Mechanisms of Enamel Mineralization Guided by Amelogenin Nanoribbons

Abstract

The nanofibrous nature and its intricate structural organization are the basis for the extraordinary ability of sound enamel to outlive masticatory forces at minimal failure rates. Apatite nanofibers of several hundreds of micrometers to possibly millimeters in length originate during the secretory stage of amelogenesis as 2-nm-thin and 15-nm-wide ribbons that develop and grow in length under the guidance of a dynamic mixture of specialized proteins, the developing enamel matrix (DEM). A critical role in the unidirectional and oriented growth of enamel mineral ribbons has been attributed to amelogenin, the major constituent of the DEM. This review elaborates on recent studies on the ability of ribbon-like assemblies of amelogenin to template the formation of an amorphous calcium phosphate precursor that transforms into apatite mineral ribbons similar to the ones observed in developing enamel. A mechanistic model of the biological processes that drive biomineralization in enamel is presented in the context of a comparative analysis of enamel mouse models and earlier structural data of the DEM emphasizing a regulatory role of the matrix metalloproteinase 20 in mineral deposition and the involvement of a process-directing agent for the templated mineral growth directed by amelogenin nanoribbons.

Keywords: amorphous calcium phosphate; apatite; biomineralization; developing enamel matrix; matrix metalloproteinase 20; self-assembly.

Figure 1.

Schematic of the polymer-induced liquid precursor (PILP) process with transmission electron microscopy (TEM) evidence. (A) PILP nanodroplets of 15 to 20 nm form when polyaspartic acid (pAsp) is mixed with Ca²⁺ and PO₄³⁻ ions in saturated aqueous solutions. (B) PILP-droplets attach to collagen fibrils comprising aligned collagen molecules with gap zones between them. Droplets attach to fibrils at the gap zones and mineral precursors infiltrate into the interstices. (C) Liquid precursors solidify into amorphous calcium phosphate (ACP), which gradually transforms into oriented apatite crystals (D) aligned with collagen molecules. Schematic adapted from Olszta et al. (2007); TEM images from Nudelman et al. (2010).

Figure 2.

Supramolecular structures of the developing enamel matrix (DEM). (A) Ribbon-like structures are predominant in secretory stage enamel in transmission electron microscopy (TEM) images and have been associated with apatite mineral (adapted from Diekwisch et al. 2002). (B) Scanning TEM analysis shows that ribbons initiate at dentin (De), which has a higher degree of mineralization compared to secretory stage enamel (En) (adapted from Smith et al. 2016). (C) TEM analysis indicated that mineral appears to form at the edges of protein ribbons at ameloblast (ambst) membrane (adapted from Pandya et al. 2017). (D) Demineralized and negatively stained section of klk4^−/− enamel shows ribbon-like protein structures previously described as “crystal ghosts.” (E) Protein ribbons measure 16 to 20 nm in width (red arrows) and show a dark central line (D and E adapted from Bai et al. 2020). (F) Section of klk4^−/− enamel-stained Congo Red for amyloid (adapted from Carneiro et al. 2016). (G) X-ray diffraction analysis of developing enamel showing reflection pattern characteristic of cross-β structure (adapted from Jodaikin et al. 1986). (H) TEM analysis of developing murine enamel stained with uranyl acetate showing electron-lucent spherical structures (black arrows) of 15 to 20 nm between fine mineral needles (adapted from Fincham et al. 1995). This figure is available in color online.

Figure 3.

Supramolecular structures of amelogenin proteins and peptides. (A) Transmission electron microscopy (TEM) analysis of recombinant full-length murine amelogenin rM180 assembled in phosphate buffer showed nanospheres of 15 to 20 nm (adapted from Fincham et al. 1995). (B) TEM analysis of recombinant full-length human amelogenin rH174 showed the formation of nanoribbons when calcium and phosphate ions were present during assembly (adapted from Carneiro et al. 2016). (C) Nanoribbons have high propensity to self-align (adapted from He et al. 2011). (D) When exposed to chelating agent EDTA, nanoribbons disintegrate and form spherical aggregates (adapted from Martinez-Avila et al. 2012). (E) TEM analysis shows a dark central line and width of rH174 nanoribbons averages at 16.7 nm with only ±1 nm variation (adapted from Martinez-Avila et al. 2012). (F) X-ray diffraction (XRD) analysis of human full-length amelogenin assembled into ribbons showed characteristic of cross-β structure of the developing enamel matrix (DEM) (adapted from Zhang et al. 2020). (G) Staining of micrometer-sized aggregates of rH174 nanoribbons by ThT indicates their amyloid-like character (adapted from Carneiro et al. 2016). (H) Amelogenin nanoribbons tend to align in parallel and will aggregate into larger bundles of 1 to 2 μm in diameter over time (adapted from Martinez-Avila et al. 2012). (I) Atomic force microscopy analysis of 14P2 peptide derived from self-assembling domain at the N-terminus of amelogenin showing randomly oriented nanoribbons of about 7 nm in width (adapted from Carneiro et al. 2016). (J) Model of the cross-β structure adapted by amyloid domain P2 shows that phosphorylation site at serine residue is able to interact with glutamic acid residue from the neighboring β-sheet, forming intermolecular ion bridges and triggering self-assembly into nanoribbons (adapted from Carneiro et al. 2016). (K) XRD analysis of 14P2 nanoribbons also shows cross-β structure (adapted from Engelberth et al. 2018).

Figure 4.

Mineral formation in enamel tissue and on recombinant amelogenin nanoribbons. (A) Enamel also follows the nonclassical pathway and mineralizes into apatite ribbons from an amorphous precursor, amorphous calcium phosphate (ACP) (adapted from Beniash et al. 2009). (B) Transmission electron microscopy (TEM) analysis of section through enamel from klk4^−/− mice shows apatite mineral ribbons of 20 to 23 nm in width with a dark central line. (C) Negative staining of demineralized enamel from klk4^−/− mice revealed matrix was composed of nanoribbons that originated at the dentin, with collagen fibrils in dentin visible. (D) Polymer-induced liquid precursor (PILP) mineralization treatments of demineralized enamel from klk4^−/− mice produced a coating of ACP on the surface of nanoribbons; enamel texture is visible. (E) Same treatment produced ACP coating at the dentinoenamel junction with only limited interaction and changes to dentin collagen. (F) Exposing PILP-treated specimens to a temperature of 50°C succeeded in phase transformation to oriented crystalline apatite, which aligned in the direction of protein ribbons. (G) TEM shows an area of incomplete phase transformation, and the transition zone indicates that ACP gradually transforms into crystalline apatite with guidance of the underlying amelogenin nanoribbons. (H) PILP treatment of nanoribbons from rH174 amelogenin did not facilitate ACP formation. (I) PILP treatment of nanoribbons from rH146 amelogenin resulted in aggregation of nanoribbons into bundles and formation of amorphous mineral rich in Ca and PO₄. (J) PILP-treated rH146 nanoribbons, after heating to 80°C, formed tens of micrometer-long bundles with oriented apatite mineral; selected-area electron diffraction (SAED) shows the c-axis parallel to the nanoribbon long-axis. (K) Bundles show mineral fibers or ribbons separated by organic templates and amelogenin nanoribbons (B to K adapted from Bai et al. 2020).

Figure 5.

Enamel microstructures in mouse models by scanning electron microscopy (SEM) analysis. (A) Characteristic pattern of decussated enamel rods in wild-type mouse incisor. (B) Amel–knockout (KO) mouse with sharply reduced thickness of enamel, absence of prismatic structure, and mineral mostly comprising octacalcium phosphate (OCP). (C) Rescue of Amel-KO mouse using M180 transgene results in characteristic prismatic structure with slightly reduced thickness. (D) Rescue of Amel-KO mouse using MMP20-cleavage product failed to produce prismatic structure. (E) Rescue of double KO-mouse lacking Amel and MMP20 by inserting M180 transgene failed to produce prismatic structure. (F) Rescue of Amel-KO mouse inserting transgene for LRAP and MMP20-cleavage product shows significant improvement of phenotype with enamel rods developing but lack of rod organization. (G) Rescue of Amel-KO mouse inserting LRAP transgene does not recover prismatic structure. (H) Enam-KO mouse shows only minimal layer of mineral on dentin, without structural organization (Fig. A–D, F, and G adapted from Xia et al. 2016; Fig. E adapted from Pugach et al. 2013; Fig. H adapted from Hu et al. 2008).

Figure 6.

Mechanistic model of templated growth of apatite ribbons in enamel development. Full-length human amelogenin (H175) is exocytosed at Tomes’s process, possibly as an antiparallel dimer. Step I: Interaction with calcium and phosphate triggers self-assembly into nanoribbons. Step II: Matrix metalloproteinase 20 (MMP20) cleaves off hydrophilic C-termini at both edges of the nanoribbons. Step III: C-terminal peptide and/or MMP20-processed enamelin form aggregates with calcium and phosphate ions, for example, polymer-induced liquid precursor (PILP) nanodroplets, which interact with remaining nanoribbons comprising mostly H147 amelogenin. Unloading of calcium and phosphate ions from nanodroplets results in the deposition of amorphous mineral (amorphous calcium phosphate [ACP]) on the ribbon surface via the PILP process. Step IV: ACP transforms into crystalline apatite (hydroxyapatite [HAP]) directed by underlying amelogenin nanoribbons. HAP adopts ribbon-like morphology matching the approximate width of the protein template of 15 nm. Protein ribbon assembly and concomitant mineral growth advance as the ameloblasts recede and continue to exocytose matrix proteins; consequently, ribbons follow the cell pathway and create the intricate microstructure of prismatic enamel.