Tooth Enamel and its Dynamic Protein Matrix

Abstract

Tooth enamel is the outer covering of tooth crowns, the hardest material in the mammalian body, yet fracture resistant. The extremely high content of 95 wt% calcium phosphate in healthy adult teeth is achieved through mineralization of a proteinaceous matrix that changes in abundance and composition. Enamel-specific proteins and proteases are known to be critical for proper enamel formation. Recent proteomics analyses revealed many other proteins with their roles in enamel formation yet to be unraveled. Although the exact protein composition of healthy tooth enamel is still unknown, it is apparent that compromised enamel deviates in amount and composition of its organic material. Why these differences affect both the mineralization process before tooth eruption and the properties of erupted teeth will become apparent as proteomics protocols are adjusted to the variability between species, tooth size, sample size and ephemeral organic content of forming teeth. This review summarizes the current knowledge and published proteomics data of healthy and diseased tooth enamel, including advancements in forensic applications and disease models in animals. A summary and discussion of the status quo highlights how recent proteomics findings advance our understating of the complexity and temporal changes of extracellular matrix composition during tooth enamel formation.

Keywords: amelogenin; amelogenin-Y (AMELY); dental anthropology; dental fluorosis; enamel peptide; enamel proteome; molar hypomineralization; serum albumin; tooth enamel.

Figure 1

Illustration of the process of enamel formation. (A) Micro computed tomography radiograph of mouse mandible (without mandibular ramus) showing lighter grey values for higher mineral density, tooth enamel in white on molar crowns and in a gradient according to increasing mineralization in the continuously forming incisor. Data acquired on a Scanco Medical μ40 instrument (Scanco, Brüttisellen, CH). (B) Schematic representation of mouse mandible showing continuous incisor growth from early stages of enamel formation with high amount of protein (blue) to mature, erupted enamel containing less than 1% by weight protein and more than 95 % by weight mineral (yellow). (C) Schematic of ameloblast morphology indicating differentiation and different stages during enamel formation (adapted from Hu et al. [11]). Color filled arrow indicates progression of time and compositional changes of the mineralizing matrix from high protein content (blue) with little mineral, to low protein and high mineral content (yellow) in later stages of mineralization and mature enamel. (D) Graph depicting decreasing protein and increasing mineral content in developing enamel, with activity period of matrix metalloproteinase 20 (Mmp20), phosphatase and kallikrein-4 (Klk4) during mineralization of rodent incisors (adapted from Robinson et al. [12]). (E–G) Scanning electron microscopy (SEM) images of the forming mouse incisor in longitudinal plane. Enamel with high protein content seen at high magnification in late secretory stage (E, scale bar 200 nm) and with some matrix protein, colored in blue, around crystal bundles in early maturation stage (F, scale bar 200 nm). At lower magnification, the decussation pattern of crystal bundles in mature, erupted enamel in (G, scale bar 10 microns). SEM—samples were fixed, dehydrated, epoxy resin embedded, polished, phosphoric acid etched, gold coated and imaged on a Zeiss SEM Ultra55.

Figure 2

Strategies for proteomic analyses of tooth enamel. Schematic of treatment steps used to characterize proteins in human tooth enamel from archeological, modern and teeth affected by molar hypomineralization (chalky tooth enamel). Porto et al. [21]; Lugli et al. [43]; Stewart et al. [44]; Castiblanco et al. [45]; Stewart et al. [46]; Jagr et al. [47]; Farah et al. [48]; Nielsen-Marsh et al. [49]; Mangum et al. [50]. Each bracket denotes alternative options and reflects the variability in published protocols.

Figure 3

Comparison of protein content between healthy and diseased tooth enamel. Shown proteins are seen in (rows) identified in any of the tooth conditions (columns) are illustrated. Healthy teeth shown as reference in light grey, Chalky/ Molar Hypomolarization (MH): enamel affected by molar hypomineralization [48,50,92], Fluorosis [93,94,95] and Amelogenesis Imperfecta (AI): hypocalcified and hypomaturation amelogenesis imperfecta enamel [88,89,90]; Range of percent by weight (wt%) of protein abundance relative to healthy enamel show in colors: light gray for healthy range of 0.1–1 wt%; 2–3 fold increase (light teal); 3–30 fold increase (dark teal); 0–30 fold increase (grey-teal gradient).

Figure 4

Overview of proteins detected in enamel from permanent molars of 6-months-old [112] and 8-months-old pigs [15]. The chart distribution is based on the protein detection in soft enamel and hard enamel, described as secretory and mature stages, respectively.

Figure 5

Schematic of potential mechanism of post-translational modifications and secretion of enamel proteins during the course of enamel formation. (1) Post-translational modifications of enamel proteins occur with the ameloblast. These modifications are used as a signal to be secreted, cleavage during the secretory stage and degraded during the maturation stage. (2) Some of the enamel proteins generated with the ameloblast lack post-translational modification and they are secreted via non-classical pathways. These proteins would be less or not degraded during the mineralization process. (3) Alternative to pathway 1, de-phosphorylation to account for the presence of non-phosphorylated enamel proteins in mature enamel. (P) phosphorylation, (G) glycosylation; Blue: enamel that is more recently deposited, younger, with higher protein content. Yellow: enamel that is further developed and more mature, older enamel, more mineralized with lower protein content.