Читать книгу Oral Cells and Tissues - Philias R. Garant - Страница 10

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During the early stage of tooth formation, the enamel organ consists of the outer enamel epithelium (OEE), the cells of the stellate reticulum (SR), the stratum intermedium (SI), and the inner enamel epithelium (IEE) (see Fig 1-6). The cells of the outer enamel epithelium are generally cuboidal. They attach by hemidesmosomes to a basal lamina separating them from the adjacent dental sac, a connective tissue of ectomesenchymal origin. Cytoplasmic organelles in the OEE include a moderate number of mitochondria, a small number of cisterns of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum, and a poorly developed Golgi complex. The presence of coated vesicles in the peripheral cytoplasm and along the plasma membrane facing the basal lamina suggests that the OEE is involved in specific endocytosis of extracelluar substances.

Soon after the onset of enamel formation, the OEE becomes convoluted by indentations of highly vascularized connective tissue. This structural change becomes pronounced during enamel maturation, when the OEE, the SR, and the SI form the papillary layer to increase the surface area between the enamel organ and the adjacent blood supply. This change is pronounced in the continuously developing incisor of the rat, the most thoroughly investigated model of tooth development.

The cells of the SR have a compact cell body with many long folds of cytoplasm that contact and partly overlap similar cytoplasmic folds from adjacent cells. Desmosomes and gap junctions are formed at points of cell-to-cell contact. These folds of cytoplasm line wide extracellular spaces rich in water-binding glycosaminoglycans.

The soft, jellylike consistency of the enamel organ is due to the hydration of the SR glycosaminoglycans. This property is believed to be important in equalizing pressure generated by cell proliferation and matrix secretion in the dental papilla. It has been suggested that, if external tissue-generated forces at the interface between the preameloblasts and the preodontoblasts are eliminated, the three-dimensional outline of the future dentinoenamel junction, and ultimately the shape of the crown of the tooth, can be molded solely by the forces of cell proliferation in both the IEE and the underlying preodontoblasts of the dental papilla. With the onset of ameloblast differentiation, the formation of a terminal web in the IEE, coupled with the assembly of a basement membrane beneath the IEE, stabilizes the shape of the future dentinoenamel junction.

The cells of the SI form a compact zone, one to two cellular layers deep, between the SR and the IEE (Figs 3-1 and 3-2). The intercellular spaces between the SI cells are relatively narrow, and the adjacent cells are held together by many desmosomes. Large gap junctions are formed with adjacent cells of the IEE. The SI cells contain many mitochondria, a characteristic shared with the distal cytoplasm of secretory ameloblasts.

Fig 3-1 Secretory ameloblasts of the rat incisor tooth. In the secretory stage, ameloblasts are tall columnar cells, characterized by a secretory process (Tomes process [TP]) and an infranuclear concentration of mitochondria (M). (BV) Blood vessels; (N) nucleus; (OEE) outer enamel epithelium; (SI) stratum intermedium.

Fig 3-2 Electron micrograph of the proximal end of secretory ameloblasts (AM) containing a high concentration of mitochondria (M). Cells of the stratum intermedium (SI), stellate reticulum (SR), and outer enamel epithelium (OEE) have not yet formed the papillary layer. Blood vessels (endothelium [E]) lie in close apposition to the OEE. (Original magnification × 1,600.)

Although the exact function of the cells of the SI is not clear, the fact that they contain a rich complement of mitochondria and stain intensely for alkaline phosphatase and adenosine triphosphatase (ATPase), suggests a possible role in ion and water transport. The presence of extensive gap junctions between the cells of the SI and the ameloblasts indicates that the two cell types act in close concert during amelogenesis.

Recent studies have shown that the regulatory activity of the enamel knot is taken up by the SI during cusp formation.¹ Growth regulatory signals mediated by Sonic Hedgehog (Shh) are activated in the SI in a wavelike manner from the cusp to cervical zone.

The IEE is a layer of columnar preameloblasts abutting the dental papilla. Organ culture studies of amelogenesis indicate that contact with the dental papilla is required for the expression of enamel protein.² Although the precise nature of the early instructive signals that originate from the dental papilla have yet to be identified, the permissive effect of the extracellular matrix of the basement membrane is required for initiating expression of amelogenin.³

The region of highest mitotic activity in the IEE is located near the cervical loop portion of the enamel organ.^4–⁶ Stem cells in the cervical loop divide to give rise to an expanding metablast clone of preameloblasts.^7,⁸ The rates of proliferation and differentiation in the blast-metablast populations vary among species, among individual teeth, and among different parts of a given tooth.

Secretion of growth factors by the enamel knot and the dental papilla regulate cell proliferation and growth of the IEE.^6,⁹ Receptors for epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor have been localized by immunohistochemistry in preameloblasts.¹⁰ Phospholipase C_γ, a downstream signaling molecule activated by growth factor–receptor interaction, was also demonstrated in preameloblasts. Preameloblasts must exit the cell cycle to begin the process of cytodifferentiation.⁴

The cytoplasm of the preameloblasts contains many free ribosomes, a small Golgi apparatus, a few cisterns of rough and smooth endoplasmic reticulum, and a small number of mitochondria. Adjacent preameloblasts form gap junctions and desmosomes. A prominent zonula adherens junction with an associated terminal web of cytoplasmic filaments forms in the proximal cytoplasm adjacent to the cells of the SI.^11,¹² This proximal junctional complex binds and stabilizes the preameloblasts prior to degradation of the underlying basal lamina.

Soon after the deposition of mantle dentin, the odontoblasts express matrix metalloproteinases (MMPs) that begin the digestion of the basement membrane. Preameloblasts subsequently remove basement membrane fragments through phagocytosis.¹³ Removal of the basement membrane allows the dentin and enamel matrices and their respective mineral phases to come into direct contact. As a result, a strong mechanical bond is formed between the enamel and dentin.

Recent studies using reverse transcription and polymerase chain reaction techniques have shown that immature enamel organ cells exist in a “protodifferentiated” state.¹⁴ Preameloblasts of the IEE produce small amounts of enamel matrix protein prior to overt morphodifferentiation as secretory phenotypes.^15,¹⁶ Some of these proteins traverse the dental lamina and are taken up in odontoblast-coated vesicles.

Although the signal transduction pathways that regulate ameloblast and odontoblast differentiation have yet to be identified, immunohistochemical evidence has shown that cytokine-activated signaling pathways, including protein kinase C activation, are involved in early amelogenesis.¹⁷

Structure of Secretion-Stage Ameloblasts

In describing the structure of the ameloblast, the term proximal is used to refer to the end of the cell nearest to the SI, and the term distal is used to identify the secretory end of the cell, next to the enamel (see Fig 3-2). The term apical is also used to describe the secretory pole of ameloblasts.

The structural changes that characterize each of the various stages of ameloblastic activity have been well documented.^18–²¹ The cytoplasm of the mature secretory ameloblast is highly polarized (Figs 3-1 to 3-4). The infranuclear (proximal) cytoplasm contains many mitochondria and a terminal web of cytoplasmic filaments associated with a zonula adherens (see Fig 3-2). Gap junctions are present between the proximal surface of the ameloblast and the overlying cell of the SI.

The supranuclear (distal) cytoplasm, which accounts for more than one half of the total cell volume, is filled with an extensive array of RER cisternae and a well-developed Golgi complex (see Figs 3-4a to 3-4c).^18,^20,²² Electron microscopic autoradiography and immunocytochemical studies have shown that enamel proteins are synthesized in the RER and glycosylated in the Golgi cisternae prior to being packaged into specific granules (see Figs 3-4a to 3-4c).^23–²⁵ Condensing vacuoles derived from the trans-Golgi network mature into smaller, dense-core secretory granules. The newly formed secretory granules are immediately transported along a microtubular network to the distal end of the cell, where they are released by merocrine secretion into the enamel compartment.^26,²⁷ Microtubule inhibitors, such as colchicine and vinblastine, block enamel matrix secretion.²⁸ A distal junctional complex, consisting of gap junctions, a zonula adherens, and a zonula occludens, bind adjacent ameloblasts and seal the lateral intercellular spaces from the enamel-forming compartment (see Fig 3-3).²⁹

The first layer of enamel matrix (about 20 μm) is secreted across the flat distal cell surface of the newly differentiated ameloblast. As new membrane is added to the distal plasma membrane by the fusion of matrix secretion granules, the distal cell surface develops a protruding cytoplasmic process, 5 to 15 μm in length (see Figs 3-1, 3-3, and 3-4). Sir John Tomes, a British dentist and histologist, first described this process in the mid-19th century. The formation and the length of Tomes process (TP) appear to be related to the quantity and speed of matrix secretion, because new secretory granule membrane is added to the secretory pole of the cell faster than it can be recovered and recycled from that region. Tomes process protrudes at an angle to the long axis of the ameloblast cell body (see Fig 3-1).

With the formation of TP, the secretory surface of the ameloblast becomes more complex, and secretory granules are directed to two regions of the distal cytoplasm.^12,^26,³⁰ Enamel matrix proteins released from the distoventral part of TP form prismatic or rod enamel (see Figs 3-3 and 3-4). Secretion from the proximal part of TP, at the point where adjacent ameloblasts abut each other, gives rise to the interprismatic or interrod enamel (see Fig 3-2). The plasma membrane is highly infolded and apparently continuous with a tubulovesicular compartment at both the proximal and distal secretory sites.^25,²⁷ The species-specific prism pattern is genetically determined by the shape and hexagonal packing of ameloblasts, the orientation of TP vis-à-vis the cell body, and the rate of enamel deposition.^31–³³

Observation of TP by electron microscopy has led to the conclusion that its surface can be subdivided into a secretory face (the concave or ventral surface) and a retrieval face (the convex or dorsolateral surface) (see Fig 3-3).³⁴ Endocytosis for retrieval of membrane is carried out by formation of coated vesicles along the nonsecretory plasma membrane. Internalization and subsequent fusion of coated vesicles form endosomes and multivesicular bodies, components of the cell’s digestive apparatus. Studies of the fate of injected tracer substances have shown that they are taken up in coated vesicles of TP, suggesting that solutes in the extracellular space, including initial breakdown products of the enamel matrix, might begin to be removed in relatively small amounts during the secretory stage of amelogenesis.

Fig 3-3 Secretory ameloblasts. A zonula adherens (ZA) junction binds adjacent ameloblasts at both proximal and distal ends. The bulk of the infranuclear cytoplasm is occupied by the rough endoplasmic reticulum and a well-developed Golgi apparatus (GA). A zonula occludens (ZO) barrier is present in the intercellular space just proximal to Tomes process (TP). Secretion granules (SG), originating in the GA, are secreted from the proximal end of TP (A), giving rise to interprismatic enamel (IPE). Additional SG discharge at the distal end of the process (B) gives rise to a single enamel prism (PE). Interprismatic enamel is contributed by several contiguous cells. Cross sections of TP (1) and the adjacent enamel (2) illustrate the relationship of the secretory surface to prismatic enamel and the endocytotic surface to the development of a prism sheath (Sh). (N) Nucleus. (inset) Relationship among the prism sheath, the prism, and Tomes process.

Figs 3-4a to 3-4c Electron micrographs of secretory ameloblasts. (a) Distal portion of the ameloblasts, containing Golgi complexes (G) and an abundance of rough endoplasmic reticulum (RER). Secretion occurs from Tomes process (TP). (Nuc) Nucleus; (IPE) interprismatic enamel. (Original magnification × 2,200.) (b) High magnification of Tomes process (TP), containing secretion granules (SG). (IPE) Interprismatic enamel; (PE) prismatic enamel. (Original magnification × 17,000.) (c) Regular spacing of developing enamel crystallites (EnCR) in the enamel matrix (Em). (Original magnification × 94,000.)

During initial formation of enamel and during the last few days of enamel deposition, ameloblasts have no TP, and thus no prismatic pattern is formed; therefore, the first few microns of enamel next to dentin, and the last several microns of enamel at the surface, are aprismatic. The crystallites of aprismatic enamel are tightly packed and aligned perpendicular to the enamel surface. Aprismatic surface enamel compromises adhesion of dental occlusal sealants and orthodontic brackets by interfering with the penetration of adhesives into the enamel.³⁵ This layer should be removed by acid etching before treatment protocols that require bonding to enamel.

Each ameloblast forms a single prism or enamel rod (see Fig 3-3).^12,^34,³⁶ The enamel prism is made up of thousands of hydroxyapatite crystallites, oriented more or less parallel to each other. Each enamel crystallite is a ribbonlike structure that is believed to extend without interruption from the dentinoenamel junction to the enamel surface.³⁷ Ultrastructural studies of enamel show that individual crystallites follow a spiral course within the prism.²⁶ In longitudinal sections, enamel prisms exhibit optical cross striations, about 3.7 μm apart, caused by slight constrictions in the width of the prisms due to a daily cyclical rhythm of enamel matrix secretion.³⁸ When human enamel is viewed in cross section, the prisms have an arcshaped outline and are arranged in offset horizontal rows (see Fig 3-3).³¹

Packing irregularities of crystallites demarcate the prismatic and interprismatic domains. This border region retains protein to form a sheathlike structure. Interprismatic crystallites have their long axes oriented at an angle to those in the prism (see Fig 3-3). The distinction between interprismatic and prismatic enamel is believed to reside solely in the orientation of crystallites. There is no evidence to suggest that the biochemical compositions of the interprismatic and prismatic matrices are different. Physicochemical forces, rather than biochemical differences in matrix proteins, act to orient the matrix and determine crystallite orientation at each of the two secretion sites.²⁶

A prism sheath (Figs 3-3 and 3-5) delimits approximately three quarters of the boundary between prismatic and interprismatic enamel. The composition of the sheath and its manner of development are not well understood. However, the shape of the sheath and its location over the convex surface of the prism suggest that its formation is associated with the endocytotic surface of Tomes process.

Fig 3-5 Cross-sectional arrangement of the prisms in human enamel. The position of each ameloblast in relation to the prism outline is represented by the superimposed boundary lines (B). Each arcade-shaped prism is surrounded by interprismatic enamel (IP), which is contributed by the secretions of seven ameloblasts. Note the offset arrangement of the horizontal rows of arcades. (P) Prismatic enamel; (S) sheath region.

No sheath is present, and prismatic enamel is in direct contact with interprismatic enamel, along the flat surface of the prism, corresponding to the secretory surface of TP (see Fig 3-3). During enamel secretion, Tomes process undergoes fragmentation at its most distal point. This may create space between the already mineralized prismatic and aprismatic matrices.³⁹ Several nonamelogenin proteins, collectively known as sheath proteins, appear to localize along TP, and in the space along prism boundaries.⁴⁰ The space created along the prism boundary may provide a route for the escape of enamel matrix degradation products during enamel maturation. Retention of enamel protein fragments in the space created by the irregular packing of crystallites at the border between interprismatic enamel and prismatic enamel may contribute to the prism sheath.

Fully matured enamel provides a hard, wearresistant surface. Its only weakness, relative brittleness or susceptibility to crack formation, is along cleavage planes that follow the border between prismatic and interprismatic enamel.⁴¹ Biomechanical analysis of the fracture behavior of teeth has shown that the dentinoenamel junction undergoes plastic deformation to help resist crack propagation into the underlying dentin.⁴² Thus, most cracks are confined to enamel. It has been suggested that coarse collagen fibrils in the dentinoenamel junction resist and deflect crack propagation.

Biology of the Enamel Matrix

Information about the composition, mechanism of action in mineralization, and maturational change of enamel matrix proteins has been difficult to obtain because many enamel proteins are present in only relatively small amounts, and most undergo proteolytic processing soon after secretion. However, through the application of molecular biology techniques, significant progress is now being made in this area.⁴³ Current understanding is that ameloblasts produce two classes of matrix proteins: amelogenin, a relatively homogenous product, which constitutes approximately 90% of newly secreted enamel matrix, and a heterogenous group of non-amelogenin proteins, including tuftelin, ameloblastin, enamelin, metalloproteinase, and serine proteinases, which make up the remaining 10%. The role of the enamel epithelium and the enamel matrix proteins in the mineralization of enamel has been the subject of several in-depth reviews.^43–⁴⁵

Amelogenins

Amelogenins are expressed as several isoforms through alternative splicing of pre–messenger ribonucleic acids (mRNAs).^12,^25,^30,^46–⁴⁹ The amelogenins are rich in proline, leucine, glutamic acid, and histidine. Upon secretion the amelogenins form aggregates (Fig 3-6). The hydrophilic carboxy terminals of the amelogenins are exposed at the surface of the aggregates, facing the water-mineral phase. The external anionic surface, containing phosphorylated serine, is believed to play a role in controlling crystal growth.^50,⁵¹ Following the cleavage of the hydrophilic terminals, the amelogenins self-assemble into supramolecular nanospheres approximately 18 nm in diameter (see Fig 3-6).^49–⁵⁴ Each nanosphere comprises 100 to 200 amelogenin molecules stabilized by intermolecular hydrophobic interactions. High-resolution electron microscopy of newly secreted enamel matrix reveals nanospheres aligned between long, ribbonlike crystals of newly formed enamel. Presumably, adjacent crystallites are prevented from lateral fusion by the intervening amelogenin nanospheres, yet are able to grow rapidly along their C-axis.^49,^53,⁵⁵ It is postulated that a scaffold of amelogenin nanospheres controls the orientation of the C-axis (long axis) of the developing hydroxyapatite crystals (see Figs 3-4 and 3-6).^49,^53,^54,⁵⁶

Amelogenins are secreted as 25-kDa molecules that undergo progressive breakdown in the extracellular space. Proteases secreted by the enamel organ carry out specific and sequential proteolytic processing of the amelogenins.^57–⁶² The heavier amelogenins aggregate to form nanospheres that provide a structural scaffold to support the rapid and lengthwise growth of the crystallites (see Fig 3-6).^63–⁶⁶ The smaller (20- and 13-kDa) fragments may slow crystallite growth in width and thickness by controlling the ionic activity of calcium in the enamel fluid.^64,^66,⁶⁷

Fig 3-6 Current concept of the role of amelogenins in the mineralization of enamel. The hydrophobic amelogenins form globular aggregates (nanospheres) on secretion into the extracellular space. The nanospheres form lattices that regulate the spacing and the orientation of the C-axis of the newly forming enamel crystallites. (Adapted from Fincham et al⁵¹ with permission from Elsevier Science.)

As the amelogenins complete their function, they are resorbed from the enamel matrix. Proteolytic enzymes from two classes of proteases, the serine proteases and the matrix metalloproteinases, appear responsible for degradation of enamel matrix (for review, see Woessner⁶⁰). Degradation of amelogenin is accomplished by specific proteinases produced by secretory and maturation ameloblasts. Cleavage of the hydrophilic carboxy terminal peptide by a serine protease initiates the degradation process.⁶⁸ The rest of the amelogenin molecule is degraded by another serine proteinase (ameloprotease I), which appears to be a component of the enamelin fraction.⁶⁹

Recent studies suggest that metalloproteinases are involved in matrix degradation during the secretory-to-transition phase and that serine proteinases function mainly during the maturation phase.⁴³ In situ hybridization and immunohistochemistry indicate that MMP-20 (enamelysin) is expressed in secretory ameloblasts and odontoblasts.⁷⁰ Secretory ameloblasts may, to a limited extent, remove amelogenin peptides by endocytosis along the dorsal surfaces of Tomes process. However, the bulk of the amelogenin breakdown products are removed by maturation ameloblasts following the completion of the full thickness of the enamel layer.

Nonamelogenins

The nonamelogenin protein fraction contains relatively large (28- to 130-kDa) proteins of a generally acidic and hydrophilic nature. Several specific gene products have now been identified in the nonamelogenin fraction. These include tuftelin, ameloblastin, enamelin, and proteinases. Proteins of the nonamelogenin fraction demonstrate high binding affinity for hydroxyapatite crystals.⁷¹ These proteins are retained in small quantities in fully maturated enamel in the prism sheaths and as thin coatings surrounding the crystallites.

Tuftelin is a specific nonamelogenin acidic protein found in high concentration near the dentinoenamel junction and within enamel tufts.^21,⁷² Enamel tufts are hypomineralized developmental defects that extend perpendicularly from the dentinoenamel junction into the enamel. Tuftelin is the first enamel protein to be expressed during IEE differentiation. Tuftelin is a glycosylated protein with serine and threonine phosphorylation sites. Because of its composition, its early secretion, and its concentration at the mineralization front, tuftelin could have a role in nucleation of enamel crystallites.⁷³ The human tuftelin gene has been localized on chromosome 1.⁷⁴

Ameloblastin, amelin, and sheathlin form a group of related “sheath” proteins that have been detected in rat, human, and porcine enamel.^16,^40,^75–⁷⁸ Ameloblastin and sheathlin proteins accumulate between the plasma membrane of Tomes process and the growth zone of enamel prisms.^16,⁷⁹ It has been suggested that sheath proteins may serve an early adhesive function in stabilizing the nonsecretory surface of TP to the enamel matrix.^77,⁸⁰

Soon after their secretion, the parent molecules undergo cleavage. The C-terminal polypeptide fragments are rapidly degraded and removed from the enamel. However, the N-terminal ameloblastin and sheathlin polypeptides are retained in prism sheaths.^79,⁸⁰ Ameloblastin degradation products are less soluble than the parent molecule.⁴³ Nonamelogenin protein fragments are believed to account for most of the remaining small percentage of protein contained in fully mature enamel.

Of additional interest is the report that amelin mRNA has been localized in preodontoblasts before it is expressed in ameloblasts.⁷⁸ It has been suggested that amelin may have a role in the presecretory epithelial-mesenchymal interaction.

Enamelin is a high–molecular weight, acidic, glycosylated protein. It is secreted as a 186-kDa entity that subsequently undergoes progressive degradation to a 32-kDa molecule.⁸¹ The later fragment binds readily to enamel crystallites.⁸² The higher molecular weight fractions are localized along Tomes process and the newly developing enamel prism. The smaller fractions are located more deeply in the enamel, in association with the mineral in the prismatic and interprismatic domains.^81,⁸³

Some acidic proteins of the enamelin fraction are now known to be precursors of a group of serine proteinases that degrade enamel matrix.^58,^68,^69,⁸⁴

Despite recent progress in the biochemical characterization of the enamel matrix, knowledge of the sequential biochemical and biophysical interactions between mineral ions and enamel matrix proteins is still very incomplete. The most recently postulated roles for amelogenin and nonamelogenin proteins in initiating and controlling the construction of enamel have been reviewed by Nanci et al,¹⁶ Robinson et al,⁴³ and Fincham et al.⁵¹

Location and Expression of Amelogenin, Ameloblastin, and Tuftelin Genes

The gene for amelogenin (AMEL) has been mapped to the sex chromosomes (Fig 3-7). In the rat, hamster, and mouse, Amel is present on the X chromosome⁸⁵; in humans, AMEL is present on both the X and Y chromosomes.^86,⁸⁷ The gene on the Y chromosome (AMELY) is located in the q11 region, and the AMELX gene is located on the distal short arm (p22.1 to p22.3 positions) of the X chromosome. Recombination errors during the duplication of the sex chromosomes can lead to amelogenesis imperfecta.

Fig 3-7 Structure of the X-chromosomal copy of the human amelogenin gene. The bar segments represent the introns and the boxes (1 through 7) correspond to the exons. The nucleotide numbers are indicated below the exons. (Adapted from Simmer et al.¹⁷³)

The human amelogenin gene has seven introns and seven exons (see Fig 3-7). Both the X and Y amelogenin gene copies are expressed during tooth development. Transcription of the AMELX message appears several times more active than that of the Y copy, and the level of X-chromosomal amelogenin mRNA has been measured to be several fold higher than that of Y-chromosomal amelogenin mRNA.

A variety of amelogenin proteins are produced by alternative splicing of pre-mRNA.^46–⁴⁸ Exons, and parts of exons, are deleted during alternative splicing. The resulting proteins all have a hydrophobic amino terminal, a large hydrophilic middle polypeptide, and a hydrophilic carboxy terminal. It is unclear if each amelogenin isoform performs a different function during enamel formation.

A small deletion in the AMELY gene permits it to be distinguished from its AMELX counterpart. This difference has proven useful in sex identification of human remains recovered from archeological sites⁸⁸ and in forensic science.⁸⁹

The human tuftelin gene is located on chromosome 1.^74,⁹⁰ The ameloblastin gene is localized to chromosome 4q21.⁹¹

Mineralization of the Enamel Matrix

At the onset of enamel formation, the first enamel crystallites are spatially separated from the smaller dentin crystallites. High-resolution electron microscopy of the dentinoenamel junction indicates that the earliest enamel crystallites form from the alignment of dotlike mineral nuclei, approximately 2 to 4 nm in diameter.⁹² Chainlike association of these nuclei, apparently controlled by the amelogenin organic matrix, gives rise to long, needle-shaped crystallites. The crystallites develop in small clusters within extracellular deposits of amelogenin matrix, having the appearance of stippled material in electron micrographs.

Biochemical and electron probe analysis of the earliest crystallites suggests that the first mineral phase to be formed is a two-dimensional octacalciumphosphate precursor that subsequently transforms into hydroxyapatite.^66,⁹³ The smallest hydroxyapatite crystal units (unit cells) are formed by the following reaction:

The hydrogen ions generated during crystal formation must be buffered to maintain a neutral pH to allow continued matrix mineralization.⁴⁴

Enamel crystal growth occurs in a compartment isolated between mineralized dentin and the zonula occludens junction of the ameloblastic layer. Elemental analysis indicates that the fluid in the mineralization compartment has a different composition than serum and extracellular fluid.^94,⁹⁵ The presence of a distal zonula occludens junction between ameloblasts and the histochemical demonstration of calcium ATPase activity in the plasma membrane of Tomes process suggest that ameloblasts might control the fluid milieu within which enamel is deposited.^96–⁹⁸

Calcium ATPase has also been demonstrated in the distal cytoplasm of maturation ameloblasts.⁹⁸ The recent localization of Ca²⁺ pump proteins in human secretory and early-stage maturation ameloblasts provides additional support for a functional plasma membrane calcium pump.⁹⁹ The highest concentration of calcium pump protein was localized in the distal ends of ameloblasts near the mineralized enamel.⁹⁹

It has been proposed that the intracellular transport of calcium could be carried out by several calcium-binding proteins localized in both secretory and maturation ameloblasts.^100–¹⁰² The recent identification of two low-affinity, high-capacity, calcium-binding proteins (calreticulin and endoplasmin) in the endoplasmic reticulum of secretory and maturation ameloblasts provides support for a new theory of calcium transcytosis involving the endoplasmic reticulum and inositol triphosphate–gated calcium channels.¹⁰³ The endoplasmic reticulum could serve as a high-volume conduit for calcium transport across ameloblasts without altering the normal cytosolic calcium concentration. This theory would also explain why a large amount of endoplasmic reticulum but low levels of secretory protein synthesis are found in maturation ameloblasts.^103,¹⁰⁴

In situ hybridization with complementary deoxyribonucleic acid (cDNA) probes for bone sialoprotein is strongly positive in secretory ameloblasts. The potential role of bone sialoprotein, a calcium-binding protein common to most mineralized tissues, in enamel mineralization remains to be determined.¹⁰⁵ It has been suggested that tuftelin and/or bone sialoprotein could trigger enamel crystal nucleation.^43,⁷³

Structure of Transition-Stage Ameloblasts

On completion of the full thickness of enamel, the secretory ameloblasts undergo cytoplasmic reorganization as they switch from a primarily protein secretory cell to that of an absorptive and transport cell. This process is characterized by extensive intracellular digestion of parts of the RER and other cytoplasmic organelles inside autophagosomes. During this stage, the ameloblasts contain high levels of acid phosphatase, indicative of increased lysosomal enzyme activity. The transition stage remodeling is so intensive that approximately 25% of the ameloblasts undergo programmed cell death.^106,¹⁰⁷

Surviving cells contain less RER, and their Golgi complexes contain many smooth vesicles and lysosomal-like structures. The development of a ruffled border, against the surface of the mineralized enamel, signifies the start of the rapid removal of water and protein from the enamel. At the completion of transition, the ameloblasts are shortened to half their previous height (Figs 3-8a and 3-8b). They are now referred to as maturation ameloblasts.

Formation of the Papillary Layer

During the final phase of secretion, and progressing through the transition stage, the OEE, SR, and SI are transformed into the papillary layer, an epithelium believed to be specialized for transport.¹¹ This conversion is preceded by a reduction in the size of the intercellular spaces of the SR and by a loss of glycosaminoglycans. The redifferentiated cells of the OEE, SR, and SI arrange themselves into numerous epithelial folds, or papillae, located between the ameloblasts and a well-developed capillary bed (see Figs 3-8a and 3-8b).

The former OEE, SR, and SI cells are no longer distinguishable as separate cell types, and are now referred to as papillary cells. Papillary cells contain numerous mitochondria, large numbers of pinocytotic vesicles, and extensive gap junctions.^108–¹¹¹ Numerous microvilli increase the papillary cell surface area several fold.

The cytoplasmic features of the papillary cells, along with their association with a rich bed of fenestrated capillaries, suggest that at this stage the enamel organ has become specialized to perform transport functions related to enamel maturation.¹¹² The fact that papillary cells form extensive gap junctions with adjacent maturation ameloblasts leads to the conclusion that these two types of cells are acting in concert during maturation.¹¹

Figs 3-8a and 3-8b Papillary layer (PL) cells situated between the capillaries and the maturation ameloblasts (MA). (Hematoxylin-eosin stain. Original magnification × 600.) (a) Cross section depicting the MA through their long axis, and the alternating arrangement of papillae and indenting blood vessels. (E) Endothelial cells. (b) Tangential section through the maturation enamel organ, illustrating the close contact between papillary cells and capillaries filled with red blood cells (rbc).

Papillary cells have been shown to endocytose exogenous tracer material and to transport it to lysosomal bodies.¹¹¹ This has led to the speculation that the papillary layer participates directly in the removal and degradation of enamel matrix breakdown products that gain access to the intercellular spaces of the papillary layer. However, there is no direct evidence that enamel matrix degradation products diffuse into the intercellular spaces of the papillary layer.

An alternative hypothesis suggests that sodium-potassium-ATPase activity in papillary cells generates an intercellular osmotic gradient across the enamel, drawing water and small matrix polypeptides toward the maturation ameloblasts.¹¹³ The polypeptide matrix fragments would undergo endocytosis and additional degradation in secondary lysosomes of maturation ameloblasts (Figs 3-9 to 3-11).

Fig 3-9 Maturation ameloblast phenotypes. Ruffle-ended and smooth-ended maturation ameloblasts cycle back and forth during the maturation phase. Cycling (C) of the two phenotypes involves extensive remodeling of the distal cytoplasm and junctional complexes at both ends of the cells. The Golgi complexes (GA) and the lysosomal (Ly) apparatus are well developed in both cell configurations. Zonula adherens (ZA) and zonula occludens (ZO) shift from a distal position in the ruffle-ended ameloblasts to a proximal position in the smooth-ended ameloblasts. Mitochondria (M) are located primarily in the distal cytoplasm. Endosomes (E) containing enamel matrix (EM) are present in highest amount in the ruffle-ended ameloblasts.

Fig 3-10a Distal part of a ruffle-ended maturation ameloblast. (Em) Enamel matrix; (Ncl) nucleolus; (Nuc) nucleus. (Original magnification × 9,000.)

Fig 3-10b Golgi complex containing numerous Golgi cisternae (Gc) in a maturation ameloblast. (Original magnification × 16,000.)

Fig 3-10c Ruffled border (RB) and endocytosis vesicles (Ev) at higher magnification. (Original magnification × 20,000.)

Fig 3-11 Proposed pathway of enamel protein (EMP) reabsorption and digestion by ruffle-ended ameloblasts. The intercellular space (ICS) is sealed by a zonula occludens (ZO). Enamel proteins are endocytosed from the labyrinthine spaces of the ruffled border into endosomes (E) that fuse with larger secondary lysosomes (SL). Lysosomal enzymes are transported to the SLs via primary lysosomal vesicles (L) originating in the area of the Golgi apparatus. H⁺-adenosine triphosphatase, expressed at high levels, is responsible for secretion of H⁺ into the enamel. High levels of alkaline phosphatase are correlated to calcium transport at the ruffled border. (CV) Coated vesicle.

Structure of Maturation-Stage Ameloblasts

During the maturation stage, water and enamel matrix degradation products are removed from the enamel, and mineralization continues until the final enamel achieves a composition (by weight) of 95% mineral and only 4% water and 1% organic matrix.^26,¹¹⁴ Biochemical analysis of enamel indicates that there is a rapid loss of matrix during the initial phase of maturation. Prior to this stage, the enamel is soft and porous, and the crystallites have yet to grow to their final thickness.¹¹⁵ During the final stages of the maturation process, water is lost as mineral continues to be added to the growing crystallites. Ever-smaller quantities of matrix proteins are released and removed by the maturation ameloblasts until the enamel reaches its mature state prior to eruption.

Maturation ameloblasts (and perhaps the secretory ameloblasts) contribute proteolytic enzymes that are involved in an extracellular enzymatic cleavage of matrix proteins into small peptides prior to removal by endocytosis.^84,^116,¹¹⁷ One such enzyme is enamelysin, a matrix metalloproteinase (MMP-20) that degrades amelogenin.⁵⁹ A serine proteinase (ameloprotease) capable of degrading the entire amelogenin molecule has been isolated from pig enamel matrix.^69,¹¹⁸ Membrane-type matrix metalloproteinase (MT-MMP) is also expressed in ameloblasts.¹¹⁹ It has been suggested that MT-MMP might function as an activator of extracellular MMPs close to the cell surface during enamel maturation.

Enamel maturation is more time consuming than the preceding secretory stage. Maturation ameloblasts remain in contact with the enamel surface for approximately two thirds more time than do the secretory ameloblasts. Failure of enamel maturation leads to the eruption of enamel that is relatively soft, porous, and easily discolored by food and/or blood and serum.

On completion of the transition phase, maturation ameloblasts develop a ruffled border, a zone of cytoplasmic folds and invaginations along the distal end of the cell in contact with the enamel (see Figs 3-9 and 3-10).^120–¹²² Freeze-fracture studies of maturation ameloblasts have revealed a high concentration of intramembrane particles, indicative of possible transport and/or receptor-ligand activity at the distal surface.¹²³ Maturation ameloblasts cycle between distal ruffle-ended and smooth-ended morphotypes (see Fig 3-9).^124–¹²⁶

Maturation ameloblasts have well-developed Golgi complexes that contain many lysosomal vesicles. Morphologic, tracer, and autoradiographic evidence suggests that resorption of the enamel matrix occurs from the zone of the ruffled border.^29,^127,¹²⁸ Unlike secretory ameloblasts, the maturation ameloblasts produce a basal lamina over the surface of the maturing enamel.¹²⁹ Matrix degradation fragments must traverse the basal lamina prior to undergoing endocytosis at the ruffled border. Endocytosis of granular material within vesicles formed in the invaginations of the distal cytoplasm has been observed in all species that have been studied at the electron microscopic level. Additional high-resolution immunocytochemical studies have shown that endocytotic vesicles and secondary lysosomes contain material that reacts with antibodies raised against amelogenin proteins.¹²⁸

It is not known if all amelogenin peptide fragments are removed via the cytoplasmic route or whether some of the small peptides simply diffuse out of the enamel and the enamel organ without passing through the maturation ameloblasts.¹³⁰ The presence of distal zonula occludens junctions between adjacent ruffle-ended maturation ameloblasts suggests that a direct intercellular diffusion pathway is blocked to the free flow of substances from the enamel, at least beneath the ruffled border (see Fig 3-9).^111,¹³¹ The absence of a proximal zonula occludens between ruffle-ended maturation ameloblasts and the absence of a distal zonula occludens between the smooth-ended maturation ameloblasts, however, permit diffusion of peptides from enamel into the intercellular spaces between the smooth-ended maturation ameloblast (see Fig 3-9). From there, degradation products could gain access to the intercellular spaces of the papillary layer by lateral movement through the spaces between the ruffle-ended maturation ameloblasts (see Fig 3-9). This indirect pathway between maturation-phase enamel and the papillary layer and its blood vessels has been demonstrated by the diffusion of tracers.²⁶

A similarity between the activity of ruffle-ended maturation ameloblasts and osteoclastic resorption of bone matrix has been noted. Mannose-6-phosphate receptors for lysosomal enzymes are present on the ruffle-ended membranes of both cell types, suggesting that the ruffled border of the ameloblast is a target for outward transport of lysosomal enzymes.¹³² Positive immunocytochemical reactivity for cathepsin B in the distal ends of ruffle-ended ameloblasts confirms lysosomal enzyme transport to that location.⁸⁹

In addition to resorption of matrix, the ruffle-ended ameloblasts engage in the transport of calcium into the maturing enamel (see Fig 3-11).¹³³ With the onset of maturation, there is a relatively sharp drop in matrix protein followed by an increase in the rate of mineral incorporation into the enamel. Peaks of mineral acquisition are associated with the presence of the ruffle-ended ameloblasts.^132,^134,¹³⁵ High levels of calcium-ATPase activity in the ruffle-ended membranes of ruffle-ended ameloblasts appear related to the transport of calcium.⁹⁸

Alkaline phosphatase activity is high in the ruffled border of maturation ameloblasts.¹³⁶ It has been suggested that alkaline phosphatase may generate PO₄, required during formation of hydroxyapatite. Smooth-ended ameloblasts, in contrast to ruffle-ended ameloblasts, occupy less surface area on the tooth surface, exhibit less intense alkaline phosphatase and calcium-ATPase activity, and are not correlated to areas of calcium incorporation.

Histochemical and immunocytochemical studies have also shown that the ruffle-ended maturation ameloblasts contain proton pumps (H⁺-ATPase) and carbonic anhydrase.^137,¹³⁸ It has been proposed that protons generated by carbonic anhydrase activity are transported into the enamel across the membranes of the ruffled border by H⁺-ATPase (see Fig 3-11). The resulting decrease in pH beneath the ruffle-ended ameloblasts might activate proteolytic enzymes required for the degradation of matrix proteins. The large concentration of mitochondria adjacent to the ruffled border could supply the adenosine triphosphate (ATP) for the energy needs of proton transport.

Paradoxically, the role of carbonic anhydrase may also be to generate bicarbonate needed to scavenge hydrogen ions generated during hydroxyapatite formation. Bicarbonate ions could also be supplied from plasma circulating through the fenestrated capillaries. Carbonic anhydrase is also found in early enamel matrix.¹³⁹ Its potential role in mineralization has yet to be clarified.

Maturation ameloblasts do not remain in the ruffle-ended configuration for the duration of the maturation process. The ruffled border is transformed into a smooth distal surface abutting the enamel. This change is accompanied by the loss of the distal zonula occludens (see Fig 3-9). Maturation ameloblasts with a flat distal cytoplasmic configuration are called smooth-ended ameloblasts. Many lysosomal vesicles and a high acid-phosphatase activity characterize the smooth-ended ameloblasts. The precise role of the smooth-ended ameloblasts in enamel maturation is unknown. They appear to participate in protein degradation.

Entire clones or cohorts of maturation ameloblast undergo cyclic change from the ruffle-ended to the smooth-ended phenotype during maturation (see Fig 3-9).¹²⁵ In the continuously developing incisor of the rat, a total of 45 modulation cycles between the ruffle-ended ameloblast and smooth-ended ameloblast modes have been measured during the maturation of enamel; a mean of 2.8 modulations occurred each day of the 16-day maturation phase.¹²⁶ In teeth that develop more slowly, as in porcine incisors, the modulations occur less rapidly and there are fewer cycles. It remains clear, however, that ruffle-ended ameloblast and smooth-ended ameloblast cycling occurs during the development of all teeth, including those of primates.

Following tooth eruption, interaction of the enamel surface with ions in the oral fluids leads to a small but significant increase in enamel maturation. This posteruptive maturation, especially if fluoride ion is present in the oral fluids, leads to additional improvement in the surface resistance of enamel to subsequent acid demineralization.

Structure of Postmaturation-Stage Ameloblasts

On completion of maturation, the maturation ameloblasts and the cells of the papillary layer undergo regression, reducing the quantity of their cytoplasmic organelles and their overall size. The postmaturation ameloblasts appear as low columnar cells, and the senescent papillary layer is reduced to one or two layers of low cuboidal cells. The reduced enamel epithelium remains in position, covering and protecting the enamel surface until the erupting tooth makes contact with the oral mucosa. At that stage, the reduced enamel epithelium fuses with the oral epithelium to form the primary junctional epithelium attachment to the cervical aspect of the crown.

Basic Science Correlations

Cytoplasmic organelles undergo rapid change during the many phases in the life cycle of the ameloblasts. These changes reflect the principal cellular functions that occur at each stage of amelogenesis. For example, the RER is most highly developed in secretory ameloblasts, lysosomes appear most abundant during the transition stage, and endocytotic coated vesicles are unusually prominent in papillary cells during maturation of enamel.

Significant changes in cell-to-cell contacts also occur throughout all phases of enamel formation. They appear to be needed for the coordination of cellular activity and for controlling the compartmentation of the extracellular space. These requirements are met by the formation of gap junctions and zonula occludens junctions. The following sections provide brief reviews of the structure and function of these two junctions.

Gap junctions

Gap junctions provide hydrophilic passageways across adjacent cell membranes for the intercellular exchange of ions and small molecules (less than 1,000 Da).^140,¹⁴¹ Special transmembrane gap junction proteins, called connexins (Fig 3-12), create the channel through the membrane. The connexin molecule has four transmembrane domains, two rather rigid extracellular domains, and two cytoplasmic domains.¹⁴² The carboxy terminal domain, larger than the amino terminal domain, contains amino acid sequences that regulate channel permeability.

Fig 3-12 Gap junction connexin protein from mammalian liver cells. The amino and carboxy terminals are located on the cytoplasmic surface. Two polypeptide loops of protein extend across the membrane to the external surface of the connexon.

Six connexin molecules aggregate in the membrane to form a supramolecular hemichannel, the connexon (Fig 3-13).¹⁴³ In forming a connexon hemichannel, the gap junction proteins assemble with their hydrophobic surfaces facing the lipid phase of the plasma membrane and their hydrophilic surfaces oriented inward (toward each other) to delimit a central fluid-filled channel across the membrane. When connexons from two adjacent cells are connected across the narrow intercellular gap, and the connexons are open, an intercytoplasmic exchange of ions and small molecules may occur. Typical gap junctions are made up of a hundred or more connexons aggregated in complementary patches in the cell membranes of a pair of participating cells (Figs 3-13 and 3-14).

Fig 3-13 Gap junction in the (A) coupled and (B) uncoupled states, showing the association of connexons in two juxtaposed plasma membranes. In the open condition (A), ions and small molecules can move through a fluid-filled pore (green arrows) from cell to cell. When the gap junction is uncoupled (B), the connexons are constricted and the pore is closed (red arrows). (Adapted from Peracchia¹⁴³ with permission from Kluwer Academic.)

Fig 3-14 Gap junction particles (arrows) are aggregated on the protoplasmic face (Pf) of the fractured plasma membrane. Pits (arrowhead) on the external face (Ef) of the membrane represent the position of the pore of the connexon particle. (Original magnification × 92,000.)

The flow of ions and small metabolites across gap junctions has been shown to be involved in coordinating and regulating cellular activity in groups of contiguous cells. For example, second messengers, such as cyclic adenosine monophosphate, calcium ions, and inositol triphosphate, have been shown to spread through gap junctions.¹⁴⁴ In cardiac muscle, gap junctions, functioning as electrotonic synapses, coordinate the contraction of the heart. Gap junctions are needed during embryonic development to coordinate sequential differentiation of groups of cells.

Gap junction proteins are to some degree tissue specific. Lens, heart, and liver gap junction proteins have different molecular weights, suggesting that their respective connexons have tissue-specific physiologic functions in addition to common properties. The family of genes that encode connexins is made up of at least 12 members.^141,¹⁴⁵ Homotypic and heterotypic assembly of connexin proteins result in gap junctions with different physiologic properties.¹⁴⁶ In addition, it is possible for a single cell type to form different types of connexons and to restrict each type to specific domains of the cell membrane.¹⁴⁷

Participating cells are coupled when adjacent gap junction connexons are open. Various substances regulate the size of the pore opening and thereby control the degree of cell-to-cell coupling (reviewed by Bruzzone et al¹⁴⁸). Cytosolic calcium, cellular pH, retinoic acid, and intracellular oxygen tension have been shown to influence coupling. Connexons close within minutes in response to increased intracellular calcium, acidification of the cytoplasm, and low intracellular oxygen tension. This decoupling represents an emergency shutdown mechanism to prevent a cell-to-cell spread of noxious stimuli.

The calcium-binding protein, calmodulin, has been shown to participate in regulating the action of calcium on connexon proteins. Cyclic adenosine monophosphate modulates the number of gap junctions by increasing the rate of connexon assembly. Since gap junction proteins have a half-life of about 6 hours, there is a constant turnover of connexons at the cell surface.

Gap junctions are present between all cells of the enamel organ, suggesting that intercellular communication is necessary during all phases of enamel development.^11,^12,^96,^149,¹⁵⁰ Immunocytochemical studies have shown that connexin 43 localizes in the SI, IEE, and preameloblasts.¹⁵¹ Information transferred across gap junctions may control cell proliferation and coordinate the activation and subsequent regulation of protein matrix secretion.

Large gap junctions are formed during enamel maturation. This may indicate that a bidirectional flow of ions from ameloblasts to papillary cells is a significant component of cellular activity during enamel maturation. Annular gap junctions are especially conspicuous in papillary cells.¹⁵⁰ The latter are believed to represent stages in the internalization and breakdown of gap junctions.

Gap junction proteins have a rapid turnover time of approximately 5 hours. The functional significance of an apparent high turnover of gap junctions during the maturation phase remains to be explored.

Tight junctional complexes

Epithelial cells that are closely juxtaposed may participate in forming zones of fusion (tight junctions) between adjacent plasma membranes. For tight junctions to form, specific proteins must migrate from cytoplasmic pools to the cell surface to be inserted into the plasma membrane at points of cell-to-cell contact. Tight junctional contacts occur either as spotlike macula occludens, larger sheetlike fascia occludens, or as beltlike zonula occludens specializations.

The exact significance of the macula and fascia occludens junctions is unclear. Although these junctions cannot compartmentalize an extracellular space, they might provide increased cell-to-cell adhesion, or they might act as intramembrane stabilizers to restrict the lateral diffusion of other integral membrane proteins. In contrast, because the zonula occludens seals the extracellular space in a beltlike zone around the entire circumference of the cell, it compartmentalizes the extracellular space.

The zonula occludens plays two important functions in the physiology of epithelial layers. It provides a variable permeability barrier in the intercellular space, thereby creating isolated compartments and delineating luminal spaces. Second, by preventing lateral diffusion of integral proteins in the plasma membrane, it maintains specific domains in the cell membrane, such as the basolateral and apical surfaces of polarized cells.¹⁵²

The transmembrane protein responsible for creating the seal is called occludin. It binds additional proteins, zonula occludens 1 and zonula occludens 2, on its cytoplasmic domain. The zonula occludens proteins are kinases that may have signaling functions involved in regulating the degree of paracellular permeability.¹⁵³

Structural analysis of the zonula occludens by electron microscopic observation of freeze-fracture replicas of the plasma membrane has shown that tight junctional proteins (occludin) assemble in linear strands or fibrils within the plasma membrane (Figs 3-15a and 3-15b). The plasma membranes of the adjacent cells are fused along the linear strands of occludin proteins. The zonula occludens contains multiple anastomosing tight junctional strands.

Figs 3-15a and 3-15b Freeze-fracture replica of the distal plasma membrane of a ruffle-ended maturation ameloblast (MAb). (a) Low magnification reveals the ruffled border (RB), components of the zonula occludens (ZO), and a gap junction (GJ). (Original magnification × 17,000.) (b) Higher magnification of the protoplasmic face (Pf). The tight junctional strands (S) of the zonula occludens are visible, as are depressions (D) created by the strands in the external face (Ef). (Original magnification × 80,000.)

Physiologic studies of epithelial permeability have shown that there is no clear correlation between the number and arrangement of tight junctional strands and the degree of intercellular occlusion. Some zonula occludens act as total barriers, while others (leaky tight junctions) permit the flow of ions and solutes through the paracellular space. Modulation of the contraction of the actomyosin ring (terminal web) associated with the zonula occludens and zonula adherens has been proposed as an explanation for differences in tight junctional permeability. Contraction of the actomyosin ring mediated by myosin light-chain kinase exerts tension on components of the zonula occludens, thereby altering the permeability of the paracellular space.¹⁵⁴

The presence of a zonula occludens at the distal end of the secretory ameloblast, just proximal to Tomes process, suggests that the space into which the enamel matrix is deposited is isolated from the intercellular spaces of the enamel organ. The enamel mineralization compartment is bounded below by mineralized dentin and above by the ameloblasts joined together by zonula occludens junctions. Analysis of the fluid contained in this compartment indicates that it has a different composition than the general extracellular fluid and serum.

In addition to creating an intercellular barrier, the zonula occludens of the secretory ameloblast may stabilize the secretory domain of Tomes process (analogous to the development of a luminal membrane compartment in other polarized secretory cells). The zonula occludens of the ruffle-ended ameloblast may have a similar role in maintaining the ruffled border and sealing the intercellular space of the enamel organ from the enamel compartment.

Microtubules and motor proteins in secretion

Microtubules (MTs) form a key component of the cytoskeleton in all cells. They provide a scaffold on which organelles, vesicles, and secretory granules are translocated by the action of motor proteins. In addition, MTs act as rigid struts involved in maintaining cell shape. During mitosis, MTs assemble to form the spindle apparatus required for chromosomal segregation.

Each MT is a hollow cylinder constructed of 13 protofilaments of tubulin. Tubulin protofilaments are assembled from heterodimers of α and β tubulin molecules (Fig 3-16).¹⁵⁵ The addition and removal of tubulin heterodimers takes place at opposite ends of an MT. The positive (+) end of an MT is the growing end, while the negative (–) end is the point of removal of tubulin. The removal of subunits at the negative end of an MT is slower than the rate of addition of new subunits at the positive end.

Fig 3-16 Microtubule assembly by parallel association of tubulin protofilaments. Each protofilament forms by binding heterodimers of α and β tubulin at the positive end of the microtubule. Heterodimers are added when they are in the guanosine triphosphate (GTP)–bound state.

Both α and β tubulins are guanosine triphosphate (GTP)-binding proteins. In the GTP-bound state, the β tubulin subunit has a high binding affinity, thereby favoring rapid addition of subunits at the growing end of the elongating protofilament.¹⁵⁶ Hydrolysis of GTP on the β tubulin subunits destabilizes the protofilament structure, causing rapid depolymerization of the MT (Fig 3-17). Microtubules continue to grow as long as the rate of addition of GTP tubulin is faster than the rate of GTP hydrolysis.

Fig 3-17 Hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). Hydrolysis of GTP on the β tubulin subunit destabilizes the protofilaments, leading to rapid depolymerization of microtubules.

Initiation of the polymerization of an MT requires the action of the microtubular organizing center (MTOC). The composition and mechanism of action of the MTOC is poorly understood. A third form of tubulin, γ tubulin, is found in MTOCs, where it performs a nucleating function. The most prominent MTOC is associated with the centrioles. Numerous MTOCs are located in the cytoplasm (the pericentriolar matrix) surrounding each pair of centrioles (Fig 3-18).

Fig 3-18 Microtubule organizing centers. Numerous microtubule organizing centers are located in the centrosomal matrix associated with the centrioles. Each microtubule organizing center nucleates the development of a microtubule and stabilizes the microtubule by capping the negative end.

The negative end of the growing MT is stabilized by components of the MTOC. This arrangement permits the polarized growth of MTs away from the MTOC and toward the peripheral cytoplasm (Fig 3-19). Microtubules radiate from the centrosome outward toward the plasma membrane, where the positive end of each MT is capped by special proteins.

Fig 3-19 Centrosome, consisting of a pair of centrioles and associated microtubule organizing centers. The centrosome regulates the polarization of the cellular microtubule network. The positive ends of the microtubules are located in the peripheral cytoplasm.

Microtubules are stabilized by interaction with capping proteins, microtubule-associated proteins, and by detyrosination (removal of tyrosine from the carboxy terminal of tubulin). Detyrosinated MTs constitute a small percentage of the total microtubular complement of the cell. They have a life span of about 2 hours. Most MTs are unstable. Unstable MTs are dynamic structures whose average life span is about 10 minutes.

Microtubules serve as conduits for the transport of organelles and vesicles.^157–¹⁵⁹ Transport requires the action of microtubule-associated motor proteins (motor MAPs) and ATP. The most widely studied motor MAPs are the kinesin and dynein families of motor proteins. Classic kinesin is composed of two heavy chains and two light chains.¹⁶⁰ The heavy chain contains a large N-terminal globular head group with binding sites for ATP and tubulin. The tail portions, stabilized in a helical conformation, contain binding sites for various integral membrane proteins that are contained in the limiting membranes of organelles, vesicles, and granules (Fig 3-20). Dynein is a multimeric complex of heavy, intermediate, and light chains.

Motor MAPs transform the chemical energy released by the hydrolysis of ATP to adenosine diphosphate into mechanical displacement of the motor protein and its cargo along the surface of the MT (see Fig 3-20). It is unclear whether the movement of the motor protein and its cargo is caused by a conformational change (rachet power stroke) in the motor protein or by some form of biased diffusion along the MT surface. In general, kinesin transports cargo from the centrosome toward the peripheral cytoplasm, while dynein transports cargo in the opposite direction. For example, dynein has been shown to be needed for early endosome to late endosome and lysosomal transport, while kinesin is necessary for the transport of secretory granules from the trans-Golgi network to the plasma membrane.

Fig 3-20 Association of kinesin motor protein to a microtubule and the limiting membrane of a cytoplasmic vesicle. Adenosine triphosphatase (ATP) activity in the globular head group of the motor microtubule-associate protein causes displacement of the vesicle toward the positive end of the microtubule.

Individual members of these two families of motor MAPs appear to be relegated to the movement of specific cytoplasmic organelles and inclusions, for example, secretory granules, mitochondria, and transport vesicles. Specificity is believed to be a function of the binding affinity of carboxy tail domains of the motor MAP to target (receptor) proteins in the membrane of the transported entity.

Microtubule-associated structural proteins (structural MAPs) help to stabilize MTs by forming bridges to other cytoplasmic proteins.^156,¹⁶¹ They are present in high numbers in axons and dendrites of nerve cells. Approximately 15% to 20% of the total proteins of the brain are made of tubulin and MAPs.

Because of the abundance of structural MAPs in the brain, the neuronal MAPs have been most highly studied. Neuronal MAPs stabilize and promote the alignment of MTs in parallel arrays in axons and dendrites. In nonneuronal cells of the body, MTs are stabilized and bundled by MAP4. Phosphorylation of serine and threonine residues on MAPs by various kinases leads to MAP inactivation and decreased ability to interact with tubulin. On the other hand, the action of various phosphatases can activate MAPs and increase the organization of MT systems.

Clinical Correlations

Enamel dysplasia

Alteration of the ionic and metabolic milieu of secretory and maturation ameloblasts leads to defective enamel (enamel dysplasia). Because enamel does not remodel, its defects are retained in the fully formed tooth. Interference with the secretion stage leads to a reduction in the amount and/or composition of the enamel matrix, a condition known as enamel hypoplasia. The resulting enamel is thinner than normal, but usually fully mineralized. If the interference affects maturation ameloblasts, the result will be hypomaturation and hypomineralization. The resulting enamel contains more protein than normal, and the hydroxyapatite crystallites fail to reach their normal size.

Early maturation appears to be a critical stage in the formation of sound enamel, because disturbances occurring during the period when transitional ameloblasts differentiate into maturation ameloblasts lead to prolonged periods of suboptimal mineralization.¹⁶² Differentiating and secretory ameloblasts appear to have a greater potential for recovering normal function following metabolic insult.

Enamel affected by hypomaturation is usually of full thickness but more porous and less mineralized. Because it is less translucent, hypomaturated enamel appears clinically as white spots, or it may appear stained because of the subsequent absorption of extraneous molecules derived from food and serum. Dysplastic enamel usually contains physical evidence that both matrix deposition and maturation have been altered. Such teeth may have horizontal rows of pits and grooves of discolored and white opaque enamel.

Enamel dysplasia can be caused by local, systemic, and hereditary factors:

1. Anoxia in premature birth

2. Congenital syphilis

3. Erythrobastosis fetalis

4. Exantematous infections

5. Fluorosis

6. Hypoparathyroidism

7. Hypothyroidism

8. Renal hypophosphatasia

9. Vitamin A deficiency

10. Vitamin D–resistant rickets

In locally acting etiologies, there is no regular pattern involving contralateral teeth and no consistent pattern with relation to timing. An example of a local factor is an inflammatory process in a carious primary tooth that affects the nearby dental germ of its permanent replacement.

In a patient with enamel defects, symmetry of the lesions usually indicates a systemic cause. Systemic agents act in a symmetric and contemporaneous manner to involve all teeth under development at the time of the insult. Based on the position, distribution, and nature of the lesions, the approximate time period over which a disease occurred can be determined. The chronology of enamel formation (Fig 3-21) reveals how a serious systemic disease, such as pneumonia or measles, affecting a 1-year-old child will cause enamel hypoplasia of the permanent incisors, canines, and first molars.¹⁶³ A similar disease occurring in a 3-year-old child will affect the maturation phase in the incisors and canines and the secretory phase of the premolars. Recurrent systemic diseases, or the medications used in their treatment, frequently produce a series of symmetric horizontal ridges and grooves across the enamel surface.

Fig 3-21 Period of amelogenesis in the permanent teeth of the human dentition. Each bar represents the duration of enamel formation from beginning to completion of maturation. (Adapted from Seltzer and Bender.¹⁶³)

Enamel defects caused by environmental factors are not uncommon. In a study of more than 1,500 schoolchildren in London, it was reported that 68% had enamel defects in the permanent dentition.¹⁶⁴ More than 10% had defects on 10 teeth or more.

Genetically acquired enamel defects are much rarer than the environmentally produced varieties. Hereditary enamel dysplasia, also known as amelogenesis imperfecta, occurs in several forms. The hypoplastic form, involving the secretion stage, leads to thin enamel. The teeth are smaller and lack contact points. Exposure of dentin and hypersensitivity are common sequelae. In the hypocalcified or hypomatured type, the enamel is soft, deeply stained, and easily chipped away from the dentin. In general, affected enamel shows an inverse relationship between mineral and protein contents.¹⁶⁵

Amelogenesis imperfecta may be inherited as an autosomal-dominant defect with variable penetrance or as a sex-linked dominant trait. It was recently shown that a mutation in the AMEX gene, deleting nine base pairs in exon 2, resulting in the loss of three amino acids and the exchange of one amino acid in the signal peptide of amelogenin, was sufficient to cause severe enamel hypoplasia (Fig 3-22).¹⁶⁶ In yet another family, a mutation on the AMEX gene, leading to the deletion of a much larger segment (5 kilobases) and the loss of entire exons, caused hypomineralization of enamel (Figs 3-22 and 3-23).¹⁶⁷

Fig 3-22 Base pair and amino acid sequences of normal and mutant signal peptide portions of the human AMEX gene and amelogenin protein. Mutation leading to the loss of a tripeptide (isoleucine, leucine, and phenylalanine) and the substitution of threonine for alanine cause severe hypoplasia. (Adapted from Lagerström-Fermér et al¹⁶⁷ with permission.)

Fig 3-23 Two mutations on the AMEX gene that cause amelogenesis imperfecta. (Adapted from Lagerström-Fermér et al¹⁶⁷ with permission.)

Enamel pits and fissures

During the development of multicusped teeth, pit and fissure defects are formed in the steep depressions separating adjacent cusps. These defects form when the enamel organ is compressed by the growth of enamel along the slope of the cusps, constricting the ameloblasts that are located in the deepest and most narrow regions of the depression (Fig 3-24). Degeneration of the ameloblasts results in the formation of a pit and/or fissure running from the surface of the crown to a level just above the dentin. A thin layer of enamel at the base of the defect is usually formed by the ameloblasts prior to their death. The space created by the degeneration of the cells of the enamel organ provides a niche that becomes colonized by bacteria as soon as the tooth erupts into the mouth.

Fig 3-24 Mineralized tooth sectioned in half to reveal caries in the enamel (chalky white) along the sides of a fissure and below it in the dentin (brownish red). (From Paterson et al.¹⁷² Reprinted with permission.)

Pits and fissures are the parts of the tooth most susceptible to caries attack. Acid production by bacteria demineralizes adjacent enamel and dentin, leading to the formation of an incipient carious lesion (see Fig 3-24). Because the amount of enamel at the floor of the defect is minimal, the caries process can invade the dentin within a short time after its initiation. Unless teeth are protected with fluoride or an occlusal sealant, there is a very high probability that they will develop clinically detectable pit and fissure caries within 2 years after eruption.

Infant malnutrition and dental disease

A growing body of evidence accumulated during the past two decades has established the importance of an adequate intake of protein during early childhood for optimal dental health. Protein malnutrition during the formative years leads to delay of tooth eruption and increased susceptibility to dental caries later in life.^168,¹⁶⁹ The combination, frequently found in underdeveloped countries, of protein malnutrition in infants and subsequent increased consumption of sucrose-containing foods by children and adolescents, leads to a widespread incidence of dental caries.

Although the specific underlying biochemical deficiencies in enamel and dentin matrix that result from insufficient consumption of dietary protein remain to be established, it is clear that ameloblasts and odontoblasts require high levels of energy as well as a wide variety of amino acids for protein synthesis. Thus they are detrimentally affected in periods of protein starvation. Furthermore, enamel matrix produced during embryonic development and early childhood cannot undergo subsequent remodeling. Therefore, any deficiency occurring during its development will lead to increased susceptibility to dental disease in later life.

Fluoride, dental caries, and fluorosis

Epidemiologic studies conducted nearly a half-century ago demonstrated an inverse relationship between the level of fluoride in local water supplies and caries experience. Children who grew up in communities with fluoride levels greater than 1.0 ppm in drinking water experienced significantly fewer dental caries than did children in neighboring towns with low fluoride concentrations (less than 0.5 ppm) (Fig 3-25).¹⁷⁰

Fig 3-25 Relationship between fluoride levels in drinking water (in parts-per-million [ppm]), the detrimental effects of high fluoride intake (fluorosis index), and dental health as measured by the number of diseased, missing, and filled teeth (DMF). (Adapted from Shaw et al.¹⁷⁰)

Fluoride enters hydroxyapatite mineral, where it substitutes for hydroxyl ions. Incorporation of fluoride into enamel and dentin occurs during tooth development. Additional fluoride is added even after enamel maturation, as fluoride is absorbed in surface enamel from tissue fluids prior to eruption and from saliva once the teeth have erupted into the oral cavity. Fluoridated hydroxyapatite is more resistant to acid demineralization than is nonsubstituted mineral. The addition of only small amounts of fluoride to the hydroxyapatite crystal greatly improves its stability by decreasing the mobility of hydroxyl ions within the crystal lattice.¹⁷¹

The addition of fluoride in drinking water supplies at the optimal level of 1.0 ppm has a great dental health benefit, reducing the incidence of dental caries by more than 50%. Furthermore, the reduction in dental caries minimizes other pathologic sequelae that result from early tooth loss.

High levels of fluoride consumed during the period of tooth development have detrimental effects on enamel formation (see Fig 3-25). Sustained consumption of fluoride at levels greater than 4.0 ppm causes dental fluorosis, a condition characterized by chalky white defects and areas of yellow-brown discoloration in the enamel. Chronic ingestion of fluoridated toothpaste or mouthrinse in areas with optimally fluoridated water can raise systemic fluoride concentrations to a level at which fluorosis may develop. Despite the enamel defects, the involved teeth are more resistant to caries. For the patient, enamel fluorosis is mainly an esthetic problem.

Analysis of fluorotic enamel indicates that it is more porous and less mineralized. There is also evidence of retention of some amelogenin protein, indicating a defect in maturation, possibly related to the inhibition of the proteolytic breakdown of enamel proteins. In addition, ultrastructural studies of ameloblasts indicate that fluoride can disrupt the secretory phase by obstructing the normal vesicular transport of secretory proteins.

References

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