MMP20-generated amelogenin cleavage products prevent formation of fan-shaped enamel malformations

John D Bartlett,Yuanyuan Hu,Rebecca C Freeman,Tian Liang,James P Simmer,Amanda H Trout,Atsushi Ikeda,Jan C.-C Hu,Mike Strauss,Ya-Hsiang Hsu,Charles E Smith,David W Mccomb

doi:10.1038/s41598-021-90005-z

Abstract

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. Secreted proteins are critical for this process. Enam−/− and Ambn−/− mice fail to form enamel. We characterize enamel ribbon formation in wild-type (WT), Amelx−/− and Mmp20−/− mouse mandibular incisors using focused ion beam scanning electron microscopy (FIB-SEM) in inverted backscatter mode. In Amelx−/− mice, initial enamel mineral ribbons extending from dentin are similar in form to those of WT mice. As early enamel development progresses, the Amelx−/− mineral ribbons develop multiple branches, resembling the staves of a Japanese fan. These striking fan-shaped structures cease growing after attaining ~ 20 µm of enamel thickness (WT is ~ 120 µm). The initial enamel mineral ribbons in Mmp20−/− mice, like those of the Amelx−/− and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. Remarkably, Mmp20−/− mineral ribbons also form fan-like structures that extend to ~ 20 µm from the dentin surface. However, these fans are subsequently capped with a hard, disorganized outer mineral layer. Amelogenin cleavage products are the only matrix components absent in both Amelx−/− and Mmp20−/− mice. We conclude that MMP20 and amelogenin are not critical for enamel mineral ribbon initiation, orientation, or initial shape. The pathological fan-like plates in these mice may form from the lack of amelogenin cleavage products, which appear necessary to form ordered hydroxyapatite.

Highlights

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane
We demonstrate by quantitative real-time PCR that mRNA transcript levels for Ambn, Amelx, and Enam are not significantly different between WT and Mmp20−/− mouse molars (Fig. 1B)
Montages of magnified images (1500 or 2500x) show 13 (~ 90 μm) segments of Amelx−/− and 24 segments of Mmp20−/− from continuously erupting mandibular incisors that were analyzed by focused ion beam scanning electron microscopy (FIB-scanning electron microscopy (SEM))

Summary

Introduction

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. The initial enamel mineral ribbons in Mmp20−/− mice, like those of the Amelx−/− and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. As ameloblasts continue to differentiate, they begin secreting a melogenin[5] and extend finger-like projections through the thinning basement membrane and into the predentin between collagen fibers[6]. Ameloblast finger-like projections initiate the formation of enamel mineral ribbons primarily on the sides and tips of mineralized collagen fibrils to form the D EJ8. These initial ribbons are part of the interrod component of e namel[10]. It was definitively demonstrated by FIB-SEM that the absence of ameloblastin or enamelin expression eliminates enamel ribbon formation, knockout of amelogenin does n ot[8]

Methods

Results

Conclusion