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...

Objective-This study was carried out to determine in vitro the effect of fluoride on 1) the demineralization of sound human enamel and 2) the progression of artificial caries-like lesions, under relevant oral conditions.Methods-Thin sections of sound human enamel were exposed to solutions undersaturated with respect to tooth enamel to a degree similar to that found in dental plaque fluid following sucrose exposure in vivo, containing fluoride concentrations (0 -0.38 ppm) found in plaque fluid. Mineral changes were monitored for 98 days, using quantitative microradiography. The effect of fluoride (1.0 -25.0 ppm) on the progression of artificial caries-like lesions was similarly studied.Results-Fluoride concentrations of 0.19 ppm and greater were found to prevent the demineralization of sound enamel in vitro. However, significantly higher concentrations of fluoride (25.0 ppm) were required to prevent further demineralization of artificial caries-like lesions. Demineralizing solutions with intermediate fluoride concentrations (2.1 -10.1 ppm) induced simultaneously remineralization in the outer portion of the lesion and demineralization in the inner portion. Simultaneous remineralization and demineralization were also observed in hydroxyapatite pellets.Conclusions-Our results show that the observed effect of fluoride on enamel demineralization is not solely a function of bulk solution properties, but also depends on the caries-status of the enamel surface. A mechanistic model presented indicates that, in comparison to sound enamel surfaces, higher concentrations of fluoride are required to prevent the progression of artificial caries-like lesions under in vivo-like conditions since the diffusion of mineral ions that promote remineralization is rate-limiting.

We report here a novel biomimetic approach to the regeneration of human enamel. The approach combines the use of inorganic pyrophosphate (PP) to control the onset and rate of enamel regeneration and the use of leucine-rich amelogenin peptide (LRAP), a nonphosphorylated 56-amino acid alternative splice product of amelogenin, to regulate the shape and orientation of growing enamel crystals. This study builds on our previous findings that show LRAP can effectively guide the formation of ordered arrays of needle-like hydroxyapatite (HA) crystals in vitro and on the known role mineralization inhibitors, like PP, play in the regulation of mineralized tissue formation. Acid-etched enamel surfaces of extracted human molars, cut perpendicular or parallel to the direction of the enamel rods, were exposed to a PP-stabilized supersaturated calcium phosphate (CaP) solution containing 0 to 0.06 mg/mL LRAP for 20 h. In the absence of LRAP, PP inhibition was reversed by the presence of etched enamel surfaces and led to the formation of large, randomly distributed plate-like HA crystals that were weakly attached, regardless of rod orientation. In the presence of 0.04 mg/mL LRAP, however, densely packed mineral layers, comprising bundles of small needle-like HA crystals, formed on etched surfaces that were cut perpendicular to the enamel rods. These crystals were strongly attached, and their arrangement reflected to a significant degree the underlying enamel prism pattern. In contrast, under the same conditions with LRAP, little to no crystal formation was found on enamel surfaces that were cut parallel to the direction of the enamel rods. These results suggest that LRAP preferentially interacts with ab surfaces of mature enamel crystals, inhibiting their directional growth, thus selectively promoting linear growth along the c-axis of enamel crystals. The present findings demonstrate a potential for the development of a new approach to regenerate enamel structure and properties.

Amelogenin, the predominant extracellular matrix protein secreted by ameloblasts, has been shown to be essential for proper tooth enamel formation. In this study, amelogenin adsorption to hydroxyapatite (HAP) surfaces, a prototype for enamel mineral, has been studied using a quartz crystal microbalance (QCM) to interrogate effects of protein phosphorylation and solution pH. Dynamic flow-based experiments were conducted at pH 7.4 and 8.0 using native phosphorylated porcine amelogenin (P173) and recombinant non-phosphorylated porcine amelogenin (rP172). Loading capacities (μmol/m2) on HAP surfaces were calculated under all conditions and adsorption affinities (Kad) were calculated when Langmuir isotherm conditions appeared to be met. At pH 8.0, binding characteristics were remarkably similar for the two proteins. However, at pH 7.4 a higher affinity and lower surface loading for the phosphorylated P173 was found compared to any other set of conditions. This suggests that phosphorylated P173 adopts a more extended conformation than non-phosphorylated full-length amelogenin, occupying a larger footprint on the HAP surface. This surface-induced structural difference may help explain why P173 is a more effective inhibitor of spontaneous HAP formation in vitro than rP172. Differences in the viscoelastic properties of P173 and rP172 in the adsorbed state were also observed, consistent with noted differences in HAP binding. These collective findings provide new insight into the important role of amelogenin phosphorylation in the mechanism by which amelogenin regulates enamel crystal formation.

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