Amelogenin-Collagen Interactions Regulate Calcium Phosphate Mineralization in Vitro

Abstract

Collagen and amelogenin are two major extracellular organic matrix proteins of dentin and enamel, the mineralized tissues comprising a tooth crown. They both are present at the dentinenamel boundary (DEB), a remarkably robust interface holding dentin and enamel together. It is believed that interactions of dentin and enamel protein assemblies regulate growth and structural organization of mineral crystals at the DEB, leading to a continuum at the molecular level between dentin and enamel organic and mineral phases. To gain insight into the mechanisms of the DEB formation and structural basis of its mechanical resiliency we have studied the interactions between collagen fibrils, amelogenin assemblies, and forming mineral in vitro, using electron microscopy. Our data indicate that collagen fibrils guide assembly of amelogenin into elongated chain or filament-like structures oriented along the long axes of the fibrils. We also show that the interactions between collagen fibrils and amelogenin-calcium phosphate mineral complexes lead to oriented deposition of elongated amorphous mineral particles along the fibril axes, triggering mineralization of the bulk of collagen fibril. The resulting structure was similar to the mineralized collagen fibrils found at the DEB, with arrays of smaller well organized crystals inside the collagen fibrils and bundles of larger crystals on the outside of the fibrils. These data suggest that interactions between collagen and amelogenin might play an important role in the formation of the DEB providing structural continuity between dentin and enamel.Dentin and enamel, the two mineralized tissues that comprise a tooth crown, are strikingly different in terms of their compositional, structural, and mechanical properties (1). Despite these differences, dentin and enamel work together for decades under severe mechanical stress, without delamination or catastrophic failure.Dentin is a mineralized tissue similar to bone, comprised primarily of fibrillar collagen type I, carbonated apatite and water (2). There are other so-called noncollagenous macromolecules present that, in total, represent less then 10% by mass of organic material, although they play important roles in the formation and function of these tissues. All bone materials share the same basic building block, a collagen fibril, in which plateshaped apatitic crystals are organized in parallel arrays with their c-axes co-aligned with the long axis of the fibril (2-4). Collagen type I triple helical molecules, are super-coiled assemblies of two identical ␣1-chains and one ␣2-chain with a different sequence (5, 6). Each chain contains more than 1000 amino acids and is primarily composed of Gly-X-Y repeats, where amino acids proline (Pro) and hydroxyproline (Hyp) predominantly occupy X and Y positions. All three chains in the molecule adopt a polyproline II (PPII)-like structure, which is stabilized by direct and water mediated inter-and intra-chain hydrogen bonds (5, 6). Collagen fibrils form via a self-assembly process in which collag...

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale

“…Our observation that full-length amelogenin possesses a unique ability to stabilize and organize prenucleation clusters is in agreement with earlier in vitro mineralization experiments (1,8,28) and studies of transgenic animals, which demonstrate that the absence of the C telopeptide leads to alterations in amelogenin assembly and structural defects of enamel (7). Our observations correlate well with the initial mineralization events in vivo.…”

“…The electron diffraction analysis of these mineralization products suggests that at this stage mineral remains amorphous (Fig. S10D) in agreement with our earlier in vitro mineralization studies, which show that the mineral crystallization occurs later (8,28). In contrast to rM179, rM166 was not able to stabilize prenucleation clusters or guide their organization.…”

“…S9B). The ability of rM179 to stabilize the prenucleation clusters and guide their assembly into linear chains that subsequently fuse into the needle-shaped mineral particles is consistent with the view that the enamel crystals form by controlled assembly of prenuclation clusters (8,28) via a nonclassical crystallization mechanism (29). Finally, after 2 h in the control experiments, large aggregates of plate-like mineral particles were observed (Fig.…”

“…S10). Similar mineral bundles formed in the presence of full-length amelogenin have been previously reported in dehydrated samples (1,8,28). A close examination of the needle-shaped mineral particles in the bundles (Figs.…”

How to control the size and morphology of apatite nanocrystals in bone

“…It is well known that the organic component of bone acts as an important regulator of lattice orientation, particle size, and size distribution in biomineralization processes, but the molecular recognition details at inorganic crystal interfaces are poorly understood (4). Recently, most investigations have been focused on the effects of macromolecules such as carboxylate-rich proteins on hydroxyapatite nucleation and growth because complex interactions and changes of conformation are involved (5,6). The role of small organic molecules such as citrate was deemphasized and was assumed to be simply that of adsorption on the crystal growth steps, resulting in inhibition of crystal growth (7,8).…”