Protein and Mineral Ribbons in MMP20^-/- and AMEL^-/- Mice

Objectives: Mineral ribbons (MR) of 2-3 nm thickness are the hallmark of secretory stage enamel. In our model, amelogenin protein assembles into nanoribbons which template the mineral growth after MMP20 cleavage. Interestingly, in mice that lack amelogenin or MMP20, MR form at the DEJ and extend for about 3-5 um in length, before the mineralization process goes awry. This study investigates the structure of the developing matrix in MMP20^-/-, and AMEL^-/- mice to gain insights into the origin of MR near the DEJ.
Methods: TEM with SAED was used to analyze mandibular incisors from Wild-type, MMP20^-/-, and AMEL^-/- mice along secretory stage of enamel before and after demineralization. Recombinant full-length amelogenin with Serine-16 phosphorylation were self-assembled and analyzed by AFM and TEM.
Results: Although the enamel matrix of AMEL^-/- mice is remarkably thinner than MMP20^-/-, their demineralized matrices share similarities close to the ameloblast membrane. Nanoribbons about 200-300 nm long can be observed emerging from the cell membrane. Ultrastructural differences are noticed as the enamel matrix deposition progresses. In the AMEL^-/- mice, curvy ribbons are enveloped by a homogeneous matrix and accompanied by granules, occasionally forming a squama-like structure. The ribbon structures vanish toward the DEJ, where they exhibit a gel-like appearance, intercepted by empty spaces, presumably the location of dissolved mineral. In the MMP20^-/- mice, fibrillar structures close to the proximal surface of the ameloblast form a mesh of short worm-like aggregates, similar to in vitro assemblies of recombinant phosphorylated amelogenin. To the contrary, the non-phosphorylated variant forms nanoribbons that resemble those observed in the matrices of wild-type enamel.
Conclusions: MR have different origin in the AMEL and MMP20-knockout mice and do not appear to be guided by protein nanoribbons, which may be a reason for their significantly reduced length when compared to wildtype enamel.