Unlike other mineralized tissues, mature dental enamel is primarily (> 95% by weight) composed of apatitic crystals and has a unique hierarchical structure. Due to its high mineral content and organized structure, enamel has exceptional functional properties and is the hardest substance in the human body. Enamel formation (amelogenesis) is the result of highly orchestrated extracellular processes that regulate the nucleation, growth, and organization of forming mineral crystals. However, major aspects of the mechanism of enamel formation are not well-understood, although substantial evidence suggests that protein-protein and protein-mineral interactions play crucial roles in this process. The purpose of this review is a critical evaluation of the present state of knowledge regarding the potential role of the assembly of enamel matrix proteins in the regulation of crystal growth and the structural organization of the resulting enamel tissue. This review primarily focuses on the structure and function of amelogenin, the predominant enamel matrix protein. This review also provides a brief description of novel in vitro approaches that have used synthetic macromolecules (i.e., surfactants and polymers) to regulate the formation of hierarchical inorganic (composite) structures in a fashion analogous to that believed to take place in biological systems, such as enamel. Accordingly, this review illustrates the potential for developing bio-inspired approaches to mineralized tissue repair and regeneration. In conclusion, the authors present a hypothesis, based on the evidence presented, that the full-length amelogenin uniquely regulates proper enamel formation through a process of cooperative mineralization, and not as a pre-formed matrix.

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Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.Extracellular matrix molecules play a crucial role in the regulation of biological mineralization by controlling crystal size, shape, and organization. An example of this exquisite regulation is in the formation of the highly organized dental enamel tissue that is regulated in part by amelogenin, the major extracellular matrix protein secreted by ameloblasts (1). Although amelogenin is processed by proteinases soon after secretion, the intact full-length parent molecule has been found to be exclusively associated with newly formed enamel mineral (2). Prior studies in our laboratory (3) have also shown that fulllength recombinant mouse amelogenin (rM179) can regulate the formation of parallel arrays of apatitic crystals (a salient feature of developing and mature dental enamel) under conditions of spontaneous precipitation in vitro. This functional capability appears to be related to the specific primary structure of the full-length amelogenin and its unique assembly properties under certain physicochemical conditions of pH and temperature (4). In particular, the conserved hydrophilic C terminus of amelogenin has been shown to play a key role in these processes (3).To date, however, most studies have utilized r...