defective enamel. We developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchoring to the natural enamel substrate through a cluster growth process. A dense interface between the newly-grown layer and natural enamel was formed and the enamel-like layer had improved hardness and elastic modulus compared to etched enamel. We anticipate that chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving co-assembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.

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