Investigating the Cooperative Interaction of Amelotin and Amelogenin for Mineralization

Objectives: Dental enamel is the hardest, most mineralized tissue in the human body. It is highly organized and consists of different layers. Unlike bulk enamel, the final surface layer is composed of aprismatic, dense crystals and forms the critical protective interface against external factors such as acid, bacteria and masticatory forces. However, the molecular mechanisms underlying the development of this compact surface enamel is not yet understood. Amelotin (AMTN) is a relatively recently discovered enamel matrix protein that was shown to promote mineralization in vitro, and is coincidently expressed during surface layer formation. Conversely, the major enamel protein, amelogenin (AMEL) has been suggested to act as a scaffold for mineralization upon self-assembling into supramolecular structures. The functional relationship between AMTN and AMEL has not yet been established. Therefore, this study aims first to describe AMTN self-assembly over time and then to determine the effect of self-assembled AMTN and AMEL, alone and in combination, on calcium phosphate mineralization.
Methods: Human recombinant AMTN and AMEL were self-assembled in calcium phosphate solutions for 25 days before being co-incubated for 20 min. Structures of AMTN, AMEL, and AMTN-AMEL were analyzed using atomic force microscopy (AFM) and transmission electron microscopy (TEM).
Results: AMTN self-assembled into ribbon-like nanostructures in calcium and phosphate solutions. Mineral deposits formed on top of these ribbons 21 days after assembly. Co-incubation of AMTN and AMEL led to the formation of needle-like mineral, resembling hydroxyapatite crystals, on AMEL structures. No mineralization was observed on AMEL alone.
Conclusions: Our results show that self-assembled AMTN can induce directional crystal growth on AMEL supramolecular structures, suggesting a similar mechanism may occur in vivo during surface enamel formation. Understanding the mechanisms of enamel formation can potentially lead to applications in enamel regeneration.