Abstract

Matrix metalloproteinase 20 (MMP20) and kallikrein‐related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild‐type, Mmp20 −/−, Klk4 −/−, Mmp20 +/− Klk4 +/−, and Mmp20 −/− Klk4 −/− mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy‐dispersive X‐ray analysis (EDX). Following eruption, fractures were observed on Mmp20 −/−, Klk4 −/−, Mmp20 +/− Klk4 +/−, and Mmp20 −/− Klk4 −/− molars. Failure of the enamel in the Mmp20 +/− Klk4 +/− molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro‐CT analyses of hemimandibles demonstrated significantly reduced high‐density enamel volume in the Mmp20 −/− and Klk4 −/− mice relative to the wild‐type, which was further reduced in Mmp20 −/− Klk4 −/− mice. bSEM images of 7‐week Mmp20 −/− and Mmp20 −/− Klk4 −/− mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20 +/− and Mmp20 +/− Klk4 +/− incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20 +/− Klk4 +/− incisors relative to Mmp20 +/− incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20 −/− and Mmp20 −/− Klk4 −/− exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple additional functions necessary for the adherence of enamel to dentin, the release of intercellular protein stores into the enamel matrix, the retreat of ameloblasts to facilitate thickening of the enamel layer, and the timely transition of ameloblasts to maturation.

Highlights

  • Two secreted proteinases are essential for dental enamel formation: matrix metalloproteinase 20 (MMP20; OMIM *604629) and kallikrein-related peptidase 4 (KLK4; OMIM *603767) (Lu et al 2008)

  • The gross morphologies of the mouse molar and incisors before and after eruption were assessed under a dissecting microscope, by scanning electron microscopy (SEM) and by backscattered scanning electron microscopy (bSEM)

  • Dental enamel formation occurs by a biologic mechanism that originated in lobe-finned fish with lungs some 400– 450 million years ago (Kawasaki et al 2009)

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Summary

Introduction

Two secreted proteinases are essential for dental enamel formation: matrix metalloproteinase 20 (MMP20; OMIM *604629) and kallikrein-related peptidase 4 (KLK4; OMIM *603767) (Lu et al 2008). MMP20 is secreted early during the secretory stage, and cleaves or “processes” enamel matrix proteins at a limited number of sites (Ryu et al 1999). Uncleaved enamel proteins are prominent in the secretory stage enamel of Mmp null mice (Yamakoshi et al 2011). A line of hypermineralization along the DEJ can be observed until the overlying enamel reaches a similar density (Hu et al 2011a). This line is absent during early amelogenesis in Mmp null mice (Hu et al 2011b), and the enamel layer in these mice fails at the DEJ (Simmer et al 2012b). Mutations in human MMP20 cause a nonsyndromic form of amelogenesis imperfecta (Kim et al 2005; Papagerakis et al 2008; Wang et al 2013; Seymen et al 2015)

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