Dental Enamel Formation and Implications for Oral Health and Disease

Abstract

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth’s epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

I. INTRODUCTION

Dental enamel is the hardest substance in the human body and serves as the wear-resistant outer layer of the dental crown. It forms an insulating barrier that protects the tooth from physical, thermal, and chemical forces that would otherwise be injurious to the vital tissue in the underlying dental pulp. Because the optical properties of enamel are also derived from its structure and composition (205), developmental defects or environmental influences affecting enamel structure are typically visualized as changes in its opacity and/or color. The impact of developmental insults on enamel is critical because, unlike bone, once mineralized, enamel tissue is acellular and hence does not remodel.

In mammals, dental enamel is the only epithelial-derived tissue that mineralizes in nonpathological situations (bone and dentin, the other principal mineralized tissues, are derived from mesenchymal cells). Enamel forms within an organic matrix composed of a unique grouping of extracellular matrix proteins (EMPs) that show little homology to proteins found in other tissues. The enamel organ is formed by a mixed population of cells. Among these are ameloblasts, which are primarily responsible for enamel formation and mineralization, and form a monolayer that is in direct contact with the forming enamel surface.

The process of enamel formation is referred to as amelogenesis. Enamel matrix proteins are secreted by ameloblasts into the enamel space, and are later degraded and proteolytically removed, also by ameloblasts. It is with a high level of precision that ameloblasts regulate the formation of a de novo hydroxyapatite-based (Hap-based) inorganic material within the enamel space. The formed enamel has a characteristic prismatic appearance composed of rods, each formed by a single ameloblast and extending from the dentino-enamel junction (DEJ) to the enamel surface, and the interrod enamel located around the enamel rods. Traces of EMP peptides are included in the fully formed enamel and are believed to contribute to the final structure, such that the fully formed (mature) enamel has unique morphological and biomechanical properties. By weight, mature enamel is ~95% mineral, ~1–2% organic material, and ~2–4% water (100, 331, 479, 509, 523, 548).

In this review, we discuss enamel from its developmental beginnings to its final structure. We will pay particular attention to the proteins comprising the enamel matrix, the role of ameloblast-mediated ion transport and mineralization, and the importance of extracellular pH regulation during enamel formation. There is also mounting information on the clinical outcomes that result from abnormal ameloblast function related to specific gene mutations, and we will summarize what is currently understood about enamel genotype-phenotype relationships.

II. DENTAL TISSUES: HUMAN, RAT, AND MOUSE TEETH

All mammalian teeth share a similar structure: 1) the enamel crown, formed by epithelial cells; 2) the dentin found underlying the enamel, formed by mesenchymal cells and containing a large collagen component; 3) the pulp, the organ generating/supplying the dentin-forming cells (odontoblasts), and also containing vasculature and nerve supply; 4) the root, comprised primarily by the dentin, but also containing the root canal and surrounded by a thin layer of mineralized cementum; and 5) the periodontal ligament, which is part of the dental socket that unites the cementum to the alveolar bone (FIGURE 1) (263, 333, 355, 403, 507, 655). Enamel is far more mineralized than the other tooth structures and serves to protect the dentin and pulp. Enamel contains no collagen, and once formed is devoid of any cells, so it cannot remodel.

FIGURE 1.

The anatomy of a human mandibular molar tooth. The major features of the mammalian tooth include the enamel, dentin, pulp contained within the pulp chamber, the root canal that carries the nerve and vasculature to the pulp, cementum, periodontal ligament, and the alveolar bone.

Humans are diphyodonts (having 2 sets of teeth) with an initial/primary dentition of 20 teeth and a secondary/permanent dentition of 32 teeth. Rats and mice are monophydonts (having one set of teeth) with a single dentition of 16 teeth. Rats and mice have become widely used animal models to study tooth formation because rodents have continuously growing maxillary and mandibular incisors. This characteristic in rodents means that throughout the animal’s lifespan, all stages of amelogenesis (see below) can be studied on a single incisor at any one time.

III. AMELOGENESIS

A. Embryological Development of Teeth

The staging of tooth formation has been studied histologically and morphologically for centuries. The principal stages include the initial development of a dental lamina comprising an inward growing band of thickened oral epithelium at specific sites determined by the localized expression of key transcription factors (FIGURE 2). The dental lamina rapidly folds and penetrates the underlying mesenchyme to form the dental placode, followed by the bud, cap, and bell stages. These stages shape the crown, which is then followed by the development of the roots. The mesenchyme immediately underlying the dental epithelium is derived from cranial neural crest cells (74). Very early in tooth formation there is epithelial-mesenchymal molecular crosstalk initially orchestrated by the mesenchyme, such that epithelial cells destined to create enamel start to differentiate to form ameloblasts, and the underlying neural crest-derived mesenchyme differentiates into cells that will form the remainder of the tooth. It is beyond the scope of this review to discuss the morphogenesis and histology of mammalian tooth formation, or the cellular origins and molecular signals used locally to direct odontogenesis; however, the reader is directed to some outstanding publications that cover all these topics (74, 262, 271, 377, 378, 403, 594).

FIGURE 2.

The principal stages of tooth formation. Thickening of the oral epithelium (blue) to form the dental lamina, the placode, bud, cap, and bell stages. The dental epithelium is shown in blue, the neural crest-derived mesenchyme in green, and all other (non-neural crest-derived) underlying mesenchyme in pink. At the cap and bell stage, the outer enamel epithelium is shown in white, the inner enamel epithelium (ameloblasts) is shown in orange, and the dentin-forming cells (odontoblasts) are shown as light brown. At the bell stage the forming enamel is yellow and the dentin dark brown. Green represents the (cranial) neural crest-derived mesenchymal cell population that migrates to the dental lamina and will eventually form the pulpal tissues seen in the bell stage.

B. Amelogenesis

1. Overview of enamel formation

Enamel development involves two major functional stages, secretory and maturation, with a brief transition between the two stages (403), although additional subdivisions may include: presecretory, early secretory, late secretory, transition, preabsorptive, early maturation, and late maturation stages (17, 273, 299, 464, 524). Throughout this review we focus primarily on the secretory and maturation stages as the bulk of data available to date, on the secretion of structural matrix proteins and proteinases, and on ionic transport, relates to these two stages.

2. The enamel organ

Amelogenesis involves the formation of a number of epithelium-derived cell types. The innermost layer, the inner enamel epithelium, is a single layer of cells that differentiate into ameloblasts. The outermost layer is also a single layer of cells, referred to as the outer enamel epithelium. The inner and outer enamel epithelium converge at a region called the cervical loop, which is a niche for dental epithelial stem cells (47, 204, 272, 336, 379, 380, 420) and thus provides a constant source of enamel-forming cells until the enamel crown is fully formed with one exception. In rodent incisors, the long teeth in the upper and lower jaws, the stem cell niche in the cervical loop is retained for life, enabling the continuous growth of these teeth.

The cells comprising the enamel organ in the secretory stage and maturation stage are morphologically very different (228, 541). Hu et al. (228) illustrated the changing ameloblast morphologies throughout amelogenesis as viewed histologically. During the secretory stage, four cell populations are easily recognized: a single layer of secretory ameloblasts; the stratum intermedium, typically one or two cell layers thick; the stellate reticulum comprised of a larger grouping of star-shaped cells; and the single-layer outer enamel epithelium (FIGURE 3). The vascular network that supplies nutrients to the developing enamel organ is associated with the outer enamel epithelium (275, 403). The anatomy of the enamel organ changes quickly and dramatically from secretory to maturation stage. Secretory ameloblasts transform, after a short transition period, and become shorter; they have frequently been referred to as squatter maturation cells (320, 403, 541). The other three epithelial cell populations identified in the secretory stage (stratum intermedium, stellate reticulum, and outer enamel epithelium) reorganize to become the papillary layer (PL) cells that are rich with blood vasculature weaving through its folds (FIGURE 3).

FIGURE 3.

Secretory- and maturation-stage ameloblasts. A: highly polarized secretory ameloblasts (Am) with the Tomes’ process (TP) projecting into the forming enamel front. B: shorter maturation ameloblasts. Connective tissue (CT), outer enamel epithelium (OEE), stellate reticulum (Sr), stratum intermedium (Si), and papillary layer cells are also identified, as is the enamel (En) or enamel space (ES) region which is in contact with the distal/apical pole of ameloblasts. Blood vessels (BV) can also be seen in the folds of the papillary layer cells.

The functional roles of these cell populations of the enamel organ, other than the ameloblasts, are poorly understood (343). The stratum intermedium has high alkaline phosphate (ALPL) activity (225, 640), suggesting that its function may be to facilitate transport of phosphate from the circulation to the developing enamel organ (640). Cells of the stellate reticulum maintain contact with each other through numerous desomosomes and gap junctions, giving them a star-like appearance (366, 367). The stellate reticulum cells express glycoaminoglycans such as perlecan, which accumulate in the intercellular spaces (245). The outer enamel epithelium is a single layer of cuboidal cells covering the entire enamel organ, thought to form a protective buffer isolating the other cells of the enamel organ (403). During the transition to maturation stage, the stratum intermedium, stellate reticulum, and possibly also the outer enamel epithelium reorganize to form the PL cells (61, 269). The PL cells are vascularized and participate in ion transport during the maturation stage, aiding the movement of ions from the blood circulation to the ameloblasts (61, 269).

3. Secretory-stage amelogenesis

During the secretory stage, ameloblasts are highly polarized cells. The height (basal-apical distance) of a secretory ameloblast can be as great as 90 µm but is generally ~70 µm, while a narrow average diameter is ~5 µm, as detailed by Smith (541). These cells synthesize and secrete a limited number of structural enamel matrix proteins (EMPs), most notably amelogenin (AMELX), ameloblastin (AMBN), and enamelin (ENAM) (316, 541, 544). A unique characteristic observed in the morphology of secretory ameloblasts is the presence of the Tomes’ processes (276, 450, 592, 621), triangular-shaped extensions of the cell found at the distal end and penetrating into the enamel matrix, giving an ameloblast monolayer a “picket-fence” appearance if viewed on a histological section (276, 403). The Tomes’ process is important for exocytosing secretory vesicles and also plays a role in determining boundaries between rod and interrod regions (160, 403).

The precursors of the enamel crystals start to form during the early secretory stage in a protein-rich extracellular environment that is maintained at near-neutral pH conditions (316, 544). Thin hydroxyapatite-like (Hap-like) crystals [sometimes referred to as either enamel ribbonlike structures (97) or enamel ribbons (236, 543)] grow almost exclusively along their c-axis and elongate perpendicular to the DEJ under the influence of EMPs, in a direction that is finely coordinated with the movement of the ameloblasts traveling away from the dentin (148, 403). Historically, it was considered that initial formation of enamel crystallites (i.e., nucleation) occurred within the enamel matrix (115). However, some recent data have challenged this notion and suggest that enamel crystal growth is initiated on mineralized collagen fibers from the dentine (236, 543). These crystals then extend through the DEJ to the ameloblast membrane, and throughout the enamel (236, 543). Almost the entire thickness and volume of enamel is laid down during the secretory stage. It is a very soft tissue (gel-like) at this point, comprised of similar amounts of EMPs, mineral, and water by weight. Adjacent secretory-stage ameloblasts are tightly opposed and are connected to each other by intercellular junctional complexes on the lateral membrane at both the proximal/basal and distal/apical poles (240, 403, 503, 504, 506). These junctional complexes can be either tight, forming a beltlike and complete seal around the cell, as is frequently observed at the apical membrane, or they can be incomplete/leaky, as may be seen at the basal pole (240, 403, 504). These junctional complexes of secretory ameloblasts form a semipermeable barrier for intercellular movement/diffusion of mineral ions from the circulation to the enamel matrix (240, 579).

4. Transition stage

The transition from secretory to maturation is brief (228, 603) and, in the rat lower incisor, spans ~170 µm (541) or ~30–40 cell widths. During this brief transition, significant morphological changes can be seen as ameloblasts become shorter and lose their secretory Tomes’ process, and the PL is formed (274). These changes are accompanied by dramatic changes in gene expression profiles (318, 527, 664). The expression of EMP coding genes AMELX, AMBN, and ENAM are downregulated during this transition, whereas many other genes including those involved in ion transport, proteolysis, and pH homeostasis are upregulated (234, 318, 615, 664). During the transition stage, ~25% of ameloblasts die (550), presumably from apoptosis, which may result from the cells being in a metastable state due to calcium overload (240).

5. Enamel maturation

Maturation-stage ameloblasts are shorter than secretory-stage ameloblasts, being ~40 µm in height. The major functions of the ameloblasts during enamel maturation encompass many activities, including ion transport (541), acid-base balance (316), EMP debris removal/endocytosis (313, 524), and apoptosis (318). To date, many of the molecular mechanisms involved in ameloblast-directed enamel maturation remain unclear (320). However, in the past decade there have been significant contributions to the literature highlighting the importance of ion transport and pH regulation during enamel maturation (reviewed in Refs. 125, 320, 410).

Although crystal growth takes place during both the secretory and maturation stages, it is during the maturation stage that the crystals greatly expand in width and thickness, giving enamel its characteristic durability and hardness (541). To add complexity during the maturation stage, ameloblasts change morphology in a unique series of modulations (cyclical changes) between a ruffle-ended (RA) appearance and a smooth-ended (SA) appearance in coordinated groups, appearing as bands of similar morphology across the circumference of the crown in an oblique fashion (466, 622). SA waves appear at ~8.5-h intervals in rat incisors, and these ameloblasts change after 2 h into RA cells, reforming their characteristic cell specializations at the distal border (545). On average, the surface of a rat incisor in any histological section shows ~70% of maturation ameloblasts in the RA phase and ~20% of maturation ameloblasts in the SA phase (466). Transitional cells can also be identified (268). RA cells are characterized by a distinct distal striated or ruffled border (468). RA cells are cytoplasmically polarized with a large concentration of mitochondria proximal to the ruffle-border and supranuclear Golgi complex (268). Intercellular spaces are noticeable along the lateral region of RA cells, but these cells are tightly bound by junctional complexes at their apical (distal) ends (167, 240, 403, 466), limiting the movement of small molecules into the enamel space. RA cells are also associated with increased endocytotic functions (313, 403, 498). In contrast, SA cells show a complete absence of the distal ruffled border (501). SA cells contain many lateral cytoplasmic projections, and they are bound at their basal ends by tight junctions whereas the apical ends of the cells may have incomplete/leaky or absent junctional complexes (240, 313, 318, 403, 504, 536, 541). It is believed that this dynamic permeability pattern allows bidirectional diffusion of small molecules into and out of the enamel via intercellular spaces (269, 541).

To briefly summarize the distinct roles of RA and SA maturation-stage ameloblasts, RA cells with their ruffled apical membrane likely have greater capacity to transport ions into and away from the enamel organ, and also to endocytose the EMP debris. SA cells with incomplete junctional complexes may allow for intercellular movements of fluids that may in turn contribute to the neutralization of pH in the enamel matrix (403). Although SA cells show little endocytotic activity (403), clathrin-coated vesicles and endocytotic activity have been identified in both RA and SA cells (499, 500, 505).

The RA to SA modulations play a role in pH regulation and bicarbonate transport, which differ at each stage (316, 541, 581). Ameloblasts associated with the Ca²⁺ chelator glyoxal bis-2-hydroxyanil (GBHA) showed SA morphology under a light microscope, indicating that neutral-alkaline conditions dominate at this stage. Alternatively, it has been proposed that extracellular pH conditions modulate the RA to SA transitions (64). Others have shown that Ca²⁺ entry to the enamel increased during the RA stage (467).

Recent immunohistochemical analyses have reported a number of differences in certain protein localizations between RA and SA ameloblasts. These include the anion exchanger AE2 (a member of the SLC4 gene family) that is differentially localized at the lateral or the basal part of the lateral membrane of primarily RA cells, and in the same location but to a lesser extent in SA cells (269). The expression of carbonic anhydrase-2 (CA2), an enzyme that is involved in the local production of bicarbonate, is upregulated in RA cells (269, 342). Protein subunits of the V-type ATPase proton pump are fairly evenly distributed in the cytoplasm of SA cells, but most highly concentrated at the apical membrane of RA cells (269, 342, 495). These data emphasize the greater capacity of RA cells to transport ions, and to influence and control changes in the extracellular pH during enamel maturation, although this is likely a simplified portrayal of the functions performed by each cell type.

C. Crystal Structure of Apatite

Calcium (Ca²⁺) and phosphate (PO₄³⁻) ions are only sparsely soluble in water and thus precipitate at rather low concentrations as a crystalline or amorphous solid (671). Under physiological conditions, apatite has the lowest solubility among the calcium phosphate minerals and is therefore the most chemically stable mineral phase. Consequently, apatite constitutes the inorganic component in all sound mineralized tissues in vertebrate animals (120).

In saturated aqueous calcium phosphate solutions with physiological range of pH (6.0 to 7.4), precipitation of stoichiometric hydroxyapatite (Hap) can occur according to the following reaction (Equation 1)

$${\text{10Ca}}^{\text{2+}}+{\text{6HPO}}_4^{2-}+{\text{2H}}_{\text{2}}\text{O}\to {\text{Ca}}_{\text{10}}{\left({\text{PO}}_{\text{4}}\right)}_{\text{6}}{\left(\text{OH}\right)}_{\text{2}}+{\text{8H}}^{+}$$ (1)

Precipitation of one mole of Hap will result in the release of eight protons, thus acidifying the solution and requiring active pH regulation by ameloblasts as will be described below.

The unit cell (the simplest repeating unit) of Hap corresponds to the chemical formula Ca₁₀(PO₄)₆(OH)₂ (289). Its crystal lattice has hexagonal symmetry and comprises PO₄³⁻ tetrahedrae coordinated with Ca²⁺ ions (FIGURE 4). There are two types of Ca²⁺ positions in the Hap-lattice, of which Ca (2) is unique as it forms channels that allow anions to move along the c-axis of the apatite crystal (FIGURE 4). Hydroxyl ions are able to diffuse and be replaced by other ions such as fluoride (F⁻), carbonate (CO₃²⁻), or chloride (Cl⁻) from aqueous solutions. This makes apatite composition highly adaptable to its solution environment, which is critical to its properties in biological apatites. Apatite crystals in bones and teeth are far from being stoichiometric. Instead, they are rich in defects and usually calcium-deficient (120, 667). To maintain electron neutrality upon calcium depletion, phosphate groups are protonated (HPO₄²⁻) and/or phosphate groups are replaced with CO₃²⁻. Carbonate ions can also replace two hydroxyl groups along Ca (2) channels. The chemical reactions below describe the formation of a calcium-deficient carbonated apatite (Equation 2) and a calcium-deficient carbonated hydroxyapatite (Equation 3)

FIGURE 4.

Crystal structure of calcium phosphate apatite shows hexagonal symmetry. Two types of calcium sites are indicated, with Ca (2) sites in triangular configuration (yellow and red lines) creating channels along which ions can move (green). Anions (green) such as OH⁻, Cl⁻, CO₃²⁻, or F⁻ can fill sites in the channels. F⁻, the smallest of these species, can fit within the triangle created by the Ca sites forming the channel; larger anion species deform the lattice significantly when occupying these sites, leading to a reduced lattice stability and therefore higher solubility.

$${\text{9Ca}}^{\text{2+}}+{\text{6HPO}}_4^{2-}+{\text{CO}}_3^{2-}\to {\text{Ca}}_{\text{9}}{\left({\text{PO}}_{\text{4}}\right)}_{\text{4}}{\left({\text{HPO}}_{\text{4}}\right)}_{\text{2}}\left({\text{CO}}_{\text{3}}\right)+{\text{4H}}^{\text{+}}$$ (2)

$${\text{18Ca}}^{\text{2+}}+{\text{10HPO}}_4^{2-}+{\text{2CO}}_3^{2-}+{\text{4H}}_{\text{2}}\text{O}\to {\text{Ca}}_{\text{18}}{\left({\text{PO}}_{\text{4}}\right)}_{\text{8}}{\left({\text{HPO}}_{\text{4}}\right)}_{\text{2}}{\left({\text{CO}}_{\text{3}}\right)}_{\text{2}}{\left(\text{OH}\right)}_{\text{4}}+{\text{12H}}^{\text{+}}$$ (3)

Carbonate substitutions for phosphate (PO₄³⁻) or hydroxyl ions (OH⁻) affect the ideal crystal structure of apatite and lower its symmetry, resulting in lower binding energies and ultimately increase the chemical solubility of the mineral phase (332). Carbonated apatite is therefore much more susceptible to acidic dissolution and dissolves at pH around 5, which is readily produced by cariogenic (caries-producing) bacteria. In contrast, F⁻ fit perfectly between Ca (2) triangles and stabilize the hexagonal symmetry and crystal lattice. Exchange of CO₄³⁻ for F⁻ therefore lowers the solubility by at least three orders of magnitude, and fluoroapatite can withstand a pH as low as 4 without dissolution. This partly explains the high benefit of fluoride supplements in toothpastes and drinking water for caries prevention and erosion reduction in teeth (393, 451, 671).

IV. EVOLUTIONARY ORIGINS OF ENAMEL AND ENAMEL MATRIX PROTEINS

The evolution of enamel is tied to the appearance of teeth and in general with the development of skeletonized structures. Early vertebrates during the Cambrian period show mineralized structures in the oropharynx that were used for feeding, and which are likely related to the origins of teeth, although they were composed of a carbonated material dissimilar to enamel (286). These structures are comparable to pharyngeal teeth found in modern fish (teleosts). Some modern fish (e.g., hagfish and lampreys) show structures resembling teeth, but these are cartilaginous and, unlike true teeth, derive from ectomesenchyme rather than ectoderm (244). Modern vertebrate teeth may have evolved from oropharyngeal denticles or from dermal denticles such as those found in sharks. However, shark teeth (and their dermal denticles) are formed from enameloid, which contains a large component of collagen, unlike true enamel. Reptiles also have teeth, but unlike mammals, reptilian enamel lacks a rod/interrod (prismatic) structure. Mammalian enamel has a highly organized prismatic structure that forms as described in section IIIB (FIGURE 5). The prismatic or rod/interrod architecture seen in mature mammalian enamel can be appreciated in FIGURE 6. Mammalian enamel is rather heterogeneous in its microstructure, even within the same species. For example, rodent incisor and molar microanatomy differ, which has been ascribed to different functional requirements by each tooth type during mastication (185, 297, 354, 472, 473). Also of note is that with the introduction of interdigitatung rods, which are angled to apatite fibers in the interrod enamel, crack resistance is increased (198), while fracture toughness of prismatic mammalian enamel is about double that of prismless reptile enamel (663).

FIGURE 5.

Transmission electron micrographs (TEM) of early enamel development. TEM images of the mouse showing early enamel formation. A: ameloblast Tomes’ process (TP; right) surrounded by enamel crystallites seen adjacent to the dentino-enamel junction (DEJ; broken line) that is located diagonal to frame, and with pre-dentin (PD; unmineralized dentin) to the left. Scale = 1 µm. B: initial enamel crystallite formation for the first 1–1.5 µm (shown in panel). TP, PD, and DEJ (broken line) are also identified. Scale = 0.2 µm. C: this panel shows a higher magnification of the DEJ area very early in enamel crystallite formation. Scale = 200 nm.

FIGURE 6.

Scanning electron (SE) micrographs of mouse enamel. A: bundles of single Hap crystals can be seen forming tubular structures known as enamel prisms or enamel rods. Each ameloblast is responsible for the formation of one enamel rod. These rods are the basic structural units of enamel. The architectural patterning of the rods forms the microstructure. Scale = 2 μm. B: enamel microstructure in a cross section of a mouse incisor. Scale = 10 μm. C: close up of outer incisor enamel in back-scattered SE mode showing the change in organization of the rods as the Tomes’ process is lost (top of image). At this point, the enamel microstructure loses the rod-interrod patterning. Scale = 2 μm. D: mouse molar enamel (same animal as in A) showing the complexity of its microstructure which reflects the movement of ameloblasts and how different tooth types may vary in microstructure. Inner enamel reflects strong decussation (crossing of ameloblasts along various planes as they move) but in the outer enamel (top of image) ameloblasts move in straight paths. Scale = 10 μm. E: close up of inner enamel showing the strong decussation of the enamel rods in this tooth. Scale = 10 μm. (All SE images by Timothy G. Bromage and Rodrigo S. Lacruz.)

The origin of the main structural proteins secreted by ameloblasts, AMELX, AMBN, ENAM, and the relatively newer amelotin (AMTN) and odontogenic, ameloblast-associated protein (ODAM), dates back to over ~600 million years ago (532). They are all members of the secretory calcium-binding phosphoprotein (SCPP) gene family derived from the ancestral SPARC/Osteonectin gene (482). ENAM appears to be the original protein from which the others, including AMBN, are derived. AMELX, the most abundant matrix protein, arose via gene duplication from AMBN (532). Additional information on the evolution of SCPP genes, and their role in tooth formation, can be found in a number of recent papers (104, 169, 170, 280–288, 532–534).

The human genome contains two amelogenin genes, one located on the X chromosome (AMELX: locus Xp22.3-p22.1) and the second on the Y chromosome (AMELY: locus Yp11) (325, 492, 496). Both the X and Y amelogenins are expressed in males; however, the X-chromosome-derived amelogenin is expressed at significantly higher levels (492). It is estimated that >90% of the amelogenin gene transcripts in male primates are derived from the X chromosome (103, 428, 492). The close proximity of SPARCL1, AMBN, ENAM, AMTN, and ODAM on the human chromosome 4q13-q21.1 has resulted in detailed investigation of this chromosome region by enamel researchers as it hosts genes responsible for inherited dental diseases (94, 165, 228, 561, 641). This region contains many of the genes responsible for the mineralization of hard tissues (285, 287, 288, 532, 534).

For significance, since amelogenin is expressed on both the X and Y chromosome in some mammals such as primates, cow, pig, horse, and sheep (175, 209, 256, 325, 400, 448, 462, 492), the nucleotide differences between the X- and Y-derived amelogenins allow for quick, PCR-based, sex determination in utero, or in forensic medicine (129, 159, 428, 611).

V. ENAMEL MATRIX PROTEINS AND WHAT WE HAVE LEARNED FROM ANIMAL MODELS

A. Overview

The most abundant of the secreted structural proteins of the enamel matrix are amelogenin (AMELX), ameloblastin (AMBN), and enamelin (ENAM) (228, 428, 430). While some studies have suggested that AMELX (112, 196, 211) and AMBN (152, 557) are expressed in nondental tissues in nonpathological states, there is wide consensus that all three proteins are most highly expressed in the enamel organ. Moreover, Amelx (178), Ambn (164), and Enam (230, 231) mutant mice show pathologies that appear to be limited only to the dental enamel, suggesting that the levels and biological roles of these two proteins in nondental tissues are negligible. Our current understanding is that AMELX, AMBN, and ENAM are the major secreted products of secretory-stage ameloblasts, while AMTN and ODAM are secreted by maturation-stage ameloblasts.

It has been estimated that amelogenin proteins contribute ~90% of the enamel organic matrix, based on both protein analyses and unbiased RNA and protein profiling (67, 305, 364, 475, 530, 591). AMBN composes ~8–10% of the enamel organic matrix (369), while ENAM appears to be present in trace amounts only (66, 369). These figures for relative protein levels also appear reasonable when looking at mRNA levels; for example, mRNA profiling from a rat enamel organ demonstrated that 20.0% of all gene transcripts were to Amelx and 2.9% of gene transcripts were to Ambn (305, 364). At the time of this study the “ameloblastin” mRNAs identified were to an unknown gene, and designated as only as clones Y224, Y243, and Y275 (364), but further characterization resulted in the cloning of a full-length Ambn transcript, and the subsequent naming of this gene (305).

Knockout or mutant mouse models for all these enamel-specific genes have been generated and all appear healthy and are fertile. The enamel of Amelx (180), Ambn (164), and Enam (230) mutant mice are severely impacted, showing disorganized enamel; these mice require a soft diet because the occlusal surfaces of their teeth wear easily. The Amtn and Odam mutant mice show either only a mild phenotype (Amtn-null mice) (402) or no apparent phenotype (Odam-null mice) (623).

B. Amelx Mutant Mice

Amelogenin (AMELX), first identified in 1983, has been the most studied of the enamel-specific proteins (553, 555). It was the first enamel-specific cDNA subclone to which a protein sequence could be identified (555), and specific antibodies against mouse Amelx were subsequently generated (537). AMELX assembles into multimeric units in vitro and possibly the extracellular space (151) that are widely referred to as “nanospheres” (51, 58, 79, 124, 136, 137, 148, 345, 493, 588). A number of three-dimensional models have been proposed for the assembly of amelogenin into nanospheres (124, 136, 138, 212, 389), and while these models vary in detail, all suggest that nanospheres are of the order of 20–30 nm in diameter and may contain ~12 (136) or more (124) amelogenin monomers.

A conventional targeted knockout approach produced Amelx-null mice, which had a dramatic phenotype limited to the enamel organ (178). While a thin layer of enamel was observed in these mutant mice, it lacked any prismatic architecture, and the thickness was ~20% (i.e., 1/5) that of normal enamel (177, 178). In addition, in the Amelx-null mice, the dimensions of individual enamel crystallites were smaller than in wild-type enamel (652).

Another feature of amelogenin gene products is the large number (>15) of alternatively spliced isoforms that have been identified based on mRNA profiling (26, 516, 528, 529, 653). Of these spliced isoforms, the most abundant in mice is referred to as M180, followed by the leucine-rich amelogenin peptide (LRAP) (176, 179, 326, 391, 529, 656) (FIGURE 7). Transgenic mice overexpressing either M180 or LRAP in the enamel organ have been generated and bred with the Amelx-null mice, and this has resulted in varying degrees of rescue of the enamel deficiencies, partially restoring both the prismatic architecture and crystallite dimensions (177, 652). M180 knockin mice show a normal enamel function and architecture as observed by electron microscopy (EM); however, the mechanical properties of the enamel were altered such that the hardness increased by 7%, and the fracture toughness decreased by 22% when analyzed by microhardness tests (556). Hardness has been considered as a surrogate for wear resistance, while toughness is a measure for fracture resistance (154, 556, 634). This M180 knockin mouse suggests that the inclusion of the other amelogenin spliced isoforms may contribute to the overall functional and structural properties of enamel under normal circumstances.

FIGURE 7.

Mouse amelogenin (Amelx) protein-protein interacting domains. The protein sequence of the most abundant Amelx isoform is shown in A (REFSEQ accession number NM_009666.4). The mature protein (after the signal peptide shown in red is removed) contains 180 amino acids and is frequently referred to as mouse M180 isoform. Domains A and B were defined using the yeast two-hybrid system. Interacting domains A and B are identified and underlined. The leucine-rich amelogenin peptide (LRAP) is the second most abundant amelogenin produced as a product from alternative splicing and is shown in B (without its signal peptide). This LRAP isoform contains the NH₂-terminal 33 amino acids and the COOH-terminal 26 amino acids of the sequence in A. This LRAP isoform has been referred to as either LRAP, or the mouse M59 isoform. The “#” indicates the spliced union of the NH₂- and COOH-terminal regions in LRAP.

Additionally, a number of transgenic mouse lines have been created to study the disruption of amelogenin self-assembly. Prior studies using the yeast two-hybrid system have shown that the M180 amelogenin proteins self-assemble, thanks to the amino-terminal 42 residues (the so-called A domain) interacting either directly or indirectly with a 17-residue domain (the so-called B domain) in the carboxy region (427) (FIGURE 7). Transgenic mice bearing mutant amelogenin transcripts that lack either the A or B domain show disruptions to the crystallite orientation and prismatic architecture (126, 425, 431).

The primary conclusion from all of the amelogenin mouse models is that while amelogenin is not responsible for hydroxyapatite (Hap) nucleation or growth, it is essential for normal and controlled enamel crystallite growth and crystallite orientation on the nanoscale, and rod/interrod or prismatic architecture on the microscale (652).

C. Ambn Mutant Mice

Ameloblastin was first identified around 1996 by three independent research groups, and given the names ameloblastin (305), amelin (72), and sheathlin (227). Current nomenclature refers to this gene as ameloblastin (AMBN). An ameloblastin (Ambn) mutant animal model was generated in 2004, and at the time was thought to be a complete knockout/silencing of any ameloblastin expression (164). However, it was later shown that this line expressed only a truncated version of ameloblastin missing exons 5 and 6 (624). This mouse model did have a severe dental phenotype (164). The ameloblasts differentiated to polarized secretory ameloblasts but then quickly detached from the enamel matrix and lost their polarity, resulting in the termination of amelogenesis and the failure to produce any enamel. These data suggest that ameloblastin plays a role in cell-matrix attachment and the maintenance of the ameloblast differential state (164). These Ambn-mutant mice have been bred with a transgenic mouse that overexpresses ameloblastin in the enamel organ, and the resulting enamel appears normal (90), suggesting a complete or near-complete rescue of the enamel phenotype.

D. Enam Mutant Mice

Enamelin was first identified in 1997 (226), and the first publication of the Enam-null mice, referred to as the Enam knockout NLS-lacZ knockin, was in 2008 (230). In addition to achieving a complete elimination of any Enam expression, the targeting vector included a lacZ (β-galactosidase) reporter with a mouse nuclear localization signal (NLS) (229, 230). Enam^−/− mice showed no “true enamel” (230) based on various imaging techniques (e.g., radiography, microcomputed tomography, and light and scanning electron microscopy); instead, a “thin, highly irregular, mineralized crust covered the dentin on erupted teeth” (230).

E. Amtn Mutant Mice

Amelotin (AMTN) was first identified in 2005 (255). Amtn-null mice have also been generated and studied recently (402). In these Amtn mutant mice, the enamel prismatic structure appeared unaltered; however, enamel mineralization was delayed, resulting in hypomineralization of the inner enamel and structural defects in the outer enamel (402).

F. Odam Mutant Mice

The odontogenic, ameloblast-associated gene (ODAM) was identified in 2006 and initially referred to as APin (385). Odam-null mice have recently been generated (623). This mutant line is a complete knockout with a functional insertion of the β-galactosidase gene with an amino-terminal nuclear localization signal (NLS-LacZ). In these mutant mice, the spatiotemporal expression of β-galactosidase relates directly to (i.e., copies) the Odam expression pattern, which is limited to late secretory-, transition-, and maturation-stage ameloblasts, and is also expressed in the cells of the dental junctional epithelium (JE) (386, 406, 623). The JE is the region where the oral epithelium unites with the tooth surface, thus forming a unique barrier or seal between the oral cavity and the underlying tissues (403, 407). These Odam-null mice have no observable enamel phenotype (623), but as they age there is an increased inflammatory infiltrate in the JE, and an apical down-growth of the JE typical of periodontal disease (407, 623). These findings suggest that ODAM expression in the dental JE helps maintain the integrity of the JE attachment (407, 623). It is therefore tempting to speculate that pathological mutations to Odam may increase the risk of periodontal disease.

VI. ENAMEL MATRIX ASSEMBLY

A. Amelogenin Self-Assemblies

The enamel matrix is composed primarily of three secreted structural proteins: AMELX, AMBN, and ENAM (359, 389, 430). AMELX has a single phosphorylated serine located at the amino-terminal region (149, 578). AMBN and ENAM are both glycoproteins (72, 153, 226, 305, 658) and likely account for reports indicating the existence of proteoglycans in the forming of the enamel matrix (77, 184, 190). Biglycan (BGN) has also been shown to be expressed in secretory ameloblasts, but at barely detectable levels (189). There have also been reports that phospholipids contribute to the enamel matrix (459, 514, 515). However, if these phospholipids are indeed extracellular (186), then it is unclear what role they play in amelogenesis.

Likely because amelogenin was the first enamel matrix protein cloned, and because of its abundance, most of the literature related to enamel matrix assembly has focused on amelogenin self-assembly properties (147, 148, 390, 423, 674). Transmission electron microscopy (TEM) of mouse, bovine, and hamster dental enamel tissues showed electron-lucent spherical structures/aggregates of (presumably) amelogenin that aligned with long axes of developing enamel crystallites (147, 148). In vitro studies confirmed the formation of amelogenin nanospheres with 10- to 15-nm radii (20–30 nm diameter) using native or recombinant amelogenins in aqueous solutions (150, 151). Further studies by Paine et al., using the yeast two-hybrid system and a series of systematic amino-terminal and carboxy-terminal deletions of a full-length Amelx, suggested that self-assembly of amelogenin occurs via two well-defined domains referred to as the amino-terminal “A” domain and the carboxy-region “B” domain (427, 431) (FIGURE 7). Such Amelx-Amelx binding domains would allow for the formation of nanospheres containing clusters of amelogenins, and hydrophobic and hydrophilic constraints could help define their shape and size. It is because of these self-assembly properties of Amelx and its hydrophobic character that it is only sparingly soluble in physiological conditions, and requires extreme pH conditions to show significant dissolution (340, 427, 523). In vitro, using recombinant amelogenins, self-assembly into nanospheres is a phenomenon that can occur at physiological or near-physiological pH values (pH range of 7.2–7.7) (40, 630, 636), but only in the absence of Ca²⁺ and PO₄³⁻ (212, 361, 362, 493) (FIGURE 8). Nanospheres disassemble in the presence of mineralizing ions and appear to attach to apatite surfaces as monomers or dimers (162, 212, 361, 362). It also seems that as soon as amelogenin is secreted into the extracellular space in vivo, it is processed by MMP20 into specific cleavage products with unknown functions. Ultimately amelogenin is severely hydrolyzed by KLK4 and the resulting peptide debris is removed from the enamel matrix through endocytosis (29, 348). There are reports that in vitro amelogenin may form microribbons a few micrometers in width and hundreds of micrometers long (124, 388, 389), although this remains controversial (123, 124).

FIGURE 8.

Micrographs of amelogenin assemblies. Micrographs of recombinant full-length amelogenin protein assembled in vitro without calcium and phosphate ions (A) and with addition of Ca²⁺ (30 mM) (B) and PO₄³⁻ (22 mM) (C). In the absence of mineralizing ions, amelogenin forms nanospheres with diameters between 15 and 30 nm (A). With the addition of calcium and phosphate ions, nanospheres disintegrate and at pH between 4.0 and 6.5 transform into nanoribbons over periods of days (B). Ribbons measure ~17 nm in width and are ~3–4 nm thick. The ribbons have a tendency to align themselves into parallel arrays and form bundles (C) that can reach several 10 s of micrometers in length. [C from Martinez-Avila et al. (361). Copyright 2012 American Chemical Society.]

While the general consensus has been that the supramolecular structures in the enamel matrix are critical to controlling the organization of apatite crystals in enamel (150, 338, 430, 431, 527), recent observations of nanoribbons developed from full-length AMELX proteins with the ability to self-organize challenge this paradigm (FIGURE 8). Amelogenin nanoribbons from recombinant human proteins form over a period of days. They are ~17 nm wide and align parallel to each other, maintaining a 5- to 20-nm space between them (212, 361). Bundles of aligned AMELX nanoribbons up to 100 µm in length develop over a period of 1–3 wk, producing an organic scaffold that mimics the appearance of apatite nanofibers in an enamel rod (70, 361, 362). This suggests that aligned AMELX nanoribbons may be a precursor to enamel rods and provide a template for guided apatite crystal growth (198). Interestingly, self-assembly of AMELX into nanoribbons is dependent on Ca²⁺ and PO₄³⁻. These mineralizing ions build ion bridges between AMELX dimers and thus stabilize the formation of anti-parallel β-sheets comparable to amyloid fibers known from neurodegenerative diseases (70, 493). This in vitro observation is in agreement with X-ray diffraction analyses of developing enamel, showing evidence of cross-β structures characteristic of amyloids (182, 266, 442, 446). The presence of a functional amyloid was further supported by positive staining for Congo Red in enamel of Klk4^−/− mice (70). High-resolution images are often dominated by filamentous structures that have been attributed to the early apatite crystal ribbons that develop during secretory stage but remain present even when the specimen is demineralized (596).

A major difficulty in deciphering the exact mechanisms of protein-controlled mineralization lies in the transient nature of the enamel matrix, which is rapidly processed soon after secretion and is almost completely removed by the end of the mineralization process. Further analysis of the biological structures in the developing matrix and revision of current models of amelogenin-guided mineralization are warranted. Models of amelogenin nanosphere formation and nanosphere-crystallite interaction have been widely used in the enamel research community and have been reviewed by others (387). A comparison of the nanosphere model (662) to a model based on new data on in vitro nanoribbon assemblies and how they might guide crystallite growth is presented in FIGURE 9.

FIGURE 9.

Proposed models of amelogenin-directed growth of apatite mineral during secretory stage of amelogenesis. Comparison of two models of amelogenin-directed growth of apatite mineral during secretory stage of amelogenesis. A: nanosphere model. Amelogenin protein is secreted into the extracellular space and assembles into nanospheres. Amelogenin stabilizes prenucelation clusters of calcium phosphates. Nanospheres align into chainlike structures along which amorphous calcium phosphate develops and through a ripening process transforms into crystalline Hap. Nanospheres act as spacers, as originally proposed by Fincham et al. (150). [A from Yang et al. (662), copyright 2010 American Chemical Society.] B: nanoribbon-directed crystal growth. Amelogenin is secreted from vesicles, possibly in the form of antiparallel dimers. Nanoribbon assembly is triggered by formation of ion bridges across dimers with Ca²⁺ and PO₄³⁻. Dimers are added to the existing amelogenin ribbons as soon as they are exocytosed and thus elongate the ribbons as the ameloblasts migrate away from the mineralization front. Hap mineral forms at a narrow distance from the cell membrane in form of thin ribbons that develop on the backbone of the protein ribbons. Full-length amelogenin does not induce mineral formation directly, and the mechanism of mineral nucleation is not known. Interaction with other non-amelogenin molecules and/or the processing by MMP20 may be required for guided growth of apatite onto amelogenin ribbons.

B. Other Protein-Protein Interactions of the Enamel Matrix

Ameloblastin has been suggested to be a cell adhesion molecule that can influence ameloblast growth and differentiation (164). Observations from Ambn-mutant mice showed that in the absence of a fully functional Ambn protein, presecretory ameloblasts could differentiate into polarized secretory cells, but these cells quickly detached from the forming enamel matrix to form multicell layers that occasionally (~20% of mice) proceeded to form odontogenic tumors (164). Some reports have also suggested AMBN may act as a signaling molecule or a growth factor (38, 152, 587, 670). Using a bacterially generated recombinant human AMBN, Wald et al. (612) have shown that, similar to AMELX, AMBN under certain nonphysiological conditions can form flat ribbon-like supramolecular structures (width and thickness of ~18 nm and 0.34 nm, respectively) and of varying length. It should be noted that eukaryotic-generated AMBN is present as a glycosylated protein, but this naturally occurring glycoprotein cannot be produced from bacteria; the self-assembly property seen in vitro may not recapitulate in vivo events. Of note also is that earlier studies by Paine et al. (422), using the yeast two-hybrid system, suggested that a eukaryotic-generated mouse Ambn had no self-interacting properties, questioning further whether Ambn ribbons may be present in vivo. These data shed some light on the role of AMBN in enamel formation, but the exact role remains unclear. What we do know is that Ambn mutant mice fail to produce any enamel, or indeed any mineralized tissue, from the enamel-producing cells (164).

A number of studies have investigated self-assembly properties of the different secreted enamel proteins and protein-protein interactions between them. As discussed above, using the yeast two-hybrid system, amelogenin-amelogenin interactions could be demonstrated (422) and interacting domains identified (427). In a paper by Holcroft and Ganss, also using the yeast two-hybrid system and cloned bovine enamel matrix protein sequences, it was shown that full-length AMTN could self-assemble, as could full-length ODAM, and ODAM could also interact directly with AMTN and AMELX (220). There is also one report, using in vivo-derived porcine enamel proteins, that suggested amelogenin interacts with the 32-kDa fragment of ENAM (659). Enam-null mice lack the formation of a mineralized layer, supporting the notion that ENAM may be critical to apatite nucleation (230). Similar to mineralizing collagen fibrils in bone and phosphoproteins in dentin, a mechanism of protein interaction can be suspected where an acidic or phosphorylated protein may act as a carrier, delivering mineral ions to nucleation sites on self-assembled protein scaffolds. In a similar fashion, ENAM may interact with AMELX supramolecular structures. However, work on the protein-protein interactions of the less abundant enamel matrix proteins remains in its infancy.

There have been two reports of either AMELX or ENAM interacting with members of the collagen family (111, 614), suggesting that AMELX interacts with COL1A1 (111) and COL5A3 (614), and that ENAM interacts with COL2A1 and COL5A1 (614). Collagens are a product of odontoblasts and present in dentin, while the amelogenins are a product of ameloblasts and found in the enamel. It has been shown that type IV collagen is expressed directly at the DEJ (371), and that type 1 collagen (341) and type VII (372) collagen pass from the dentin through the DEJ and into the enamel. The significance of such amelogenin-collagen or ENAM-collagen interactions, or the extension of dentin collagens into the inner enamel matrix, could help explain the significance and unusual structural and biomechanical properties of the DEJ (249, 371, 430, 633, 635). The DEJ is a unique structure of the tooth that functions to hold the enamel onto the dentin surface (34, 75, 166, 403, 635). While the DEJ is a critical component of the tooth, the biology and developmental mechanisms involved in its formation are not well understood and are beyond the scope of this review.

VII. ENAMEL-SPECIFIC PROTEOLYTIC ENZYMES

A. Overview

Although a number of proteinases have been described in amelogenesis, including matrix metallopeptidases 2, 3, and 4 (MMP2, MMP3, and MMP9) (187, 199), chymotrypsin C (CTRC) (322) and cathepsin C (CTSC, also known as dipeptidyl peptidase I) (601), most of the information on proteinase expression during amelogenesis relates to MMP20 (formerly known as enamelysin) and kallikrein-related peptidase 4 (KLK4) (27, 30, 235, 348). MMP20 expression dominates during the secretory stage (28, 29, 56, 233), and KLK4 expression during the maturation stage (29, 232, 233, 348). Two noteworthy reviews on enamel proteinases have been published (25, 348). Autosomal recessive forms of nonsyndromic amelogenesis imperfecta (AI) have been documented for mutations associated with both MMP20 and KLK4, and mutant animal models attest to the important role both enzymes play in amelogenesis.

B. Mmp20 Mutant Mice

Mmp20-null mice were first reported in 2002 (71), and work on this animal model continues today (454, 521). These mice have a severe phenotype, with the enamel being hypoplastic and delaminating from the dentin soon after the tooth erupts into the oral cavity (71). The ameloblast morphology is clearly disrupted early during secretory-stage amelogenesis, and the normal rod-interrod pattern of fully formed enamel is also disturbed (71). Thus it is clear that MMP20 is critically important not just for proper enamel formation, but also for the integrity of the DEJ. Amelogenins form the bulk of the enamel matrix (29, 103, 428, 492); thus AMELX is seemingly an obvious substrate for MMP20 in the developing enamel. Multiple in vitro studies, using in vivo-derived or recombinant proteins, have confirmed that AMELX is indeed a major substrate of MMP20, suggesting this is also the case in vivo during amelogenesis (344, 488, 524, 656). MMP20 has also been shown to effectively cleave AMBN in vivo (91, 257). It is unclear today how the third major enamel protein, ENAM, is processed and degraded post-secretion (25, 657).

C. Klk4 Mutant Mice

Klk4-null mice were first reported in 2009 (525) and showed an enamel hypomaturation phenotype (normal thickness but poorly mineralized) that, post-eruption, either quickly abrades, or fractures just above the DEJ (525). This suggests that KLK4 plays no role in the integrity of the DEJ. Although a rod-interrod morphology could be observed in Klk4 mutant mice, individual enamel crystallites failed to pack tightly with neighboring crystallites, and gave the impression that they “spilled out” following controlled enamel fracture (525). KLK4 has broad substrate specificity and readily degrades the known enamel matrix proteins (348, 399). KLK4 expression in the mouse incisor starts during the transition stage and continues throughout the maturation stage; thus mice that do not express Klk4 retain much of the enamel organic matrix, resulting in hypomineralized enamel (525, 660).

VIII. RESORPTIVE ACTIVITIES IN AMELOGENESIS

A. Overview of Endocytotic and Other Resorptive Processes

Endocytosis can be either receptor mediated or fluid phase (4, 541), with receptor-mediated endocytosis most typically defined as a clathrin-dependent process (4). This is in part because the endocytotic cellular uptake of extracellular proteins frequently involves clathrin assemblies and clathrin adaptor protein (AP) complexes that are generally activated and assembled by a membrane-bound receptor-mediated event such as ligand binding. Fluid-phase endocytosis involves multiple, relatively low-energy and nonspecific, cellular activities that allow for the uptake of fluids from the extracellular environment, and do not appear to be initiated by receptor-ligand activities (9, 119, 291). Fluid-phase endocytosis involves a number of molecular activities of clathrin-independent pathways, including the CLIC/GEEC endocytotic pathway, the arf6-dependent pathway, flotillin-dependent endocytosis, micropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis (119). As the molecular mechanisms for each of these fluid-phase activities are being better defined, it has become clear that they have some specificity as to what molecules and extracellular debris they each target for cellular uptake (9, 49, 119, 291, 327, 404).

B. Early Concepts of Resorptive Activities of the Enamel Organ

Endocytotic activities in amelogenesis have not been extensively studied. However, on the basis of EM observations, papers of the late 1970s and early 1980s described coated pits and/or vesicles on the cytoplasmic surface of the apical pole of ameloblasts in both the secretory (160, 499, 500) and maturation stages [including both the smooth-ended (505) and ruffle-ended (498) phases] of amelogenesis. Clathrin was first discovered in 1976 associated with coated vesicles (443); thus the coated pits and vesicles described in these earlier enamel studies likely represent the clathrin-coated vesicles that are recognized today (313). Other earlier studies relating to the active and passive resorptive functions of ameloblasts have recently been discussed by Lacruz et al. (313), and the reader is referred to this paper for a historical perspective and relevant citations.

C. Adaptor Protein Complex 2 (AP-2) Endocytosis in Amelogenesis

Earlier EM observations in the 1970s and 1980s of coated pits and/or vesicles forming on the inner surface of ameloblast cells (160, 498–500) were suggestive of clathrin-associated endocytotic activities being a feature of amelogenesis, and more recently published immunolocalization and real-time PCR data indicate the same conclusion (313). Lacruz et al. (313) clearly established that active, AP-2 mediated, clathrin-dependent endocytosis occurs during amelogenesis and that during amelogenesis the greatest expression of AP-2 and clathrin is noted at the apical poles of maturation ameloblasts.

AP-2-mediated endocytosis is a clathrin-dependent activity and is widely considered to be receptor-mediated (324, 584–586). Known receptors include transferrin receptor (Tfrc), the low-density lipoprotein receptor (Ldlr), and the epidermal growth factor receptor (Egfr) (50, 368, 394). When comparing the transcriptomes enamel organ cells in the rat incisor, it was noted that Tfrc transcripts increased significantly (∼60-fold) from the maturation to the the secretory stage (319, 664). It has also been shown that the iron transport protein transferrin (Tf) is a potential protein binding partner of Enam (614). Although significantly more work is required in this field, it is reasonable to suggest that the removal of the enamel matrix debris could occur through direct protein-peptide interactions between Tf and the EMP debris, resulting in an EMP/Tf/Tfrc-initiated AP-2 endocytotic pathway (313). This scenario is feasible as multiple protein partners of Tf, in addition to enamelin, have already been identified; these include the GABA(A) receptor-associated protein (Gabarap) (195), leukocyte cell-derived chemotaxin 2 (Lect2) (82), insulin-like growth factor 1 and 2 (Igf1 and Igf2) (566), and insulin-like growth factor binding proteins 1–6 (Igfbp1-6) (629).

Lamp1, Lamp2, and Cd63 have individually and collectively been suggested as possible membrane-bound protein receptors initiating the AP endocytotic pathway by direct interaction with the various AP complexes (43, 50, 223, 444, 480, 483). LAMP-AP complex formation and the subsequent trafficking of Lamp1, Lamp2, and Cd63 from the cell membrane to the lysosome is initiated by a direct protein-protein interaction between a lysosomal targeting motif (GYXX∅; where X is any amino acid and ∅ is a bulky hydrophobic amino acid) located at the cytoplasmically contained carboxy terminus of all three LAMPs (Lamp1, Lamp2, and Cd63), and the mu/μ subunit of AP-2 (Ap2m1) (113, 223, 248, 324, 452, 483). Zou et al. (675) have previously shown that Amelx, through a proline-rich region (PLSPILPELPLEAW), interacts directly with Lamp1, Lamp2, and Cd63 through an extracellular 20-amino acid domain with high homology common to all three LAMP proteins. In Cd63 this binding domain is contained within the externalized “EC2” domain (675). This proline-rich Amelx binding region is hydrophobic, largely disordered, and accessible to the external environment (675). The externalized EC2 domain of Cd63 also interacts with full-length Enam and Ambn (614, 675); thus it is feasible that the LAMP proteins could act as ameloblast receptors for AP-2, clathrin-dependent endocytosis, but this needs further investigations. A schematic of the two scenarios presented for EMP initiated endocytosis is presented in FIGURE 10.

FIGURE 10.

Hypothetical model for the initiation of AP-2, clathrin-mediated endocytosis of the degraded enamel matrix protein (EMP) debris in maturation ameloblasts. It is apparent that greater endocytotic activity is seen in the maturation ameloblasts, when compared with secretory ameloblasts. Endocytosis is likely a feature of both the ruffle-ended (RA) and smooth-ended (SA) ameloblasts. The figure illustrates that the endocytotic uptake may be initiated by direct receptor-ligand interaction, such as the EMP debris interacting directly with LAMP1, LAMP2, or CD63. Alternatively, EMP debris may bind to another EMP protein, such as TF, and then this complex may bind to the TFRC to initiate the uptake of the extracellular enamel matrix peptides. A dark gray double capsule represents a tight junctional complex at the apical region of RA, and a light gray double capsule represents a “leaky” junctional complex at the apical region of SA.

Another established pathway for the uptake of Tf located at the apical pole of some polarized epithelia (e.g., the small intestine, renal proximal tubule, visceral yolk sac, and placental cytotrophoblasts) is through the megalin-dependent cubilin-mediated endocytotic pathway (87, 88, 304, 384, 489), although to date there are no published data suggesting that this endocytotic pathway is active in the enamel forming cells (313, 318, 319).

D. Summary

Recent data indicate that AP-2-mediated, clathrin-dependent endocytosis is a prominent feature of maturation-stage amelogenesis (313), and while other resorptive processes, such as micropinocytosis, may also be active in amelogenesis, they are yet to be investigated at the molecular level. The process of enamel matrix removal is a significant topic worthy of investigation because the failure to properly remove the organic enamel matrix results in a hypomineralized enamel that is mechanically inferior and wears and fractures rapidly, as seen in Mmp20-null and Klk4-null mice (71, 525, 660).

IX. IMPORTANCE OF pH MAINTENANCE

A. Regulation of pH

Nucleation events leading to the development of enamel crystals require the formation of a stable cluster of ions that can organize and grow (127). For every unit cell of hydroxyapatite crystal, approximately eight H⁺ protons are released into the extracellular environment, thus lowering the pH in the immediate surroundings (541). This calculation is based on the stoichiometric equation shown in Equation 1 (above) (523, 541). To modulate the effects of these free protons, enamel cells use active bicarbonate (HCO₃⁻) transport systems to regulate the extracellular pH (269, 316, 317, 429, 541, 542). Here we describe changes in pH during amelogenesis and review key components of the HCO₃⁻ transport system.

B. Changes in pH During Amelogenesis

Much of the available data on pH in enamel derive from chemical tools that in some cases are outdated. For example, injections of [¹⁴C]dimethyl-oxazolidinedione (DMO), a compound that concentrates in areas of high pH, showed higher extracellular pH ~8.0–8.5 in the more matured areas of mouse enamel than in less mineralized areas where pH ~7.3–7.4 (352). This difference was associated with increased calcification as Ca²⁺ binding to protein matrix generates high local pH, which in turn allows for the accumulation of PO₄³⁻ and OH⁻ ions enabling the initiation of crystal nucleation (352).

Using GBHA, Takano et al. (581) showed a pattern of red bands on the surface of matured enamel marking alkaline conditions. GBHA positively stained around bands of SA cells (581). More recently, Sasaki et al. (497) used three different pH indicator solutions to assess pH changes in unerupted whole bovine incisors after the removal of the enamel organ and showed alternating bands of acidic to neutral extracellular pH along the crown. These acidic and neutral zones were examined by suspension in distilled de-ionized water to measure pH using a glass-electrode pH meter. A number of halved incisors were stained with GBHA. Results from each technique were consistent showing extracellular pH conditions ranging from acidic (pH 5.5–6.0) to neutral zones (pH ~7.2) with the acidic zone located in the occlusal half of the crown. It was hypothesized that the acidic conditions observed related directly to the release of protons by the forming crystals (497).

Analysis of developing bovine incisors with the use of pH indicator solutions identified four different and alternating stages of acidic and neutral pH along the crown (577). The purified protein content from each stage showed that neutral zones of enamel were characterized by the presence of full-molecular-weight forms of AMELX and ENAM, whereas acidic zones showed low-molecular-weight forms of both proteins. The composition of crystals changed between the alternating acidic-neutral stages based on ratios (Ca²⁺ + Mg²⁺)/P with erupted enamel having a higher ratio than either the acidic or neutral unerupted enamel (577). Moreover, freeze-dried strips of rat incisor enamel organ isolated from various stages of amelogenesis were assessed based on a distance from the tooth apex (using a molar reference line) and provided relatively uniform pH during secretory stage with values clustering around ~7.23, whereas maturation stage samples showed greater variability in extracellular pH values ranging from near-neutral to weakly acidic conditions (pH values 6.2–7.2) (544). Recently, the immersion of rodent incisors with the enamel organ exposed in pH indicator solutions has become common practice to determine the presence of alternating bands of RA and SA cells. Low pH is associated with RA cells.

Taken together, these studies suggest that pH in enamel oscillates from neutral to acidic during maturation-stage amelogenesis, whereas in secretory stage the pH remains near the physiological levels.

C. Regulation of Extracellular pH

Ameloblasts use a number of acid-base transport mechanisms to modulate extracellular pH. These include bicarbonate transporters, carbonic anhydrases, and chloride channels as well as other ion pumps and exchangers. As we have previously discussed (316), the acidification of the extracellular microenvironment associated with the release of H⁺ is a complex event.

D. Proteins Involved in pH Balance in Enamel

1. Bicarbonate transporters

Two genes of the SLC4 (solute carrier 4) gene family, the anion-exchanger (AE2) encoded by SLC4A2 and the electrogenic bicarbonate cotransporter (NBCe1) encoded by SLC4A4, are expressed in enamel cells and associated with pH modulation (61, 258, 269, 317, 350, 429). Both are membrane proteins that play an important role in regulating intra- and extracellular pH in eukaryotic cells (460). In addition, five members of the SLC26A gene family (SLC26A1, SLC26A3, SLC26A4, SLC26A6, and SLC26A7), all membrane-bound ion exchangers (or HCO₃⁻/Cl⁻ exchangers), have recently been described as being expressed at the apical pole of maturation ameloblasts (60, 259, 665).

The first reports of AE2 and NBCe1 expression in enamel cells were by Paine et al. (429) and Lyaruu et al. (350). In the study by Paine et al. (429), NBCe1-B (the alternatively spliced B isoform of NBCe1) expression in ameloblasts was found primarily at the basolateral pole of maturation-stage ameloblasts, whereas AE2 showed a more apical distribution. Other reports have confirmed the localization of these proteins, albeit showing variation in the localization of the different NCBe1 isoforms, with some isoforms being found primarily in the adjacent enamel papillary layer cells (258, 269). It has also been suggested that NBCe1 expression might be associated with the developmental stage of ameloblasts (258). Paine et al. (429), using the immortalized ameloblasts-like cell line LS8, found that the mRNA expression levels of both NBCe1 and AE2 changed depending on extracellular pH.

Paine et al. (429) observed AE2 in the apical pole of secretory ameloblasts in frozen-unfixed tissues, while Lyaruu et al. (350) and Yin et al. (665) reported a basolateral localization in maturation ameloblasts, leaving open the question of AE2 function (527). If AE2 is localized at the basolateral pole rather than at the apical end of the cell, the latter being closest to the enamel-forming zone, the HCO₃⁻ that has moved out of the basolateral cell membrane needs to find its way to the enamel across tight apical cell junctions to perform its putative pH buffering role. This path of movement is not necessary if AE2 is localized to the apical end (see below). However, a number of studies suggest that NBCe1 likely plays a role in mediating basolateral HCO₃⁻ import with apical bicarbonate secretion mediated by AE2 or other HCO₃⁻ export pumps/channels/exchangers, working in tandem to buffer the proton load generated by apatite formation (258, 269, 316, 320, 429). More recently, a number of members of the anion exchanger SLC26A gene family (SLC26A1, 3, 4, 6 and 7; HCO₃⁻/Cl⁻ exchangers) have been localized to the apical membrane of maturation ameloblasts (259, 665), and this large number of exchangers with similar or identical molecular activities ensures abundant opportunity for HCO₃⁻ export to the enamel matrix during enamel maturation.

Mutations to SLC4A2 and SLC4A4 result in enamel abnormalities in humans and/or mice (117, 172, 251). Lyaruu et al. (350) showed that mice lacking two of the five AE2 spliced variants (AE2a/b) have abnormal enamel in incisors, but this defect is less severe in molar teeth. There are three variants of NBCe1 (NBCe1-A, NBCe1-B, and NBCe1-C) with mutations occurring in all variants. The incisors of mice lacking NBCe1 have a chalky white appearance and fracture easily (171, 317). In patients with loss of NBCe1 function, enamel defects have been described as showing white chalk-like spots (251). Mouse models deficient for AE2 or NBCe1 both showed decreased mineral content in their enamel (65, 317, 350).

The function and role of ameloblasts as HCO₃⁻ transporting cells, and in particular that of the Na⁺/HCO₃⁻ cotransporter NBCe1, have been enhanced by in vitro studies using the ameloblast-like cell line HAT7 (52). In these studies, HAT7 cells were manipulated to form a polarized two-dimensional culture system from which transepithelial electrical resistance, immunocytochemistry, and microfluorometry data could be collected. Polarized HAT7 cells expressed NBCe1, a number of anion exchangers already discussed (Slc4a2/AE2, Slc26a4/pendrin and Slc26a6/Pat1), and Cftr. Active transcellular vectorial basolateral-to-apical HCO₃⁻ transport was recorded, and this vectorial movement of HCO₃⁻ was dependent on Na⁺ cotransport (52). One of the conclusions from this study was that “a basolateral HCO₃⁻ transporter, most probably NBCe1/SLC4A4, has an important role in HCO₃⁻ uptake.” A similar conclusion was also published almost a decade earlier by Paine et al. (429) who, based on immunolocalization data, stated that “NBCe1 is expressed on the basolateral membrane of secretory ameloblasts” and that “AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.” Both studies by Bori et al. (52) and Paine et al. (429) strongly indicate that the basolaterally expressed Na⁺/HCO₃⁻ cotransporter NBCe1 is either fully or partially responsible for the import of HCO₃⁻ into polarized ameloblasts, and is most active during maturation-stage amelogenesis (317, 318).

2. Chloride transport

Chloride transport in epithelial cells is an important regulator of salt and water (513). Chloride channels in the apical surface of the cells’ plasma membrane allow the flow of Cl⁻ across the cell membrane via an electrochemical gradient. In cystic fibrosis (CF), an autosomal recessive disease affecting 1 in ~3,000 births, the cystic fibrosis conductance transmembrane regulator protein (CFTR), which regulates water and chloride transport, is disrupted (122, 558, 564, 565). Chloride (Cl⁻) therefore accumulates inside the cells, leading to abnormal and thick mucus secretion in the airways. Mutations to the CF gene also affect the dentition (21, 23, 63, 76, 96, 143, 445, 646, 650).

Mineralized enamel contains ~0.0065 mol/g of Cl⁻, totaling 0.23% of enamel by dry weight (128). The role of Cl⁻ in forming enamel crystals is poorly understood, but it has been suggested that it may act as a transmitter of charge (412) and as a regulator of pH (59). The first reports on abnormal enamel in CF patients were inconclusive of cause and effect as CF patients typically received heavy doses of antibiotics (such as tetracycline, which can disrupt amelogenesis), masking the etiology of these enamel defects (646, 650). Wright and co-workers reported a series of studies of enamel deficiencies in Cftr-deficient mice (21, 572, 646, 650), noting that during late secretion/early maturation, their ameloblasts become cuboidal cells and prematurely transition to a squamous epithelial stage (294). In Cftr-deficient mice, the microstructure and thickness of crystallites appeared normal but showed a more porous appearance in TEM, and overall, the enamel was softer and less mineralized with reduced Cl⁻ levels compared with controls (21, 646, 650). The enamel defects of Cftr-deficient mice could result from a loss of the ameloblasts’ capacity to process extracellular matrix proteins during the maturation stage. Incisors of Cftr-deficient mice showed yellow surface stainings when immersed in pH indicator solution, pointing to acidic pH in the transition and maturation zones of enamel (572). The enamel of the incisor teeth of Cftr-deficient mice wears rapidly (59). It should be noted that these data on enamel deficiencies of Cftr-null mice are derived from the analysis of incisors, whereas molar teeth from the same mice did not show alteration in enamel, a fact that remains unexplained (59, 572). A porcine model for CF was also studied by our group, reporting that molars of CFTR-null and CFTR-delta F508 mutant pigs showed hypomineralized enamel, with the most severe pathology in the CFTR-null pigs (76). The CFTR delta-F508 mutation is the most common one found in human CF patients.

Bronckers et al. (59) have shown that Cftr is localized to the apical end of maturation-stage ameloblasts. This localization pattern places Cftr in close proximity to the enamel zone and is thus consistent with a putative role as a modulator of enamel matrix pH, as noted above, buffering the protons released during crystal formation (63). Bronckers’ group (63) and others (320) proposed that Cftr might be associated with releasing Cl⁻ into the enamel zone as part of an electrogenic exchange for HCO₃⁻. A number of exchangers could be involved in this process including anion exchanger 2 (AE2) and members of the SLC26A gene family, all of which can transport HCO₃⁻ in exchange for Cl⁻ (259, 665). It has been proposed that K⁺ and Na⁺ accumulate in enamel when Cl⁻ is low, which suggests the possibility that Na⁺-K⁺- Cl⁻ cotransporters (NKCCs) are also important during amelogenesis, although no direct evidence of NKCC expression in ameloblasts is currently available (61). Besides the presence of CFTR and its role in enamel formation, other Cl⁻ channels have been identified in ameloblasts, including the Ca²⁺-dependent Cl⁻ channels (197, 313, 320, 631). In ameloblasts a number of Cl⁻ channels are expressed, and in addition to Cl⁻ export, likely play a role in endocytosis (313). For example, Clcn7/ClC7 is expressed on the lysosomal membrane in ameloblasts (313).

3. Carbonic anhydrases

Carbonic anhydrases (CAs) are enzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate (Equation 4)

$${\text{CO}}_{\text{2}}+{\text{H}}_{\text{2}}\text{O}\stackrel{\text{CA}}{\leftrightarrow }{\text{HCO}}_3^{-}+{\text{H}}^{+}$$ (4)

CAs also participate in pH homeostasis, CO₂ and HCO₃⁻ transport, and bone resorption (539, 573). Many of the abundant CA isozymes are expressed in enamel cells but differ in cellular distribution (e.g., Refs. 315, 463). CA2 is the most widely expressed isozyme localized to the cytoplasm of many cells (316). Ameloblasts express CA2, as reported in a number of studies (118, 269, 298, 463). The earliest of these reports was made in cell homogenates from adult rat incisors (298), later confirmed by histochemical analysis of unerupted hamster molars which found CA2 signals in more mature ameloblasts (118). A similar expression pattern was later reported in rat incisors by Reibring et al. (463). RA cells were more strongly stained for CA2 than SA cells (269). The similar expression of H⁺-ATPase prompted interpretations that RA cells pump H⁺ ions into the enamel, acidifying the microenvironment in a similar fashion to that described in osteoclasts (269), but this remains untested.

Carbonic anhydrase CA6 appears to be the only CA isozyme that is secreted from cells (433, 441, 539). Complementary DNA (cDNA) library screens of rat incisor enamel-forming cells first identified a fragment that matched CA6, further characterized by RT-PCR and Northern blot analysis (549). We have since confirmed the high expression of CA6 in enamel cells (321), and a recent study found high expression of CA6 in maturation-stage ameloblasts (463). Smith et al. (549) have proposed that the function of CA6 in maturation might be associated with local buffering, supplying bicarbonate ions or recycling excess levels of carbonic acid.

In a survey of mRNA expression of all CA isozymes in mouse enamel cells, Lacruz et al. (315) identified that in addition to CA2 and CA6, other isozymes, notably Car11-15, were also expressed. More recently, Reibring et al. (463) investigated the localization of these isozymes and reported the expression of CA4, CA9, and the related isozyme CARP11 in the distal-ruffled border of RA cells, and a widespread localization in SA cells CA13 appears to be associated with the lysosomal pathway, as evidenced by similar punctate distribution of this protein with the lysosomal marker LAMP1 in ameloblasts. It should be pointed out, however, that presently human mutations or animal models deficient for CAs showing enamel defects are poorly described.

E. Modeling pH Regulation in Enamel

It is widely recognized that during the formation of enamel Hap crystals, protons (H⁺) are released in the microenvironment (523, 541); the export of protons at the apical ends of ameloblasts by the action of the V-type ATPase proton pump may contribute to this (269, 342, 495). Accumulation of H⁺ may negatively impact the formation of additional crystals by lowering local pH, thus resulting in the dissolution of crystal surface structure. It has been shown that during the secretory stage of enamel formation, the extracellular pH values are close to neutral and crystal growth at this stage is limited (541, 544). This neutral pH value during secretory stage amelogenesis has been attributed to the abundance of matrix proteins which modulate and/or buffer against changes in pH (541). However, during the maturation stage, pH values are acidic, associated with increased expansion of crystals and concomitant release of H⁺. Thus H⁺ must be removed from the enamel zone to restore physiological pH conditions. It should be highlighted that a number of proteins associated with pH homeostasis increase in expression during the maturation stage; these include NBCe1, AE2, CFTR, multiple SLC26A gene family members, and many CAs (318, 664). This marked increase in gene and protein expression of certain ion exchangers, pumps, and enzymes has been linked to an increase in their activities to counteract the rise in H⁺ observed during maturation stage amelogenesis.

A generalized model for pH maintenance in maturation stage must thus take into account the removal of H⁺. Bicarbonate can perform this function by absorbing these H⁺. In this model HCO₃⁻ can be incorporated into the ameloblasts, via NBCe1 located at the basolateral membrane, and is released apically via exchange of Cl⁻ facilitated by CFTR, AE2, and SLC26A family members. HCO₃⁻ can also be produced via the function of CA6 in the extracellular domain combining CO₂ and H₂O. However, as previously highlighted by Simmer and Fincham (523), carbonic anhydrase activity is not an effective system as this chemical reaction, generating HCO₃⁻ via CA enzymes, removes only one H⁺ locally.

The cytosolic localization of CA2 suggests that HCO₃⁻ can also be produced by the ameloblasts (269, 595). The removal of intracellular H⁺ might be mediated by the antiporter NHE1, a Na⁺/H⁺ exchanger (269). NHE1 was expressed along the plasma membrane of secretory ameloblasts, as well as both RA and SA cells (269), likely removing H⁺ into intercellular spaces. As noted earlier, if AE2 is localized along the lateral membrane, the transport of HCO₃⁻ into that space might then buffer the removal of H⁺ (61, 269). At the apical pole of RA cells, the V-type ATPase proton pump also moves H⁺ into the enamel space, contributing to acidification of this region (269, 342, 495), and a number of anion exchangers of the SLC26A gene family (SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/pendrin, and SLC26A6/PAT1) are also located at the apical pole of RA cells (259, 665). The coexpression of both the proton pump and bicarbonate channels responsible for the extrusion of H⁺ and HCO₃⁻ suggests extracellular pH is very strictly regulated during the process of enamel maturation (269). It is thus apparent that in enamel maturation ameloblasts use multiple mechanisms to lower and raise extracellular pH as needed.

The cyclical changes from RA to SA cell morphology also bear on the capacity to modulate pH as described above. While a number of proteins already discussed differ in expression profiles between these two stages, the functional interpretation of these differences is still limited. However, it has been suggested that this cycle allows changes in crystallization conditions to occur, which would impact the stability of crystals so that only the more stable would pass through each cycle (269).

X. ION TRANSPORT

A. Overview

Enamel is an almost fully mineralized tissue composed of a substituted hydroxyapatite (Hap) of primarily calcium (Ca²⁺) and inorganic phosphate (P_i). Disruptions to Hap formation can result in hypomineralized (soft) enamel, which is more prone to acid attack and caries (135, 335, 637). Recent reports suggest that Ca²⁺ and HCO₃⁻, originating from the circulation and largely diffusing across the papillary layer, are actively transported into polarized ameloblasts through ion exchangers and pumps located at their basolateral membrane (320, 429). While passive paracellular ion movement may occur during amelogenesis (403, 540), recent reports clearly suggest that the active transcellular ion transport dominates the process of enamel formation and maturation (52, 60, 269, 316, 317, 320, 321, 350). The differences between active and passive transport pathways are illustrated in FIGURE 11. Intracellular CAs are also present in ameloblasts such that HCO₃⁻ is generated within the cytoplasm (315, 316, 320, 595). Calcium (see below for Ca²⁺ uptake) and HCO₃⁻ ions are then transported across ameloblasts (409) and eventually extruded through a different series of ion exchangers and pumps located at the apical membrane to be delivered to the enamel matrix (234, 416, 615). Mutations in many of the ion exchangers associated with Ca²⁺ and HCO₃⁻ transport result in enamel pathology (117, 125, 171, 251, 314, 317, 350). These findings suggest that the enamel organ epithelium and associated Ca²⁺ and HCO₃⁻ transporters are critically important to enamel mineralization. While recent studies are starting to define Ca²⁺ and HCO₃⁻ transcellular transport in amelogenesis, there is scant information related to P_i transport associated with amelogenesis. As-yet-unidentified P_i ion channels are likely to be located on ameloblast plasma membranes, thus allowing for the transcellular transport of P_i.

FIGURE 11.

Modes of ion transport from the circulation at the basal (proximal) region to the enamel space beyond the apical (distal) pole of secretory and maturation ameloblasts. Black solid arrows indicate active transcellular transport involving ion transporters, channels, and pumps (curved arrows); black broken arrow indicates a passive paracellular/intercellular movement through “leaky” junctions (light gray double capsule) but not through tight junctions (dark gray double capsule); and a red arrow indicates a passive transcellular pathway. Dark gray double capsule indicates a tight junction at the apical region of maturation ameloblasts.

B. Bicarbonate

1. Overview

In 1998, Smith (541) published a review paper on enamel maturation clearly making the case that buffering is an essential part of the process, and that bicarbonate (HCO₃⁻) was the main buffering system used by ameloblasts. This was based primarily on the knowledge that cytoplasmic carbonic anhydrase 2 (CA2) was highly expressed during maturation, and significant expression was noted at the apical ends of ruffle-ended ameloblasts (RA) (595). It was felt that if large quantities of HCO₃⁻ were produced in the cytoplasm, then one would expect membrane-bound “carrier/transporter proteins” (i.e., ion transporters) to regulate both intracellular and extracellular pH (541). During this time Wright and co-workers were examining the enamel pathologies seen in the CFTR mutant mice (21, 572, 646, 650) and proposed that CFTR, working in conjunction with a chloride/bicarbonate exchanger, and both located at the apical ends of polarized ameloblasts, were responsible for the export of cytoplasmically generated or cytoplasmically located HCO₃⁻ to the enamel matrix (572). Wright et al. (572) also suggested transcellular movements of HCO₃⁻ were likely if a sodium bicarbonate cotransporter was located on the basolateral membrane of ameloblasts. In 2008, Paine et al. (429) identified the anion exchanger AE2 (SLC4A2) located at the apical ends of polarized ameloblasts, while the electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) could be identified at the basal pole of these same cells. More recent data however suggest AE2 is located on the basolateral membrane of maturation ameloblasts (269, 351, 665). The past decade has seen significant progress in identifying the genes and pathways involved in the buffering capabilities of ameloblasts and how HCO₃⁻ enters the enamel space, summarized in FIGURE 12.

FIGURE 12.

Generation and active transcellular movements of ameloblast-associated bicarbonate and associated ion movements during enamel formation. Ion transporters, channels, and pumps are identified either by their name or gene symbol. Nucleus (Nu) and lysosome (Ly) are identified. The transport pathways are identified in a ruffle-ended ameloblast (RA). Many of these channels have also been shown in secretory ameloblasts (not shown) and smooth-ended ameloblasts (SA) but at significantly lower levels of expression. A dark gray double capsule represents a tight junctional complex at the apical region of RA, and a light gray double capsule represents a “leaky” junctional complex at the apical region of SA. Black broken arrow indicates the movements of small molecules through leaky junctional complexes.

2. The enamel matrix buffering system

The atomic structure of enamel crystals is a variation of a pure calcium/phosphate-based hydroxyapatite (Hap) lattice which incorporates other types of ions (i.e., carbonates); thus enamel can be referred to as a nonstoichiometric carbonated calcium Hap (188, 528, 668). Despite this clarification, enamel is most frequently identified as a Hap-based mineralized tissue, and as stated above, it has been calculated that approximately eight H⁺ protons are released for every unit cell of Hap crystals formed in the extracellular environment. This generation of protons significantly lowers the enamel matrix pH (541, 544). These free protons may diffuse away from the matrix, but based on recently published data this seems unlikely. It appears that ameloblasts primarily use acid-base transport systems to regulate extracellular pH, with the bicarbonate buffer system being the primary (or perhaps the only) extracellular buffering mechanism that they employ (542). Neutralization of the increasingly acidic enamel environment is achieved with the generation of intracellular and extracellular HCO₃⁻ through the action of CAs (see Equation 4) (118, 315, 316, 318, 523, 541, 549, 595), and by the active transport of blood-derived bicarbonate through specific ion channels (transcellular) (52, 259, 320, 350, 429, 665).

As described above, HCO₃⁻ plays an important role in enamel formation (316). A number of CAs and HCO₃⁻ transporters associated with enamel formation have been described recently. The most notable are CA2, CA3 (cytoplasmic), CA6 (secreted/extracellular), and CA12 (membrane bound) (118, 315, 316, 318, 549, 595). Membrane-bound CA12 is a type I membrane protein with a catalytically active enzyme domain located in the extracellular space (254, 598). Bicarbonate ion transporters identified in maturation-stage ameloblasts include the electrogenic sodium bicarbonate cotransporter SLC4A4 (NBCe1) and multiple anion exchangers; these are coded by the genes SLC4A2 (AE2), SLC26A1 (SAT1), SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1), and SLC26A7 (SUT1) (65, 258, 259, 350, 429, 665). The large number of molecules involved with either the synthesis or transport of HCO₃⁻ in ameloblasts suggests that the entire process of pH regulation during enamel maturation is under tight molecular control.

3. The solute carrier (SLC) gene families

The solute carrier (SLC) gene series contains 52 families (SLC1 to SLC52) that include 395 unique transporter genes (214). SLC proteins transport a large number of solutes including both charged and uncharged organic molecules, in addition to inorganic molecules and gasses. The SLC proteins can be further classified as belonging to one of the following groups: facilitative transporters, secondary (coupled) active transporters, primary active transporters (requiring energy from ATPase hydrolysis), ion channels, and aquaporins. Many of the solute carriers are associated with genetic disease, and a significant number of these solute carriers are expressed in ameloblasts.

4. The SLC genes and CFTR in enamel mineralization

All of the anion exchangers identified in the enamel organ (i.e., SLC4A2 and SLC26A1, 3, 4, 6 and 7) are localized to either the apical or lateral membrane of polarized maturation-stage ameloblasts (259, 350, 429, 665), and this is a similar localization profile as CFTR in the same population of cells (59, 63, 665). Based on co-immunoprecipitation (Co-IP) data (665), there is evidence that physical interactions exist between CFTR and the SLC26A gene family members SLC26A1, SLC26A6, and SLC26A7 in maturation-stage ameloblasts (665). These functional complexes are likely localized to the apical membrane of ameloblasts where the expression of these genes is identified (665). The phenomenon of several ion transporters/exchangers, coupled to CFTR, with similar physiological functions and cellular localizations, interacting with one another to form united protein complexes, has been reported in multiple areas of biomedical research. In most cases when CFTR interacts with SLC26A family members, CFTR seems to serve as a hub for these potential interaction complexes (46, 78, 210, 216, 221, 531, 619). One example is that SLC26A3, SLC26A6, and SLC9A3R1 (the sodium/hydrogen exchanger regulatory factor or NHERF1) colocalize with CFTR in the midpiece of mouse sperm, and the protein complex formed by CFTR with SLC26A3, SLC26A6 and SLC9A3R1 functions primarily to mediate transmembrane transport of chloride, which is critical for sperm capacitation (i.e., the destabilization of the mammalian sperm head to allow for binding between the sperm head and oocyte) (78). In cochlear outer hair cells (OHCs), the physical interaction between CFTR and SLC26A5, which is localized to the lateral membrane of OHCs, has potential electrophysiological significance (221). Additionally, in human bronchial cell lines, functional CFTR contributes to the functions of SLC26A9 as an anion conductor (46). CFTR might also interact with a broader range of pH regulators, so pH regulation during enamel maturation might be achieved by the coordination of functional protein complexes that are far more sophisticated than expected. Thus the interactions noted between CFTR and members of the SLC26A family during amelogenesis warrant further investigation to determine their functional importance.

5. Carbonic anhydrases and bicarbonate transporter dysfunction and enamel pathologies

a) carbonic anhydrases.

There are 15 carbonic anhydrases isozymes/genes in the human genome (CA1-4, 5A, 5B, and 6–14) and 16 in the mouse genome (Ca1-4, 5a, 5b, and 6–15) (315, 573, 574). However, the isoforms CA8, 10, and 11 (also Ca8, Ca10, and Ca11) do not contain one or more of the required histidine residues of the catalytic domain that binds a zinc ion; thus these three isozymes are devoid of any catalytic activity. Because of this, CA8, 10, and 11 are sometimes classified as CA-related proteins, or CA-RPs. Referencing the Online Mendelian Inheritance in Man website (www.OMIM.org), disease states for humans or rodents have been described for only CA2 (cytosolic), CA5A (mitochondrial), CA8 (573), and CA12 (membrane bound). To the knowledge of the authors, dental defects have only been associated with mutations to CA2 in humans and include abnormal teeth and malocclusion (22, 55, 398, 567); however, dental anomalies in the Ca2-null mice were not reported (57).

b) SLC4A2.

The bicarbonate/chloride anion exchanger SLC4A2/AE2 is expressed widely, being localized on the basolateral membranes of most epithelial cells (481). The functional role of AE2 is notable in many cell types, including gastric parietal cells (568), choroid-plexus epithelial cells (6), surface enterocytes in colon (5), and renal collecting duct cells (7, 569). In humans, primary biliary cirrhosis has been described for SLC4A2 mutations (3, 270, 375). However, because of the wide distribution of expression of AE2, it is surprising that a greater number of human pathologies have not been linked to SLC4A2; this suggests that many mutations that impact AE2 function may be early embryonic lethal.

Mice null for Slc4a2 display a changed immune response and achlorhydria (172, 491), and squamous metaplasia of the epididymal epithelium leaves the male mutants infertile (376). Slc4a2-null mice also have significant dental pathology limited to the enamel organ (350). AE2 is most highly expressed along the lateral membranes of maturation ameloblasts (65, 269, 321, 350, 665), although our earlier reports showed a more apical expression pattern related to secretory ameloblasts (429). Gene expression array and real-time PCR data from the enamel organ show that a significant increase in Slc4a2 mRNA expression from secretory ameloblasts to maturation ameloblasts (318, 321), and it may be that this transition from secretory to maturation stage results in a redistribution of AE2 localization (i.e., from the apical to lateral membranes). However, in the maturation stage, data from Lyaruu et al. (350), Josephsen et al. (269), and Yin et al. (665) clearly show AE2 expression along the lateral/basolateral membranes. These data are also most consistent with the basolateral localization of AE2 seen for most other epithelial cell types (481). The enamel of Slc4a2-null mice had a disorganized prismatic architecture and wore significantly more quickly than that of normal control mice, while the dentin (dentin-producing cells are of mesenchymal origin) was unaffected (350).

c) SLC4A4.

Mutations to SLC4A4/NBCe1 can result in complex disease in humans and mice including proximal renal tubular acidosis (pRTA), growth delay, heart failure, mental retardation, as well as ocular and dental enamel defects; all these pathological states clearly impact on morbidity and mortality (105, 117, 246, 247, 251, 292, 436, 481). Similar disease states have been shown in the Slc4a4-null mice (171). Slc4a4-null mice died soon after birth (mean age of ~12 days), attributed to severe metabolic acidosis with blood HCO₃⁻ concentrations of 4.0–7.6 mM and pH values of 6.80–6.93 (171).

NBCe1 is expressed at the basal pole of polarized ameloblast cells (317, 429), and it is upregulated by three- to fourfold as ameloblasts transition from secretory to maturation stage (318, 321). During maturation stage, significant NBCe1 expression is also noted in the papillary layer cells (269). The gross appearance of the dentition of Slc4a4 null mice was of a hypomineralized enamel (i.e., chalky white and opaque), while an electron microscopic examination showed that the enamel had a pitted surface, had lost its prismatic structure, and was both hypoplastic and hypomineralized (317). The dentin of these NBCe1 mutant mice was normal (317). These data highlight the importance of NBCe1 activity in amelogenesis (320, 604).

d) SLC26A3.

In humans, mutations to SLC26A3/DRA result in congenital chloride diarrhea and can be associated with hypokalemia, increased serum bicarbonate, and high aldosterone (86, 217–219, 357). The Slc26a3/Dra-deficient mouse displays a similar pathology to humans (511). In mice, Slc26a3 is expressed at the apical pole of maturation ameloblasts (259). To the authors’ knowledge there have been no reports of dental pathologies related to human or mouse SLC26A3 mutations.

e) SLC26A4.

Pendred syndrome, which involves sensorineural hearing loss and goiter, results from mutations to SLC26A4/Pendrin/PEN (2, 85, 93, 134, 253, 300, 301, 365, 605, 606). Although a case report does identify a patient with Pendred syndrome as having dental disease (periodontal attachment loss and hypercementosis) (517), enamel defects have not been reported with Pendred syndrome or with any of the known SLC26A4 mutations. With the use of a normal mouse model, it has been shown that, as is the case with Slc26a3, Slc26a4 is expressed at the apical pole of maturation-stage ameloblasts (60, 259). While Slc26a4 mutant mouse models have been developed and studied (121, 133), dental pathologies have not been reported in the mice.

f) SLC26A6.

Referencing the Online Mendelian Inheritance in Man website (OMIM.org), disease states for human SLC26A6 mutations have not been documented, although an in silico analysis suggests that certain single nucleotide polymorphisms (SNPs) in SLC26A6 may be a risk factor for kidney stones (347). Slc26a6/Pat1 mutant mice form calcium oxalate urolithiasis, with this being the only noticeable pathology (265); however, in a second Slc26a6 mouse mutant model, changes in the physiology of Cl⁻/base exchange in the kidney proximal tubules and oxalate stimulated NaCl absorption of the duodenum were noted (620).

C. Calcium

1. Overview

In mineralized enamel, Ca²⁺ is the most abundant ion and can be incorporated rapidly into the enamel zone from blood (541). Ca²⁺ represents ~36% of enamel by weight, about twice as much as the next ion represented (392). Ca²⁺ is largely incorporated during the maturation stage, with ~86% of the Ca²⁺ found in enamel entering the tissue during this stage (541). During the secretory stage, the concentration of calcium measured in pig enamel fluid was ~5 × 10⁻⁴ M, lower than in the serum (~3 x 10⁻³ M), suggesting that the enamel zone represents a specialized micro-compartment (17) and also pointing to the presence of an active transport system. However, the cyclical nature of morphological changes from RA to SA in maturation may indicate that both passive and active transport systems are available to these cells (see below) (541).

Ca²⁺ is also important to ameloblasts during the secretory stage. The bulk of Ca²⁺ in the enamel fluid in the secretory stage is bound, likely to 11- to 13-kDa amelogenin-derived fragments (392, 475). Amelogenin-Ca²⁺ binding can potentially be a Ca²⁺ reservoir, acting as a regulator of mineralization in particular at the secretory stage as there are limited adsorption sites for Ca²⁺ giving a smaller crystal size (17). It is estimated that one molecule of amelogenin can bind ~6.4 Ca²⁺ (328). Reith and Boyde (469) suggested that the binding of Ca²⁺ to amelogenins provides a route for the diffusion of Ca²⁺ into the deeper layers of enamel; however, this might seem paradoxical because of the increased bulk of an amelogenin-Ca²⁺ complex compared with Ca²⁺ alone. Saturation of the enamel fluid and precipitation of the mineral phase appears to be largely determined by the concentration of Ca²⁺ (392, 523).

The mechanisms associated with the transport of Ca²⁺ by the enamel organ have been the subject of a number of important reviews; however, much of our previous understanding of Ca²⁺ transport is currently being challenged and redefined (32, 240, 541, 579).

2. Classic hypotheses for Ca²⁺ transport

Ca²⁺ travels across the barrier formed by ameloblasts in the basolateral to apical direction into the enamel, rather than from the underlying dentin (469). Transport of Ca²⁺ is considered to proceed largely via a transcellular route rather than a paracellular or passive route as secretory and maturation stage ameloblasts form tight cell cohorts bound by junctional complexes (502). Perfusion studies using lanthanum observed that this tracer leaked across the proximal but not the distal intercellular junctions of secretory ameloblasts (580). In RA, tight junctions are only found near the basolateral pole, but in SA, these junctions can be found at the apical pole (268). It was found that lanthanum and horseradish peroxidase (HRP) did not leak across the distal junctions of RA or the proximal junctions of SA (579, 580, 583). The cyclic nature of RA and SA indicates that during the smooth phase, intercellular spaces are opened to the enamel area, allowing fluids to move passively in a proximal direction, but remaining in that intercellular space until the RA stage, when fluids can move towards the papillary layer area and be cleared via the vascular system. About 70% of all maturation-stage ameloblasts are ruffle-ended, limiting the passive movement of Ca²⁺ (268, 541). Studies using radiolabelled Ca²⁺ (⁴⁵Ca) suggest that Ca²⁺ is incorporated through the RA (467, 582).

Considering the transcellular transport of Ca²⁺, most prior studies have suggested that Ca²⁺ entry into ameloblasts is passive (32, 579). Influx occurred at the basolateral pole via concentration gradient differences between the lower intracellular [Ca²⁺] and higher [Ca²⁺] in the extracellular compartment (32). Leaked Ca²⁺ into the basal pole increased intracellular [Ca²⁺], exposing cells to potentially toxic Ca²⁺ levels. To prevent this while enabling transfer across the cytosolic compartment towards the apical pole, it was proposed that ameloblasts use a number of common cytosolic Ca²⁺ buffers (32, 541, 579) including parvalbumin and calretinin, as well as others with lower binding capacity acting also as Ca²⁺ sensors, including calmodulin and calcineurin (98, 239, 241). The most abundant buffers are the Ca²⁺-binding proteins known as calbindins (members of the S100 gene family). Calbindin 9kD/S100, calbindin 28kD/CALB1, and calbindin 30/CALB2 have been the subject of a number of studies (41, 42, 238, 239, 241, 243, 312, 330, 599). Binding of Ca²⁺ to buffers occurs on the sub-second scale and thus is a key process to maintaining intracellular Ca²⁺ homeostasis. The role of mobile buffers was considered to be of importance as it was postulated that cytosolic [Ca²⁺] near the apical pole is lower than at the basal pole so that calbindins can safely transfer bound Ca²⁺ across the cell (240). In addition to cytosolic buffers, Hubbard’s group also identified the expression of the sarco/endoplasmic Ca²⁺-ATPase (SERCA) (161) whose main function is to pump cytosolic Ca²⁺ into the lumen of the endoplasmic reticulum (ER), thus playing a role in Ca²⁺ homeostasis. Moreover, ER luminal buffers including calreticulin/CALR, endoplasmin/HSP90B1, and ERp72/PDIA4 were also identified in enamel cells (238, 242). The safe transit of Ca²⁺ also involves transport via secretory vesicles and along the inner leaflet of the plasma membrane through phospholipids (469).

Concerning Ca²⁺ extrusion, the plasma membrane Ca²⁺-ATPases (PMCA) were considered the main clearing mechanism (32). Four genes code for PMCA proteins: ATP2B1-4. PMCAs, which exchange protons for one Ca²⁺ in each ATP hydrolysis, were identified throughout the membrane of secretory-stage ameloblasts. In maturation stage these pumps were predominantly localized to the ruffled border of RA cells (579), thus suitably positioned to extrude Ca²⁺ while removing protons from the enamel area and indirectly participating in pH regulation (63).

Hubbard proposed a revised version of the Ca²⁺ transport system by the enamel epithelium largely based on his group’s findings that the ER played a role in transiting Ca²⁺ across the cell (238, 240). In addition to the findings of SERCA in enamel cells, Hubbard (238) also identified the expression of inositol receptors (IP₃R) which enable the release of ER Ca²⁺ pools. These findings and others related to protein buffers in the ER allowed Hubbard to put forth a model he termed the “transcytosis hypothesis,” which gained traction in the literature as a potential model for Ca²⁺ transport in enamel epithelium (238). Considering that mouse models deficient for the two calbindins found in enamel and mentioned above showed no abnormal enamel phenotypes (243, 599), the ER-based transcytosis model proposed that Ca²⁺ remained buffered within the ER lumen where it was moved across the cell to be extruded by PMCA pumps at the apical pole (240). This model, however, also envisioned that Ca²⁺ entry was likely a passive process (e.g., Refs. 32, 579). This is a key point revised in recent publications showing that this passive influx of Ca²⁺ is not a likely scenario (318, 409, 411).

3. Current ideas on calcium influx during amelogenesis

a) calcium influx.

A genome-wide study which compared rat enamel organ cells from maturation and secretory stages identified Stim1 and Stim2 transcripts as being upregulated in maturation, which was also supported by Western blot analysis (318). STIM1 and its homolog STIM2 are single-pass proteins with an ER lumen amino terminus and a cytosolic carboxy terminus. They are key components of a mechanism involved in Ca²⁺influx in many cell types known as store-operated Ca²⁺ entry (SOCE), of which the Ca²⁺ release-activated Ca²⁺ (CRAC) channels is the best characterized. STIM proteins are largely localized to the ER membrane, acting as sensors of changes in luminal ER [Ca²⁺]. When the [Ca²⁺] in the ER lumen decreases, Ca²⁺ dissociates from the EF-hand motif in the amino terminus, promoting the oligomerization of STIM (146). STIM1 oligomers directly interact with the plasma membrane pore subunit of the channel known as ORAI1, resulting in sustained Ca²⁺ entry (146). ORAI is a plasma membrane-bound protein with three homologs in mammals (ORAI1-3), but human mutations impacting on SOCE are only known for ORAI1 (314). Patients with mutations in the STIM1 and ORAI genes present with amelogenesis imperfecta (AI) (163, 314, 370, 615), linking CRAC channel function with enamel formation.

Nurbaeva et al. (411) recently demonstrated that CRAC channels are functional in enamel cells. Using the ameloblast-like LS8 cells (494), a murine enamel organ-derived immortalized cell line, we adapted protocols used in many other cell types to modulate CRAC channel function using thapsigargin to inhibit SERCA pumps, thus passively depleting the [Ca²⁺] in the ER to activate SOCE (411). Readdition of extracellular Ca²⁺ showed a marked rise in cytosolic [Ca²⁺] demonstrating that SOCE is expressed in LS8 cells (411). Some cells were exposed to inhibitors that have been previously used to block CRAC channels (i.e., Synta-66, BTP2, 2-APB), all of which hampered Ca²⁺ entry, thus demonstrating that Ca²⁺ uptake in LS8 cells is via CRAC channels (411).

This research was extended to primary enamel cells, taking advantage of the fact that enamel organs can be isolated from the secretory and maturation stages using commonly accepted protocols (547). STIM1 and STIM2 and all three ORAI homologs were detected in ameloblasts with high expression (409). STIM1 and ORAI1 showed cytosolic and plasma membrane localization, respectively (409). Stimulation of isolated enamel organ cells from secretory and maturation stage with thapsigargin showed that both cell types are equipped with SOCE. In primary cells pretreated with the CRAC channel inhibitor Synta-66, Ca²⁺ entry was significantly impaired, demonstrating that CRAC channels are functional in primary enamel cells (409). These measurements were carried out using a Flexstation-3 which records the average fluorescence of a pool of cells. A schematic model for the entry (and exit) of Ca²⁺ in enamel cells is presented in FIGURE 13.

FIGURE 13.

Proposed model for Ca²⁺ handling by ameloblasts. Secretory ameloblasts (Sec.) have either passive and/or SOCE for Ca²⁺ uptake. Extrusion at this stage is mediated largely by plasma membrane Ca²⁺-ATPases (PMCAs) or sodium/calcium exchangers (NCXs). Ca²⁺ uptake in maturation (Mat.) stage RA occurs largely via SOCE. STIM1 has a wide distribution throughout the endoplasmic reticulum (ER), and ORAI1 is found in the plasma membrane of RA. Sarco/endoplasmic reticulum SERCA2 pumps sequester cytosolic Ca²⁺ into the ER lumen, whereas inositol 1,4,5-trisphosphate receptors (IP₃R) might be the main ER Ca²⁺ release channels although ryanodine receptors (RyR) have also been identified. As Ca²⁺ pools are depleted in the ER, STIM1 clusters enable Ca²⁺ entry via the ORAI1 channel. Extrusion in RA is mediated principally by NCKX4, with NCX1, NCX3, and PMCA also playing a lesser role. In SA cells, STIM1 is nearly absent, and the localization of NCKX4 changes becoming internalized which is predicted to alter Ca²⁺ transport during SA phase. A dark gray double capsule represents a tight junctional complex at the apical region of RA, and a light gray double capsule represents a “leaky” junctional complex at the apical region of SA.

b) evidence for Ca²⁺ signaling in enamel cells.

It is well known that a rise in cytosolic [Ca²⁺] has important signaling effects over a wide range of processes (461). It has been shown that stimulation of the enamel cell line PABSo with different concentrations of extracellular Ca²⁺ resulted in an increase in cytosolic [Ca²⁺] measured by fura-2 ratios (363). The expression of the Ca²⁺ sensing receptor (CaR/CASR) in these cells was linked to this process (363). A later study investigated the effects of exposing primary enamel organ cells to external Ca²⁺ and found that media containing concentrations of 0.1–0.3 mM Ca²⁺ resulted in mRNA increase of AMELX and AMTN, suggesting that Ca²⁺ played a role in modulating their expression (83). More recently, the effects of Ca²⁺ were investigated more directly by stimulating the murine cell line LS8 with thapsigargin to induce SOCE (411). Stimulation of LS8 cells for 0.5 h resulted in a marked increase of Amelx, Ambn, and Enam mRNA levels but not when cells were pretreated with the CRAC channel inhibitor Synta-66 (411). These data indicate that SOCE-mediated Ca²⁺ entry is important in regulating enamel gene expression in LS8 cells. A similar result was found in primary enamel cells dissected from mouse enamel organs after stimulation with thapsigargin (411). As this effect was in response to a decrease in ER Ca²⁺ stores, it suggests that SOCE mediates the expression of enamel genes. Protein changes analyzed by Western blot showed that Ambn expression increased in primary cells after only 1 h following stimulation with thapsigargin, supporting the relatively fast action of SOCE and enamel protein expression.

c) calcium extrusion: roles of ncx1, ncx3, and nckx4.

Two families of exchangers expressed in enamel cells have helped redefine Ca²⁺ extrusion in ameloblasts. The first of these reports focused on the bidirectional Na⁺/Ca²⁺ exchanger family NCX (416) which exchange one Ca²⁺ for three Na⁺. The second family is the Na⁺/K⁺/Ca²⁺ (NCKX) exchanger, bidirectionally cotransporting one K⁺ and one Ca²⁺ inwardly and four Na⁺ outwardly. In most cells, [Na⁺] and [Ca²⁺] are higher outside the cell while [K⁺] is higher inside.

NCX1 and NCX3 are expressed in ameloblasts with an apical or apicolateral distribution with NCX1 also showing basal staining (416), and based on the histological analyses, it appears both secretory and maturation ameloblasts express NCX1 and NCX3 at similar levels (416). Electrophysiological analysis of whole cell recordings and pharmacological inhibitors for NCX further demonstrated NCX activity in Ca²⁺ transport (416). More recently, Lacruz et al. (321) assessed by RT-PCR expression of NCX1 and NCX3 during amelogenesis and showed that neither of these genes increased expression during the maturation stage, supporting the data that both genes are expressed at similar levels during both the secretory and maturation stages of amelogenesis.

Results from a genome-wide study comparing secretory- and maturation-stage enamel organ cells found that NCKX4 was upregulated in maturation (318). There are six NCKX gene family members, NCKX1-6, coded by genes SLC24A1-6, respectively. RT-PCR analysis identified all members of the NCKX family in both secretory and maturation stages, but NCKX4 was the most highly upregulated, suggesting an important role for NCKX4 in enamel formation (234, 615). NCKX4 is localized to the apical end of maturation stage ameloblasts and thus likely associated with Ca²⁺ extrusion (234, 320). Subsequent reports identified severe enamel defects in patients and in mouse models with mutations to SLC24A4 (439, 615), strengthening the link between NCKX4 and enamel formation.

d) calcium extrusion: plasma membrane Ca²⁺-ATPases.

Borke and co-workers (53, 54, 669) have reported the presence of plasma membrane Ca²⁺ pump (PMCA) proteins in the enamel organ cells, and further investigation is warranted based on the current interest in ion transport and amelogenesis. The PMCA proteins are coded by four unique genes: the ATPase plasma membrane transporting genes 1–4 (ATP2B1, ATP2B2, ATP2B3, and ATP2B4). Borke and co-workers used antibodies that recognized the PMCA proteins, but it is unclear if they could distinguish between the four gene products/isoforms. The majority of the staining was located at the apical membrane of both secretory- and maturation-stage ameloblasts, with the greatest reactivity seen in early maturation (669). Expression was also evident in the papillary layer cells (669). In situ hybridization showed that both Atp2b1 and Atp2b4 were expressed in ameloblasts, with the highest signal being noted during early maturation (54). With the exception of a single paper looking at the expression of Atp2b1 in zebrafish bone and teeth (183), no other papers could be identified that have extended the prior work on the role of PMCA proteins in amelogenesis.

Taking the above studies together, a number of calcium exchangers and pumps have been identified primarily on the apical membrane (and occasionally the lateral membranes) of secretory- and maturation-stage ameloblasts, with these being NCX1, NCX3, and the PMCA proteins. Expression of the NCKX4 is restricted to the apical membrane of maturation-stage ameloblasts. A summary schematic of these calcium extruding exchangers and pumps is shown above (FIGURE 13).

4. A revised model for Ca²⁺ influx/efflux in enamel organ cells.

Current evidence suggests that Ca²⁺ entry into enamel cells is modulated by CRAC channel proteins STIM1 and ORAI1 (314, 409). Stimulation of CRAC channels in most cells is mediated by the action of an agonist binding to a cell surface receptor, which results in intracellular production of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP₃) (455). IP₃ binds to its receptor in the ER membrane, releasing Ca²⁺ into the cytosol and activating SOCE (158, 435). Given that IP₃ receptors (IP₃Rs) are highly expressed in ameloblasts (409) and that IP₃R expression predominates in nonexcitable cells (559), it is likely that this receptor family is associated with SOCE in enamel cells (409). The increase in cytosolic [Ca²⁺] resulting from SOCE activation is buffered via a number of cytosolic proteins, although it is as yet unclear whether these buffers, in particular the calbindins, can move Ca²⁺ safely across ameloblasts or whether the extensive tubular network of the ER mediates this process (240). Ca²⁺ homeostasis can also be monitored by Ca²⁺-ATPases, SERCA2, and exchangers extruding Ca²⁺ including NCX1, NCX3, NCKX4, and PMCAs (32, 234, 416) (see FIGURE 13).

Overall differences between secretory- and maturation-stage ameloblasts in the expression of these proteins tend to suggest that all of them are expressed in both stages, but during maturation many of them show increased expression. These data are in keeping with the increased transport of Ca²⁺ during maturation reported by others (240, 541), so the expectation would be that as Ca²⁺ mineral uptake increases in maturation, the Ca²⁺ handling machinery is also upregulated (318).

The contribution of SA cells to Ca²⁺ transport in enamel cells during the maturation stage remains unclear. While tracer analyses (⁴⁵Ca) indicate rapid movement of Ca²⁺ from blood to the enamel area in ways that would seem more appropriate to a passive (intercellular) movement of Ca²⁺ (541), it can also be speculated that injection of ⁴⁵Ca might overload the active system associated with RA cells, facilitating rapid and perhaps artificial passive diffusion across the leaky SA cells. Until the functional differences between these two cells types are more clearly explored, this will remain unresolved. However, as pointed out by Smith (541), it is also a strong possibility that ameloblasts use the RA-to-SA modulation to enhance Ca²⁺ transport so that a constant supply is maintained regardless of the cell type involved.

D. Phosphate

1. Overview

At least two studies have used a ³²P_i-labeled pulse (following intraperitoneal injection) to investigate P_i incorporation into enamel (279, 477). Labeled ³²P_i in the enamel matrix was observed within 10 min, with a greater incorporation found in the secretory-stage enamel matrix when compared with maturation stage. While the data presented by Robinson et al. (477) show a very complex pattern of P_i movements into the enamel matrix, when viewed over 24 h there seems to be a peak of ³²P_i inclusion at ~4 h (times examined from the initial injection were 10 min and 2, 4, 8, and 24 h), again with the majority of P_i moving into the secretory-stage matrix. Similarly rapid (minutes) radiolabeled Ca²⁺ uptake into the enamel space has also been shown (202, 396, 602). The incorporation of P_i from the circulation to the enamel matrix in as little as 10 min suggests the possibility of a paracellular/intercellular route, but as discussed in a prior review paper (541), while P_i (and Ca²⁺) movement from the circulation to the enamel space occurs quickly, likely via intercellular spaces, there does appear to be a level of cellular control and facilitated movement offered by ameloblasts (541).

Lacruz et al. (318) and Yin et al. (664) have recently performed a whole-genome array analysis for rat incisor secretory-stage and maturation-stage enamel organ cells and identified that sodium-dependent P_i transporters (cellular transporters for the coupled import of Na⁺ and HPO₄²⁻ or H₂PO₄⁻) feature prominently in amelogenesis. Our array data indicate that Slc20a1 and Slc20a2 are expressed at high levels throughout amelogenesis, while Slc34a2 increases ~30- to 60-fold as enamel organ cells transition from secretory stage to maturation stage (318, 664). This is of particular interest because the expression of Slc34a2 is pH sensitive (156, 382, 610), being upregulated at lower pH, which is known to characterize the enamel environment during maturation (316, 318, 544, 664). The SLC20 gene family comprises SLC20A1 and SLC20A2, which code for the proteins referred to as PiT-1 and PiT-2, respectively (157). PiT-1 and PiT-2 are ubiquitously expressed in all tissues and are thought of as “housekeeping” transport proteins, although differences in function in various tissues are noted (157).

2. Sodium-dependent phosphate (P_i) transporters of the solute carrier (SLC) class of genes

The sodium-dependent (or sodium-coupled) phosphate (P_i) transporters are members of the SLC17A (SLC17A1-4), SLC20A (SLC20A1-2), and SLC34A (SLC34A1-3) gene families. In an initial genome-wide array analysis of the enamel organ cells (318), the expression levels of all of the SLC17 gene family members were negligible while members of both the SLC20 and SLC34 families were expressed at high levels, as was the alkaline phosphatase (Alpl) gene (318). SLC20A and SLC34A are known classically as electrogenic transmembrane proteins that cotransport P_i and Na⁺ from the extracellular space/fluid into the cell (157, 174). SLC20A moves two Na⁺ and one H₂PO₄⁻ across the cell membrane, while SLC34A moves three Na⁺ and one HPO₄²⁻ (157, 174, 508).

3. Phosphate export in amelogenesis

There are no published data that add to our understanding of P_i export to the enamel matrix once it is internalized in the enamel-forming cells. Once P_i is internalized in ameloblasts it then may be transported by an as-yet-unknown mechanism to the mitochondria and stored as polyphosphates, as has been proposed for bone cells (418, 419). From the mitochondria, the polyphosphates would then need to be transported in specialized secretory vesicles to the plasma membrane to be released to the extracellular matrix as needed (418, 419). Polyphosphates would then be enzymatically cleaved into orthophosphates allowing for Hap formation. For such an activity to occur, the expression of alkaline phosphatase (ALPL) would be required, and it has been shown previously that ALPL activity is expressed at the apical pole of polarized ameloblasts, both during secretion and maturation (311).

4. SLC20 and SLC34 gene families and their roles in health and disease

The SLC20 gene family comprises SLC20A1 and SLC20A2, which code for the proteins referred to as PiT-1 and PiT-2, respectively (157). PiT-1 and PiT-2 are ubiquitously expressed in all tissues and are thought of as “housekeeping” transport proteins, although differences in function in the various tissues are noted (157). For example, PiT-1 is involved with bone P_i homeostasis and is under the regulation of bone-specific signaling factors such as IGF-1 and BMP2 (155, 381). Pit-1 is essential for liver development (35), and in cell culture Pit-1 has been shown to play a role in cell proliferation, cell cycle regulation, mitosis, and cytokinesis (36). Slc20a1 knockout mice are embryonic lethal (35), and to date, no mutations in SLC20A1 have been linked to human disease. Humans with mutations to SLC20A2 (613) and mice null for Slc20a2 are viable and in both species show a similar neurological pathology limited to basal ganglia calcifications (261). The function of the SLC20 gene family members has not been investigated in dental tissues.

The SLC34 gene family is composed of three members, SLC34A1-3, which code for proteins NaPi-IIa, NaPi-IIb, and NaPi-IIc, respectively. Expression of NaPi-IIa and NaPi-IIc is somewhat limited to the kidney proximal tubules (155, 405). NaPi-IIb is not expressed in the kidney and mediates P_i absorption in the gut (155, 405). SLC34A2 is also expressed in lungs, testes, salivary glands, thyroid, liver, and mammary glands (405). In humans, mutations in SLC34A1 are linked to nephrolithiasis and osteoporosis (457), autosomal recessive Fanconi syndrome, hypophosphatemic rickets (356), nephrocalcinosis, and hypercalcemia (290). SLC34A2 mutations are linked to alveolar and testicular microlithiasis (381), and SLC34A3 mutations are linked to hypophosphatemic rickets and hypercalciuria (44).

Several mutant mouse models have been studied for Slc34a1 (593), Slc34a2 (413), and Slc34a3 (593) and generally bear nonlethal phenotypes similar to those noted in humans. In enamel organ tissues, levels of Slc34a1 and Slc34a3 mRNA are negligible (318). The levels of Slc34a2/NaPi-IIb mRNA are negligible in the secretory-stage enamel organ cells, and significantly upregulated during maturation-stage amelogenesis (318, 664). A recent paper in the dental literature shows, using immunolocalization with two distinct antibodies against mouse Slc34a2/NaPi-IIb, expression is limited to the apical pole of maturation ameloblasts (61). The authors suggest that NaP_i-IIb may secrete/export P_i and Na⁺ into the enamel space (61). This would require that NaPi-IIb flip such that the amino terminus and carboxy terminus, normally located in the cystoplasm, relocate to the enamel space. This would be a novel functional role for NaPi-IIb in cells; however, based on the localization data presented by Bronckers et al. (61), this positioning of NaPi-IIb at the apical membrane of maturation ameloblasts is worth investigating further.

E. Fluoride

Fluoride is incorporated into the developing enamel crystallites during enamel formation, and also after the enamel is completely formed. During amelogenesis, fluoride ions (F⁻) can substitute randomly for hydroxide ions (OH⁻) or carbonate ions (CO₃²⁻), usually at very low levels, in the hydroxyapatite (Hap) crystallite structure (15, 16, 45, 62, 107, 131, 476, 523). Some dental researchers thus refer to dental enamel as a fluorohydroxyapatite-based structure (99, 329, 523). The inclusion of low levels of F⁻ enhances crystal growth rates and makes the resulting enamel more stable than pure Hap (523), thus improving its resistance to dental caries by decreasing its acid solubility (141, 487, 597). This property has made fluoride a staple of modern dental health care. Nevertheless, if fluoride were absent during amelogenesis, teeth would form without any significant deficiencies, pathologies, or changes in morphology and function.

Once a tooth erupts, F⁻ can still be incorporated into the enamel crystallites, but F⁻ must diffuse into the enamel from the enamel surface to substitute or replace OH⁻ that may be lost, for example, as a consequence of demineralization during the process of dental caries. In an incipient carious lesion, demineralization is initially seen just below the enamel surface (20, 89, 252), and the chemical events that contribute to the demineralization/remineralization process result from the effusion and infusion of ions to restore the damaged Hap-based enamel. Topical fluorides are an effective approach to prevent early carious lesions from progressing and hasten the remineralization process (1, 14, 224).

Excessive exposure to F⁻ during enamel formation can result in dental fluorosis or mottled enamel (11, 13, 62), which is hypomineralized. Fluorosed enamel has a greater amount of retained matrix proteins (62, 108, 110, 476, 643); thus some researchers have speculated that F⁻, above a certain concentration, may have a negative impact on the function and activity of the secreted enamel proteinases such as MMP20 and KLK4 (15, 62, 107). However, recent studies suggest this may not be the case, given that a high concentration of fluoride ions does not impact the enzymatic activities of either MMP20 or KLK4 in vitro (173, 600). In vitro, excess levels of F⁻ have been shown to decrease MMP20 expression, but not the expression of AMELX (673). There is, however, an in vivo-based (rodent) study showing that increases in ingested F⁻, to levels that cause dental fluorosis, result in a decrease in TGF-β1/Tgfb1 expression in enamel cells and this in turn inhibits Klk4 expression (575). These data may suggest that decreased Klk4 activity results in retained protein matrix in surface enamel that is visualized as dental fluorosis (or white spot lesions) (575). Some of the most current research on dental fluorosis and amelogenesis suggest that there is a connection between high levels of F⁻ and the initiation of both oxidative and ER stress pathways (306, 518, 522, 576). It has been shown in ameloblasts and other cell lineages that both oxidative and ER stress result in a decrease in overall protein synthesis and secretion (215, 306, 309, 310, 346, 628), including the enamel proteinases (522). However, secretion of the major enamel structural proteins (i.e., AMELX) during secretory-stage amelogenesis appears relatively unaffected to exposure of high levels of F⁻ (62). Thus one likely cause of hypomineralization as it relates to fluorosis appears to result from lesser quantities of secreted enamel proteinases acting on a “normal” quantity of enamel protein substrates, rather than decreased kinetic activity in the presence of a normal quantity of proteinases (106, 110, 522).

While there has been extensive research directed at discovering the pathogenic mechanism behind dental fluorosis, and much of this work has been done either using cultured human enamel cells, or using mice or rats exposed to various levels of F⁻, there needs to be caution translating these findings to humans. In rats and mice, long-term exposure to F⁻ at 25–30 ppm in the drinking water would be required to produce visible sign of enamel fluorosis (12, 109), while F⁻ at 100 ppm is a typically used dose to induce dental fluorosis in both rats and mice (62, 576, 607, 608). This dose is high when compared with the dose of ~6 ppm that would result in enamel fluorosis (with most individuals being severely affected) in close to 100% of the human population (see below).

While work continues on the biological, physical, and chemical properties of F⁻ inclusion in dental enamel, the pathways that import and export F⁻ in ameloblasts remain unknown. Some researchers believe F⁻ itself is unable to diffuse across the plasma cell membrane, but can readily pass through the membrane in the form of hydrofluoric acid (HF) (522). To the authors’ knowledge, ion channels that selectively transport F⁻ in mammalian cells have not been described; however, in bacteria, yeast, and some plant cells, a number of ion channels have been described that effectively transport F⁻ (264, 323, 551, 563). For example, in bacteria, members of the voltage-sensitive chloride channel (ClC) superfamily have been shown to transport F⁻ (323, 339, 562). It should be noted that mammalian cells express nine members of this ClC family (Clcn1-7, plus Clcnka and Clcnkb) (37, 313); thus future studies may make a connection between the role of certain chloride channels and/or pumps, or even channels specific to fluoride, and the transmembrane movements of F⁻ in mammalian cells, including ameloblasts.

F. Iron

Iron is seen at its highest concentrations at the labial surface or rodent incisors and gives the surface of these teeth a yellowish appearance (374, 632). Over a century ago, it was noted that hypoparathyroidism resulted in loss of pigment in rat incisors (130). Early studies have led to differing conclusions regarding the role of iron in surface enamel. Stein and Boyle (560) suggested that iron pigmentation does not impact enamel structural properties based on the observation that, after surgically destroying the pigment-containing part of the enamel organ, the integrity of the underlying enamel is not affected. In contrast, Prime et al. (458) showed that an extended deficiency in dietary iron caused loss of pigmentation, resulting in enamel hypoplasia and aplasia, suggesting that an iron deficiency was associated with severe enamel structural defects.

Iron is essential to all living organisms. The most abundant iron-containing proteins are hemoproteins that are involved in oxygen transport and delivery. The ability of iron to shuttle between ferric iron (Fe³⁺) and ferrous iron (Fe²⁺) makes it especially useful in electron transport and enzyme catalysis. However, unregulated fluctuations in iron concentration can cause cellular damage by catalyzing reactions leading to the production of toxic oxygen radicals (10, 181). Excess iron that is not for immediate use is stored in ferritin, a shell-like protein structure with a central cavity containing Fe³⁺. Mammalian ferritins are 24-subunit heteropolymers made of two different subunit types, a heavy and light chain, coded by the FTH and FTL genes, respectively. The early embryonic lethality in Fth knockout mice suggests a critical role for ferritin during development (144). The expression of FTH and FTL is influenced by iron concentrations in the immediate environment (206).

Iron is actively involved in numerous biological functions by serving as a cofactor for many proteins, including catalases and peroxidases in oxygen metabolism, hemoglobins in oxygen binding and transport, cytochromes in oxidative phosphorylation and in electron transport (434). Energy-requiring events, such as active ion transport, and water and matrix protein removal from the maturing enamel, demand a high level of ATP production through mitochondrial oxidative phosphorylation (414). It is therefore conceivable that iron is required by maturation-stage ameloblasts to assist in cellular energy production.

Rodent incisors are characterized by yellowish pigmentation on the labial side due to an iron content of ~0.030% in the whole upper incisor and 0.027% in the whole lower incisor (449). Electron microscopy has shown that iron is found primarily in the region of the enamel organ associated with maturation (465). The functional significance of iron in rodent incisor enamel is not understood, but it has been proposed that iron can decrease the solubility of crystallized Hap because iron density positively correlates with acid resistance of outer enamel (415). In addition, many knockout or transgenic animals targeting the silencing or overexpression of enamel gene products result in an enamel with a chalky white appearance and structural defects (230, 237, 317, 525, 526, 646, 650), suggesting the incorporation of iron into enamel is linked to the normal process of enamel formation (424, 666). Given the high iron content in mature enamel, not surprisingly, Fth has been identified as one of the genes most highly upregulated in maturation ameloblasts when compared with secretory ameloblasts (318, 319).

To date, published reports on the presence of iron and ferritin in teeth have been primarily limited to observations in ameloblasts and in the enamel of rodent incisors (383, 465, 632); however, limited iron uptake in developing rat molars has also been observed using autoradiographic (33) and immunolocalization approaches (632). Data from Wen and Paine (632) indicate that iron is present in ameloblasts of continuously growing incisors, and also evident in ameloblasts of molars (albeit at significantly lower levels), and favor the idea that iron is an integral component for enamel formation. Iron is not released from the ameloblasts and deposited into the enamel until the Ca²⁺ and phosphorus contents of the enamel have reached a maximum level (201). In addition, there is an inverse relationship between the iron and Ca²⁺ content in the outer enamel layer (303). One hypothesis proposed by Halse and Selvig (201) is that enamel mineralization advances only to a point, leaving room for subsequent incorporation of iron accompanied by removal of Ca²⁺. Therefore, iron incorporation may represent the final refinement of surface enamel mineralization to provide extra strength or acid resistance. This would help explain the wear pattern of rodent incisors, where the surface enamel wears less than underlying enamel, resulting in “bladelike” occlusal surfaces that allow for gnawing of hard foods.

G. The “Other” Ions: Magnesium, Sodium, and Potassium

Mineralized enamel contains, in addition to the main components Ca²⁺, PO₄³⁻, OH⁻, and Cl⁻, a small amount of various trace elements including Mg²⁺, Na⁺, and K⁺. These elements are represented in different concentrations across the enamel layer; variation exists across species and even across individuals. This individual variation is associated with external factors such as diet and the chemistry of the water consumed during tooth formation. Young (668) reported concentrations (as percentage by weight) for these elements in mineralized enamel as follows: Mg²⁺ (0.22%), Na⁺ (0.70%), and K⁺ (0.03%), which are far lower than the values reported for Ca²⁺ (36.6%) or P_i (18%). The millimolar concentrations for Mg²⁺ and Na⁺ measured by Aoba and Moreno (17) in the enamel fluid just adjacent to the apical pole of ameloblasts were lower than in serum, whereas K⁺ was higher, suggesting that there is a system at the apical pole modulating the movement of these ions.

Mg²⁺ concentration in mineralized enamel increases from the outer enamel towards the deeper layers near the dentin (519). Being a divalent cation like Ca²⁺, it sometimes competes with the latter for space in the crystal structure (19). However, Mg²⁺ has a smaller atomic radius than Ca²⁺ as well as a greater affinity for water molecules, which limit its incorporation into the enamel crystals (523). In many cells, cytosolic Mg²⁺ concentration is higher than extracellular levels. Transcellular transport of Mg²⁺ in enamel has been poorly understood until recently as only Ca²⁺-Mg²⁺-ATPases had been implicated in enamel physiology (490). More recently, it was reported that mutations in the cyclin and cystathionine-beta-synthase (CBS) domain divalent metal cation transport mediator 4 (CNNM4) gene associated with Jalili syndrome also showed enamel deficiencies (438). Patients with CNNM4 mutations are characterized by ocular deficiencies associated with retinal dystrophy and also present hypomineralized, thin enamel. The enamel from patients with CNNM4 mutations showed lower Ca²⁺ but increased Mg²⁺ in enamel than that from healthy patients (349). In ameloblasts, CNNM4 is found at the basolateral pole and has been linked to Mg²⁺ extrusion (661). Patients with hypomagnesemia also present with dental defects (hypoplasias), adding to the relevance of Mg²⁺ in enamel formation (73).

Mg²⁺-deficient enamel phenotypes are also associated with the ion channel for divalent metal cations, TRPM7, which has been shown to play a critical role in enamel mineralization (401). TRPM7 is upregulated during maturation stage and expressed in maturation-stage ameloblasts (401, 672). Trpm7 mutant mice show an enamel phenotype that is very similar to the Alpl-null mice, with severe hypomineralization, which suggested that the activity of one protein may be influenced by the other (373, 401). Prior studies have shown that Mg²⁺ enhances the activity of ALPL (102, 213, 470). The hypomineralized enamel phenotype seen in the Trpm7 mutant mice could be rescued with supplemental dietary Mg²⁺, indicating that in amelogenesis, TRPM7 allows for the inward passage of Mg²⁺ in ameloblasts which in turn enhances the activity of ALPL (401).

In 2009 Beniash et al. (39) identified that, in newly forming enamel, the presence of amorphous calcium phosphate (ACP) is a precursor for the formation of the Hap crystals. Despite the fact that the presence of ACP in newly forming enamel had been suggested many years earlier (e.g., Refs. 48, 114, 474), the data presented by Beniash et al., showing that ACP could be detected in structures with similar shape and dimensions as more mature Hap crystals (39), were a paradigm shift in how dental researchers viewed initial enamel crystallite growth. In mature enamel, Mg²⁺, in a Mg-substituted ACP (Mg-ACP), can be detected at its highest levels at the boundaries of individual enamel crystals (192, 193), suggesting Mg²⁺ may play a role in ACP stabilization (116), or that Mg-ACP could play a significant role in the dynamic demineralization/remineralization properties of enamel, for example, as seen in initial carious lesions or the incorporation of F⁻ after the tooth has erupted (192, 193).

The amount of Na⁺ in enamel, like that of Mg²⁺, increases towards the deeper enamel layer, whereas that of K⁺ appears to be steadily represented throughout (519). There has been a renewed interest in Na⁺ transport in ameloblasts because of the recent identification of a number of exchangers and contransporters that move Na⁺ in and out of these cells (61, 234, 317, 318, 320, 416). The critical role of these proteins in enamel is evidenced by the severity of the abnormal dental phenotypes found in patients with mutations to some of these exchangers (NCKX4) and cotransporters (NBCe1), as discussed in the previous sections. NCKX4 exchanges one K⁺ and one Ca²⁺ for four Na⁺. The similar exchange of K⁺ and Ca²⁺ and the much higher abundance of the latter in enamel suggests that there is likely a rapid removal of K⁺ soon after secretion (61). The exact roles of Na⁺ and K⁺ in forming enamel crystals are poorly understood.

XI. FLUORIDE AND DENTAL HEALTH

Dental caries is a disease caused by biofilm on the tooth surface metabolizing carbohydrates and generating acids that dissolve the tooth mineral. As noted above, incorporation of fluoride into enamel occurs during development and enamel mineralization, and also after the dental crown is fully formed and has erupted into the mouth due to continued environmental exposure (250, 626). Reduced enamel solubility due to fluoride incorporation is one of the mechanisms whereby fluoride helps reduce the risk for developing dental caries. Fluoride ions are incorporated into the hydroxyapatite (Hap) molecular structure through substitution for hydroxide or carbonate ions creating fluoride-enriched Hap (45). Fluoride is not uniformly distributed throughout the dental crown and is most abundant in the outer layers of enamel compared with the enamel closer to the dentin (625). As the fluoride level in enamel increases and the carbonate level decreases, the enamel becomes less acid soluble.

Another caries prevention mechanism for fluoride occurs through exposure of partially demineralized and damaged enamel crystallites to F⁻, Ca²⁺, and PO₄³⁻ (200). Fluorine is highly reactive, allowing fluoride ions to attach to the partially demineralized enamel crystallites and then react with Ca²⁺ and PO₄³⁻ ions that are present in saliva or provided via therapeutic agents designed to control dental caries. Through this process, known as remineralization, fluoride is able to help repair the damaged crystallites and assist in replacing mineral content lost during acid exposure or demineralization (140).

Because of these properties, the acquisition of fluoride in the diet of pregnant and nursing mothers, and young children up to the time when enamel is fully formed on all permanent teeth except the third molars (circa 8 yr of age), is widely recommended by dentists and pediatricians. Fluoride is often present in, or added to, drinking water. Natural concentrations vary depending on the ground water aquifer, but the current optimal recommended level in the United States is 0.7 ppm or 0.7 mg/liter (8, 69, 139, 277, 426). If fluoride is not present in the drinking water, or present at suboptimal levels, fluoride can be given as a dietary supplement in the form of sodium fluoride salt, with the dosage (0.25–1.0mg F⁻/day or 0.55–2.2mg NaF/day) being predicated on factors such as risk for developing dental caries, the individual’s age and stage of tooth development, and consideration of their overall fluoride exposure (69).

The inverse relationship of fluoride exposure in drinking water to dental caries prevalence was discovered by evaluating populations exposed to naturally occurring variations in drinking water fluoride concentrations (101). These early epidemiological studies led to community water fluoridation studies that confirmed the reduction in tooth decay when water had 0.7–1 ppm fluoride ions compared with no fluoride. Epidemiological studies also revealed that exposure to fluoride levels in excess of 1 ppm during enamel formation increased the risk of developing dental fluorosis (142). The inverse relationship between dental caries and dental fluorosis with respect to drinking water fluoride content is illustrated in FIGURE 14. As stated above, in the United States, the Department of Health and Human Services recommends community drinking water have an optimal fluoride level of 0.7 ppm. Worldwide most dentists recommend that everyone, throughout their lifetime, continuously expose their teeth to fluoride from sources such as fluoride-containing drinking water (at 0.7 ppm) and fluoride-containing toothpastes.

FIGURE 14.

This graph shows that the level of dental caries, as measured by Decayed, Missing and Filled (DMF) Teeth (shown on left y-axis), increases as the drinking water fluoride level decreases from 6 ppm to 0 ppm as shown on the x-axis. Conversely, the prevalence of dental fluorosis increases in the population from ~0% affected individuals when there is no fluoride in the water to ~100% of individuals being affected when the drinking water fluoride concentration is 6 ppm. The severity of fluorosis increases as the drinking water fluoride concentration increases.

Enamel fluorosis is an irreversible pathological condition characterized by hypomineralization of the enamel due to excessive exposure to fluoride during enamel development/mineralization. The level of hypomineralization and clinical appearance of the fluorotic enamel varies from mild to severe (FIGURE 15) and is partially determined by the amount of fluoride in the individual’s serum (471). Individuals have differing risk and resistance to developing dental fluorosis based on their genetic makeup and health. Studies indicate there are likely multiple genes that are important in defining variance for dental fluorosis risk (132). Fluoride has a variety of actions that contribute to the development of dental fluorosis including direct effects on the ameloblasts, the developing matrix, and processing of the matrix, altering the proton release during mineralization and how these protons are handled during pH regulation (18, 24, 106, 351). The combined effects on these processes during amelogenesis cause a dose-dependent response to excessive fluoride that results in changes in the enamel crystallite morphology and packing, presenting ultimately as decreased enamel mineral content.

FIGURE 15.

Clinical appearance of hypomineralized teeth resulting from high fluoride exposure during development. In milder cases, the enamel has an opaque white appearance (A) while in moderate to severe cases the enamel will be yellow brown in color and have areas that break or wear away from the tooth, often leaving round “punched-out” areas (B). These changes in enamel color and strength as a result of hypomineralization are seen in this thin section of a tooth viewed with light microscopy that shows the outer opaque hypomineralized enamel in contrast to the more normal translucent enamel (C). Changes in enamel crystallite morphology and packing (i.e., increased spacing) resulting from dental fluorosis are illustrated in this high-resolution electron micrograph of a fractured enamel sample with no etching before imaging (SE 80,000K) (D).

XII. DEVELOPMENTAL ANOMALIES IMPACTING ENAMEL

Enamel development can be perturbed by many different environmental influences and genetic alterations. Amelogenesis is a highly regulated process and can be negatively influenced by pathological/medical conditions such as fever, infection, trauma, changes in oxygen saturation, antibiotics, and many other factors (TABLE 1) (397, 571). The enamel phenotype resulting from different types of insults during amelogenesis will vary depending on the type of stress as well as duration and intensity of the influence. In general, the resulting enamel defects can be classified as defects in the amount of enamel (hypoplasia) or deficiencies in the mineral content (hypomineralization). Enamel hypoplasia can be generalized throughout the dentition or it can be localized. Environmental stressors that are of short duration (e.g., fever) often cause localized defects, whereas chronic stressors (e.g., elevated fluoride exposure) are more likely to be associated with generalized defects (TABLE 1).

Table 1.

Environmental influences on enamel formation

Condition	Enamel Phenotype
Fever	Hypomineralization to marked hypoplasia
Starvation	Enamel hypoplasia
Excess fluoride exposure	Hypomineralization
Trauma	Hypomineralization to marked hypoplasia
Hypoxia (e.g., severe cardiac defect)	Hypomineralization to marked hypoplasia
Infection (congenital syphilis, cytomegalo virus, congenital rubella)	Hypomineralization to marked hypoplasia
Tetracycline	Hypoplasia
Low birth weight	Hypoplasia

Enamel defects are common in the general population, with reports suggesting that between 20 and 80% of the world’s population have enamel defects (538, 570). The broad range of reported enamel defect prevalence is largely due to inclusion criteria for what constitutes an enamel defect (e.g., actual hypoplasia or a deficiency in the amount of enamel vs. a color change indicating hypomineralization). Not all teeth, or even all surfaces of teeth, are affected equally by enamel defects even though they form at the same time. Children having more frequent and serious illnesses are more likely to have enamel defects.

Not uncommonly, children (2–15%) have enamel defects of the facial or front surface of their primary canine teeth (395, 535). It has been hypothesized that these lesions occur due to the thin or often fenestrated bone over the developing canine teeth during infancy that predisposes them to minor trauma, leading to enamel defects (535). Another common enamel defect involves any or all of the first permanent molars (prevalence range from 5 to 25% of children) and can vary markedly in severity of hypomineralization (168, 627). The more severely hypomineralized the molars, the more likely it is that there will be defects of the permanent incisor enamel as well. This condition is called molar incisor hypomineralization (MIH); in its mildest form the enamel shows only a minor color change and altered opacity, while in the severe forms the enamel breaks away from the tooth during eruption, leading to loss of the enamel and not uncommonly the tooth (FIGURE 16). Teeth affected by MIH are often very sensitive to thermal or chemical stimulation as they do not have an adequately mineralized enamel-insulating layer. The etiology of MIH is not fully understood, but it is known to be more prevalent in children that have had more significant illness (609). There is some evidence that there could be a genetic predisposition or increased sensitivity to certain environmental stressors (308). The affected areas of enamel have an increased protein content and decreased mineral content.

FIGURE 16.

Molar incisor hypomineralization (MIH). The first permanent molar (A) has a creamy yellow brown coloration that is mostly contained on the biting occlusal surface and has not resulted in any enamel loss in this mild to moderate case. In contrast, this first permanent molar (B) from the same individual has severe enamel loss over much of the tooth. This child had a relatively marked enamel phenotype of the permanent central incisors (C) while the permanent lateral incisor (left area of the panel) appears normal.

XIII. GENETIC DISEASES IMPACTING ENAMEL

There are thousands of genes expressed by ameloblasts (318, 664) and ~100 different hereditary conditions associated with an enamel phenotype (642). The majority of hereditary conditions affecting enamel formation are syndromes that have more generalized clinical manifestations and phenotypes extending beyond the dental enamel. Many of these conditions are caused by genes that have a known function in ameloblasts. The pathophysiology resulting from these genetic alterations and ameloblast dysfunction most commonly results in a hypoplastic enamel phenotype. For example, junctional epidermolysis bullosa is caused by mutations in genes that are expressed by ameloblasts and are important in cell-to-cell adhesion (e.g., COL17A1, LAMB3, IGTA6, and ITGB4) (645). The abnormal proteins produced from these genes result in fragile skin that blisters. In ameloblasts, abnormal function of these proteins results in cells that do not adhere to each other or to the stratum intermedium, resulting in cell separation and enamel hypoplasia (FIGURE 17). Interestingly, some mutation in genes such as LAMB3 can be associated with a syndrome such as epidermolysis bullosa or can result in only an enamel phenotype (296, 453, 618).

FIGURE 17.

Abnormal enamel resulting from junctional epidermolysis bullosa. Mutations in genes associated with cell-to-cell adhesion, such as LAMA3, LAMB3, LAMC2, and COL17A1, can result in junctional epidermolysis bullosa and abnormal enamel formation. The clinical phenotype resulting from mutations in these genes is a deficiency in amount of enamel that can be localized in the form of pitting (A). The variation in size of the pits is thought to be a reflection of the numbers or groups of ameloblasts that are lacking appropriate cell-to-cell attachment (B). [B from Wright et al. (649a), with permission from Elsevier.] In other cases the phenotype presents as a generalized thinning of the enamel, as seen in the teeth of this individual with the Herlitz form of junctional epidermolysis bullosa (C). This individual also had failure of tooth eruption of the permanent dentition as seen in their panographic radiograph (D). This is illustrative that the developing tooth epithelium plays a role in tooth eruption as well as enamel formation.

A review of the known hereditary conditions associated with enamel defects, identifying mutations in genes coding for transcription factors, growth factors, matrix proteins, ion channels, and proteinases, has been presented by Wright et al. (642). For example, mutations in the DLX3 homeobox gene, which functions as a transcription factor, cause the tricho-dento-osseous syndrome that is associated with kinky curly hair at birth, dense-thick bone, generalized thin and or pitted enamel, and large pulp chambers (651). Mutations in the p63 gene, which is important in cell growth, cause a variety of ectodermal dysplasia syndromes (e.g., Rapp Hodkins syndrome, ectrodactyly, ectodermal dysplasia cleft syndrome), all of which can have enamel defects (302). Many of the genes known to be critical for enamel formation were first identified by determining the molecular basis of hereditary conditions that had associated enamel phenotypes. A recent example of this was identification of the ROGDI gene that is associated with Kohlschütter-Tönz syndrome (510). This syndrome has a distinct phenotype where all teeth exhibit a yellow-brown discoloration of the enamel and a decreased level of mineralization. The ROGDI gene was not known to be critical for enamel formation before this discovery. This gene is thought to code for a leucine-zipper protein that is highly expressed in the brain and spinal cord. Leucine zipper motifs are a structural feature common to transcription factors and other types of proteins implicated as negative transcription regulators. Another interesting association occurs in Jalili syndrome where affected individuals have cone-rod dystrophy in the eyes and enamel that has a brown appearance and is hypomineralized. This condition is caused by mutations in the cyclin and CBS domain divalent metal cation transport mediator 4 gene (CNNM4) that may play a role in metal ion transport (438). Cystic fibrosis (CF) is caused by mutations in the CFTR gene that is involved in regulating ion movement and pH regulation. Clinical evaluation of humans with CF found a high percentage of affected individuals had associated enamel defects of varying severity. In the CF mouse model (Cftr knockout), the incisors had an opaque-white hypomineralized phenotype that was 100% penetrant (59, 572, 646). This led to the discovery that the Cftr gene was expressed by and important in the regulation of pH in the developing mouse enamel organ and provided a likely explanation for the enamel defects seen in people with CF (63, 572). Other genes involved in ion transport associated with hereditary conditions resulting in immune dysfunction include the members of the Ca²⁺ release-activated Ca²⁺ channels STIM1 and ORAI1 (145, 447). Mutations in the Na⁺/K⁺/Ca²⁺ exchanger NCKX4 (coded by SLC24A4) are directly linked with AI, but less is known about other disorders caused by mutations to SLC24A4 that include heart disease (222, 439).

Hereditary nonsyndromic conditions primarily affecting the enamel are referred to as AI (639). The prevalence of AI varies around world and is thought to occur in ~1/8,000 people, although there has only been one epidemiological study ever conducted in the United States (638). AI describes a variety of disorders that have been classified based on their clinical phenotype and mode of inheritance, as well as based on the perceived mechanism leading to the enamel defect, i.e., deficient matrix formation leading to hypoplasia, deficient crystal growth and mineralization during the maturation stage causing a decreased level of maturation and mineralization, or abnormal initiation of the enamel crystallites with subsequent abnormal mineralization or hypocalcification (639). Both hypomaturation and hypocalcification are characterized by the predominant phenotype of decreased enamel mineral or hypomineralization (see FIGURE 18). Enamel affected by these types of AI has greater amounts of protein present compared with normal enamel (647).

FIGURE 18.

Amelogenesis imperfecta. Amelogenesis imperfecta (AI) is caused by mutations in numerous genes that lead to deficiencies in the amount of enamel and a hypoplastic (A) and/or hypomineralized enamel phenotype (B). [A from Nusier et al. (411a), with permission from Elsevier.] The hypomineralized forms typically have retained protein in the enamel, such as seen in this defective fractured enamel resulting from a FAM83H mutation (C), that is not normally seen in sound enamel. Lacy protein (arrow) is seen on top of the enamel crystallites that make up the underlying enamel prism or rod. In the hypomaturation AI types, the amino acid content in the retained protein resembles that of amelogenin having a high percentage of proline residues, as seen in this enamel affected with AI caused by a C4orf 26 mutation (D).

The first gene to be associated with these conditions was AMELX, which codes for the most abundant enamel matrix protein, amelogenin (552–555). There are now known to be many allelic mutations in the AMELX gene that cause different alterations in the protein and result in different phenotypes (648). Missense mutations resulting in single amino acid changes are often associated with a hypomineralized enamel phenotype. Mutations causing a loss of the carboxy terminus of the amelogenin protein all result in a hypoplastic enamel phenotype. This is presumably due to the critical functionality of the carboxy terminus in amelogenin aggregation and orientation to the developing enamel crystallites. These genotype-phenotype associations result from changes in the protein function due to the alteration of specific functional domains with importance in cellular processes such as protein self-assembly, protein-mineral interactions, and changes in proteolytic processing of the mutant protein (423, 520).

Mutations causing AI have been identified in many of the known enamel extracellular matrix proteins and proteinases, along with a number of other genes (TABLE 2), and there will likely be additional AI-associated genes identified in the future. The phenotypes are quite diverse, with some causing more localized enamel defects, while others are associated with generalized phenotypes affecting all the teeth and areas of enamel (641). Variability in phenotypes associated with some AI types is thought to result from mutations causing proteins to have a dominant negative effect while others cause haploinsufficiency. This is the case with mutations in the ENAM gene that can result in generalized thin enamel hypoplasia (dominant negative effect) or bands of pitted enamel (haploinsufficiency) (208, 293, 358). When Enam is knocked out in the mouse, there is a complete absence of any organized and mineralized enamel layer (229). This is thought to be illustrative of the critical role the enamelin protein plays in growth of the enamel crystallites along the C-axis.

Table 2.

Amelogenesis imperfecta: OMIM designations, genes, and phenotypes

Amelogenesis Imperfecta	Gene/Locus	Enamel Phenotype	Mode of Inheritance
#301200, Amelogenesis imperfecta, type IE; AI1E	AMELX	Hypoplasia/hypomaturation depending on mutation and protein effect	X-linked
#301201, Amelogenesis imperfecta, hypoplastic/hypomaturation, X-linked 2	Xq22-q28	Hypoplastic and/or hypomaturation	X-linked
#104500, Amelogenesis imperfecta, type IB; AI1B	ENAM	Localized hypoplastic/generalized hypoplastic	Autosomal dominant
#204650, Amelogenesis imperfecta, type IC; AI1C	ENAM	Generalized hypoplastic	Autosomal recessive
#204700, Amelogenesis imperfecta, hypomaturation type, IIA1; AI2A1	KLK4	Normal enamel thickness: hypomineralized orange brown color	Autosomal recessive
#612529, Amelogenesis imperfecta, hypomaturation type, IIA2; AI2A2	MMP20	Normal enamel thickness: hypomineralized orange brown color	Autosomal recessive
#130900, Amelogenesis imperfecta, type III; AI3	FAM83H	Localized or generalized hypomineralized enamel	Autosomal dominant
#613211, Amelogenesis imperfecta, hypomaturation type, IIA3; AI2A3	WDR72	Hypomaturation: creamier/opaque enamel upon eruption, discoloration and loss of tissue posteruption	Autosomal recessive
#104510, Amelogenesis imperfecta, type IV; AI4	DLX3	TDO-thin pitted hypoplastic	Autosomal dominant
#614253, Amelogenesis imperfecta AND gingival fibromatosis syndrome; AIGFS	FAM20A	Generalized hypoplastic and failure of tooth eruption, gingival hypertrophy	Autosomal recessive
#104530, Amelogenesis imperfecta, hypoplastic type IA, AI1A	LAMB3	Hypoplastic: failure to erupt and calcification of pulp	Autosomal dominant
#614832, Amelogenesis imperfecta, hypomaturaltion type, IIA4; AI2A4	C4ORF26	Hypomaturation AI	Autosomal recessive
#616270, Amelogenesis imperfecta, hypoplastic type, IF, AI1F	AMBN	Generalized hypoplastic	Autosomal recessive
#615887, Amelogenesis imperfecta, hypomaturation type, IIA5, AI2A5	SLC24A4	Hypomineralized: mottled appearance	Autosomal recessive
#Not listed, amelogenesis imperfecta, hypomaturation type	AMTN	Hypomineralized	Autosomal dominant
#Not listed, amelogenesis imperfecta, hypomaturation type	ACPT	Generalized hypoplastic	Autosomal recessive
#Not listed, amelogenesis imperfecta, hypomaturation type	GPR68	Hypomaturation	Autosomal recessive

The protein functions for several of the more recently identified causative genes (e.g., C4ORF26, WDR72, FAM83H) are not fully understood, and their involvement in enamel formation was discovered by evaluation of families having AI (TABLE 2). FAM83H mutations cause autosomal dominant hypocalcified AI that is thought to be the most common type of AI in North America (295). Phenotypes associated with FAM83H mutations also can vary from generalized to localized enamel defects depending on the location and type of mutation and, presumably, the altered function of the resulting protein (644). FAM83H interacts with casein kinase 1 (CK1) and is thought to play a role in keratin cytoskeleton organization and desmosomes in ameloblasts (307). Mutations of the WDR72 gene result in hypomaturation defects of the enamel that are thought to be caused by abnormal endocytic vesicle trafficking of matrix proteins and subsequent enamel mineralization (278, 616). The C4orf26 gene is now thought to be tooth-specific but not enamel-specific, and it may play a role in Hap nucleation through its phosphorylated carboxy terminus (437).

Mutations in genes coding for the enamel proteinases MMP20 and KLK4 both result in hypomineralized enamel that has an orange to brown discoloration (207, 421). Abnormal function of these proteinases results in an increased retention of protein in the affected enamel, as is common to most of the hypomineralized forms of AI (647). Diminished processing and removal of the enamel matrix proteins, such and AMELX, AMBN, and ENAM, results in abnormal crystallite growth and a decreased final mineral content of the enamel (649).

As can be appreciated by the reader, discovering new genes, and identifying genotype/phenotype relationships, is ongoing. During the writing of this review paper three additional genes were been associated with AI (TABLE 2). These are mutations to amelotin (Amtn) (546), a protein expresses exclusively during maturation-stage amelogenesis (255, 402), the pH-sensing G protein-coupled receptor GPR68 (440), and the testicular acid phosphatase ACPT (512) (TABLE 2).

XIV. ENAMEL BIOMIMETICS

While enamel cannot regenerate itself or remodel like most bones do, it is nevertheless an ideal candidate to apply novel materials chemistry without the use of biology for its regeneration as it is completely acellular. Conventional restorative methods in dentistry use artificial materials like composites, ceramics, and amalgam to restore functional properties and are not discussed here. There are, however, a series of approaches available or currently being tested in the laboratory that can rebuild lost enamel structure through remineralization (92). These so-called biomimetic methods are designed to rebuild the intricate apatite crystallite structure by application of calcium phosphate chemistry that stimulates the regrowth of the natural tissue and ideally restores the mechanical and optical properties of enamel. Due to the high organization and alignment of fibrous apatite crystallites, enamel almost acts like a single crystal. It reflects only a portion of visible light and is therefore translucent (267). To mimic the translucency of enamel, the restorative process will need to rebuild the highly organized structure of enamel to perfectly blend in with the surrounding healthy tissue (267).

Initial damage on the enamel surface is often observed as “white spots” which illustrate that the organized mineral layer in enamel has been altered by a demineralization/remineralization process that most likely has been triggered by cariogenic acids produced by bacteria in the oral environment (20, 456). White spot lesions or incipient caries consist of a demineralized zone that is covered by a superficial mineral layer often comprised of larger apatite crystals, which reflect light and therefore appears white. The common treatment for those lesions is the use of fluoride, often in the form of an acidulated gel or dentifrice (68, 334, 360, 426, 589, 590). The mildly acidic composition of the delivery system will dissolve the surface mineral layer and allow fluoride ions to penetrate into the demineralized zone, which leads to an increase in saturation levels towards apatite. The reaction of this fluoride with calcium and phosphate ions then leads to the nucleation of flouroapatite crystals (191). The growing apatite crystals rebuild the structure of dental enamel fairly successfully and often fully restore its mechanical and optical properties. The simple chemistry of this approach, however, only works well on shallow lesions that are in the order of tens of micrometers deep (334, 353). Deeper lesions, and lesions that are infected with bacteria or result from secondary caries underneath a restoration, require a more sophisticated approach for recovery of form and function by the reintroduction of apatite mineral. A number of studies are currently exploring novel methods for biomimetic remineralization of such lesions (92, 337).

A fundamental difficulty with the clinical application of remineralization systems is the low solubility of calcium phosphates, particularly in the presence of fluoride ions, which makes it difficult to remineralize deeper lesions (478). In saturated solutions, mineral will precipitate randomly and thus not rebuild the enamel structure. Therefore, stabilizing agents have been developed to extend the lifetime of calcium and phosphate ions in solution (417) and thus allow for a controlled mineralization of apatite ideally through epitaxial growth of mineral onto the damaged crystalline structure in carious enamel (337). Casein, a protein present in milk, is such a stabilizing agent that facilitates the formation of ACP (95). The casein phosphopeptide-ACP complex has successfully been used for the remineralization of white spot lesions and is recommended to treat deeper lesions when a restorative treatment is not desired (408). Its success is based on the delivery of casein-stabilized ACP droplets that transform into apatite crystallites when in contact with enamel crystallites (95). Based on these studies, a number of other natural proteins, peptides, and synthetic molecules have been developed that are able to stabilize the saturated solutions and thus prevent heterogeneous nucleation while promoting homogeneous nucleation (84, 92).

The delivery of such stabilizing agents is challenging in a clinical setting as aqueous solutions will be washed out of the site and saliva can interfere with the remineralization process. The development of gels or cements that provide sustained release of mineralizing agents is desirable. A chitosan gel has been explored that has shown great ability to crystallize apatite nanofibers with similar orientation to that observed in natural enamel (486). The ability of the gel was further enhanced when amelogenin protein was added as the thickness of the rebuilt enamel layer increased in in vitro tests, taking advantage of the growth-promoting effect of amelogenin (484, 486).

In addition to these methods that attempt to regenerate enamel on decayed teeth, there are numerous studies that emphasize the generation of enamel or an enamel-like tissue in vitro and de novo (485, 654). These approaches promote oriented crystal growth of fibrous apatite that mimics the structure of enamel. EDTA is known as a chelating agent and has been used to stabilize high concentrations of calcium phosphate ions that upon water evaporation produce a layer of nanometer-sized apatite crystals with high alignment, mimicking the structure of nonprismatic dental enamel very well (80, 81). Clinical studies are currently underway to explore this method by direct application to demineralized teeth (617). A common challenge in testing and evaluating these systems, however, is the lack of suitable standards of evaluation that allow for systematic comparison between the various approaches in vitro, leaving the ultimate litmus test to the clinical trial which requires a large number of subjects for statistical validation (353).

XV. CONCLUSIONS

In mammals, in nonpathological conditions, dental enamel is the only epithelial-derived tissue that mineralizes. Ameloblasts are primarily responsible for the formation of enamel, which is essentially a Hap-based material containing less than 5% organic material by weight. It is because of its cellular origins that an extracellular matrix has evolved, comprised of proteins with little homology to any other animal proteins, to be capable of guiding the formation of a unique hierarchical structure with a highly ordered and very repetitive patterning. In normal situations, enamel’s distinctive biomechanical properties allow it to function for the entire lifespan of an animal, despite the amount of wear resulting from mastication, clenching and grinding, and disease such as caries. Ameloblasts have a very short lifespan, relative to the life of the organism, to produce the avascular enamel that, once formed, has no reparative abilities. Amelogenesis involves a large number of activities including the formation of a temporary proteinaceous matrix conducive to mineralization followed by the removal of this matrix by endocytosis, ion transport and pH regulation, and apoptosis. Failure during any one of these stages of amelogenesis may result in pathologies impacting enamel health. We have summarized the literature related to amelogenesis, with a greater emphasis on mineralization events occurring largely during the maturation stage. We have also reviewed enamel pathologies that have been linked to known genes and discussed the role of fluoride-based and biomimetic approaches to enamel repair and conservation.

There are, however, many voids in our understanding of amelogenesis. There is an increasing need to define experimentally the transport and movement of ions in the enamel organ and to gain a deeper understanding of key functions as they relate to amelogenesis. For example, phosphate transport activities and molecular mechanisms responsible for pH regulation remain poorly understood. There is also limited information to explain ameloblast movements that result in enamel’s remarkable prismatic architecture, although recent studies partly attribute this to the extensive remodeling of ameloblast junctional complexes during amelogenesis (31). It is also unknown if the small amounts of protein retained in mature enamel are ordered or not, and a recent paper has provided evidence that individual enamel crystals occlude some matrix protein (454), which may also contribute to enamel’s unique biomechanical properties. It appears reasonable to suggest that remnant proteins help to achieve stiffness and hardness required for the longevity of the tooth (198). Finally, there is current interest in generating an enamel biomimetic for future dental clinical applications, with some of this work investigating amelogenin-based peptides in mineralizing solutions (194, 203, 260, 387, 432, 485). Future research in these areas will have an immensely positive impact on understanding enamel biology and disease.

GRANTS

This work was supported by National Institute of Dental and Craniofacial Research Grants R00 DE022799 and R01 DE025639 (to R. S. Lacruz), R01 DE025709 (to S. Habelitz), R01 DE016079 (to J. T. Wright), and R01 DE019629 and R21 DE024704 (to M. L. Paine).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.