12. Dental tissues. I

Chapter 12. Dental tissues. I

Enamel: structure, composition and development

Enamel structure142
Physical properties 142
Composition 142
Enamel prisms 143
Incremental lines 143
Surface enamel 144
Enamel–dentine junction 144
Enamel formation144
Presecretory stage 144
Secretory stage 145
Transition stage 145
Maturation stage 146
Post-maturation stage 147
Self-assessment: questions (Enamel structure) 148
Self-assessment: answers (Enamel structure) 151
Self-assessment: questions (Enamel formation) 155
Self-assessment: answers (Enamel formation) 157

Enamel structure

Overview

Enamel is the hardest of the mineralized tissues and covers the crown of the tooth. It differs significantly from other mineralized tissues in the nature of its organic content as a result of its epithelial origin. It is non-vital and non-sensitive, and cannot be repaired once lost. It has an underlying prismatic structure. Enamel may be lost through various processes such as dental caries, attrition, erosion and abrasion, and can be built up by numerous restorative procedures.
Learning objectives

You should:
• know the composition and structural features of enamel
• appreciate the nature of surface enamel and the changes that take place with age
• have a detailed knowledge of how structural features in enamel relate to clinical practice.

Physical properties

Enamel covers the crown of the tooth. It is thickest over cusps and incisal edges (about 2.5 mm) and thinnest at the cervical margin. Enamel is the hardest of biological tissues and is very resistant to wear. It has little tendency to deform, has a low tensile strength and is brittle. However, any tendency to fracture is avoided by the more flexible support of the underlying dentine. Surface enamel is harder, denser and less porous than subsurface enamel. Enamel is the least porous of the dental hard tissues, its porosity representing about 5% of the volume.

Composition

Concerning the composition of enamel, 96% by weight (and 88–90% by volume) is comprised of mineral in the form of crystallites of hydroxyapatite, Ca 10(PO 4) 6(OH) 2, but in a form that contains impurities. The remaining non-mineral component of enamel is comprised of about 3% water and 1% organic material.

Calcium hydroxyapatite

Calcium hydroxyapatite is present as large crystals approximately 70 nm in width and 25 nm thick, which may extend across the whole width of the tissue. They are regular hexagonal structures when viewed in cross-section and the core may be more soluble than the periphery. Solubility of enamel decreases as development proceeds. Each unit cell of the crystallite consists of a hydroxyl group surrounded by three calcium ions. These ions are surrounded by three phosphate ions. Six calcium ions in a hexagon enclose the phosphate ions.
Crystal formation is a slow process, usually involving several different intermediates, meaning that the structural arrangement and stoichiometry of ions in the initial formed solid is different to that in the final formed crystal. Numerous different forms of calcium phosphate mineral can be found in enamel. Octacalcium phosphate crystals are thought to be precursors of the final formed hydroxyapatite.

Ionic substitution

Ionic substitution regularly takes place within the enamel surface. The hydroxyapatite crystal is highly uniform, regular and organized; however, some substitutions and variations can occur. Other ions may replace the ‘normal’ ones. In this case, carbonate may substitute for a phosphate or hydroxyl (most occurs at the phosphate site) and has a destabilizing effect. This depends upon local pCO 2 concentration and occurs exclusively during development (2% at the surface and 5% towards the dentine–enamel junction); it is one reason for the much higher solubility product of enamel compared to pure hydroxyapatite. Magnesium may also replace calcium ions and this has a destabilizing effect on the hydroxyapatite crystal lattice, but is a very limited substitution.
Of clinical importance, fluoride may substitute for hydroxyl ions, making the lattice more stable and therefore increasing resistance to acidic dissolution. Fluoride levels are greater at the outer enamel surface but fall dramatically through the tissue towards the dentine; this is probably due to it being acquired during enamel maturation.

Water

Water accounts for about 3% by weight of enamel (approximately 5–10% by volume). Water is related to the porosity of tissue. Some lies between the crystals and surrounds the organic material; however, some may become trapped within crystal defects and the remainder can form a hydration layer coating the crystals.

Organic matrix

The organic matrix in mature enamel mainly comprises two unique groups of proteins. About 90% are grouped as non-amelogenins (such as enamel and tuftelin), with small traces or fragments of amelogenins. In developing enamel, these ratios are reversed. The organic matrix of enamel is considered in more detail on pages 146–147.

Enamel prisms

The basic structural unit of enamel is the enamel prism (rod), running from the enamel–dentine junction to the surface. In a cross-section of human enamel, the prisms may be seen to be keyhole-shaped and alternate, so that the tail of a prism lies between the heads of two prisms in the row below (i.e. pattern 3 enamel). The prisms are 5–6 μm in diameter. Adjacent prisms are delineated by the prism boundary, an optical feature produced by sudden changes in crystallite orientation at that site. No such sudden changes are present in the prism core.
Although in the outer third of enamel prisms run parallel to each other when viewed in a longitudinal section of the crown, in the inner two-thirds adjacent bands of enamel approximately 50 μm wide (and containing groups of about 10–20 prisms) show prisms running in different directions as they spiral outwards; some groups of prisms are cut more transversely, others more longitudinally. When viewed in polarized or reflected light, this produces the optical phenomenon known as Hunter–Schreger bands. This complex pattern of prisms may limit crack propagation.
The surface layer of enamel in newly erupted permanent teeth is non-prismatic. Here, the enamel crystallites are all aligned at right angles to the surface and parallel to each other. Non-prismatic enamel occurs as a result of the absence of Tomes processes from the ameloblasts in the final stages of enamel deposition.

Incremental lines

During development, enamel is formed rhythmically, periods of activity alternating with periods of rest. This is associated with slight changes in the enamel resulting in the appearance of incremental lines. There are two main types of incremental line: short period (cross-striations) and long period (enamel striae).

Cross-striations

The cross-striations are diurnal, being formed every 24 hours. Cross-striations appear as lines that cross enamel prisms at right angles to their long axes, being approximately 4 μm apart.

Enamel striae

Enamel striae represent approximately weekly incremental lines and are seen in longitudinal sections of the crown as prominent lines that run obliquely across the enamel prisms to the surface. In horizontal sections they form concentric rings. They represent the successive positions of the enamel-forming front and for this reason do not reach the surface in the initial layers of enamel deposited over the tip of cusps or incisal margins. The periodic nature of this feature may be assessed by counting the number of cross-striations between successive enamel striae, the average being 7 days (range 6–10). Enamel striae in the middle portion of enamel are about 25–35 μm apart. In cervical enamel, where enamel is formed more slowly and cross-striations may be only about 2 μm apart, the striae are closer together.

Perikymata grooves and ridges

Over the whole of the lateral enamel, enamel striae reach the surface in a series of fine grooves running circumferentially around the crown. These grooves are known as the perikymata grooves and are separated by ridges, the perikymata ridges. In deciduous teeth, enamel striae and perikymata are only ever clearly seen in the cervical enamel of deciduous second molars.

Neonatal line

Enamel striae are less pronounced or absent from enamel formed before birth. A particularly marked stria is formed at birth – this is the neonatal line and reflects the metabolic changes at birth. Prisms appear to change both direction and thickness at the time of this event.

Surface enamel

The surface of enamel is, perhaps, its most clinically significant region as it is here that dental caries is initiated, restorations are attached and bleaches and fluoride remineralization preparations applied. Compared with subsurface enamel, surface enamel is harder, less porous, less soluble and more radio-opaque. It is richer in some trace elements (especially fluoride) but contains less carbonate. The enamel surface presents a variable appearance, exhibiting features such as aprismatic enamel, perikymata, prism-end markings, cracks, pits and elevations.

Enamel–dentine junction

The boundary between enamel and dentine is known as the enamel–dentine junction. It is scalloped and this feature is particularly evident beneath cusps and incisal edges. The convexities of the scallops project from the enamel into the dentine. Features seen at the enamel–dentine junction include enamel spindles, enamel tufts and enamel lamellae.

Enamel spindles

These are narrow, club-shaped structures extending up to 25 μm into the enamel; they may represent odontoblast processes that, during the early stages of enamel development, insinuate themselves between the ameloblasts. Enamel spindles are most commonly seen beneath cusps and, due to their alignment, are best viewed in longitudinal sections of enamel.

Enamel tufts

These are more extensive than enamel spindles and are seen in the inner third of the enamel. Resembling tufts of grass, they appear to travel in the same direction as the prisms. The prism boundaries in the tufts are hypomineralized and contain more enamel protein. They recur at approximately 100 μm intervals along the enamel–dentine junction and, owing to their alignment, are best visualized in transverse sections of enamel.

Enamel lamellae

These are thin, sheet-like faults that run through the entire thickness of the enamel. Like the enamel tufts, they are hypomineralized and best visualized in transverse sections of enamel. Enamel lamellae may arise:
• developmentally, in which case they would be filled with enamel proteins
• after eruption as cracks produced during loading of enamel, in which case they would be filled with saliva and oral debris.

Enamel formation

Overview

Being epithelial in origin, enamel formation differs in many respects from that associated with the other mineralized dental tissues. When initially formed, young enamel is only lightly mineralized (about 20–30%) and contains a high proportion of unique enamel proteins. However, it subsequently undergoes a process of maturation whereby its very high level of mineral content (96%) is attained and excess enamel proteins and water are removed. The more complicated pattern of development of enamel is reflected in the changing morphology of the ameloblast during development.
Learning objectives

You should:
• know the different stages that occur during enamel formation and be able to relate the changing structure of the ameloblast with its changing functions
• appreciate the composition of the organic matrix and how this changes during enamel formation
• understand how the structural features observed in the adult tissues are related to the development of the tissue
• be capable of comparing and contrasting enamel and dentine formation.
Enamel formation (amelogenesis) commences at the late bell stage of tooth formation, the earlier changes having been described in Chapter 10. During the early bell stage, the enamel organ comprises four distinct layers:
• External enamel epithelium
• Stellate reticulum
• Stratum intermedium
• Internal enamel epithelium.

Prior to the formation of dentine and enamel, the shape of the tooth has already been outlined following epithelial/mesenchymal interactions during tooth morphogenesis. The peripheral cells of the dental papilla adjacent to the internal enamel epithelium are undifferentiated, while the internal enamel epithelial cells have assumed a columnar appearance. The two groups of cells are separated by a basement membrane.

During the formation of enamel, the internal enamel epithelial cell undergoes a number of changes in its morphology, each being related to different functions. These can be considered for convenience in five stages: presecretory, secretory, transitional, maturation and post-maturation.

Presecretory stage

With the initial formation of dentine, there is breakdown of the basement membrane separating the pre-ameloblasts from the adjacent dental papilla, probably resulting from the release of enzymes from the pre-ameloblast, which may also phagocytose the breakdown products.

Secretory stage

The secretory stage is characterized by the synthesis and secretion of the enamel matrix and its initial light mineralization. At the ultrastructural level, there is an increase in endoplasmic reticulum as well as vesicles containing material representing the organic matrix of enamel. The contents of the vesicles (secretory granules) are discharged into the extracellular space at the distal end of the cell against the surface of the first-formed dentine. Almost as soon as the enamel matrix is released extracellularly, the initial calcium hydroxyapatite crystallites appear within it as thin, needle-like crystallites. The cells can now be termed ameloblasts. As the ameloblasts migrate outwards (centrifugally), small processes from the odontoblasts may get caught up between them. When the early enamel starts to mineralize around them, these processes will become entrapped as enamel spindles.

Prismatic structure of enamel

The first few microns of enamel at the site of the enamel–dentine junction are aprismatic (prismless/non-prismatic), as the distal end of the ameloblast is flat and the initial crystallites do not show sudden changes in orientation. As the ameloblasts continue to move away from the dentine surface, a cone-shaped process (Tomes process) soon forms at the distal, secretory end of the ameloblasts. As the forming enamel crystallites align at right angles to the surface of the ameloblasts, it is the Tomes process that is responsible for producing sudden changes in crystallite orientation and the resulting prismatic structure of enamel. When this is considered in three dimensions, it can be seen that four ameloblasts contribute to each enamel prism and that each ameloblast contributes to four prisms. Numerous cell contacts are present between adjacent ameloblasts. At the ends of the ameloblast, these form the terminal bar apparatus. Additional cell contacts are present between ameloblasts and cells of the stratum intermedium. The boundary of the prism is formed ahead of the central prism core, giving the forming enamel surface a ‘picket-fence’ arrangement. Increments of enamel are deposited on each other and enamel formation extends from the cusp-tips down the sides of the tooth. Just before the enamel reaches its final thickness, the Tomes process disappears and the distal surface of the ameloblast becomes flattened, so that the final 20–100 μm at the surface is prismless. The composition of ‘young’ immature enamel at this stage comprises up to about 30% organic matrix and about 20–30% mineral in the form of thin crystallites of hydroxyapatite, the remaining 40–50% comprising water.
The complex paths traced out by the ameloblasts as they move outwards are responsible for generating the Hunter–Schreger bands, with groups of prisms in adjacent layers of enamel moving in different directions. However, towards the surface they move in similar directions so that Hunter–Schreger bands are not evident in the outer third of the crown.
The secretory phase ends once the full thickness of enamel matrix has been laid down.

Incremental markings

Periodic changes in the nature or orientation of the enamel crystallites or enamel matrix or enamel prisms produce short-period or long-period incremental markings. A diurnal rhythm produces a daily cross-striation across each prism approximately 4 μm apart, while approximately every 7 days (range 6–10), an enamel stria (of Retzius) is produced outlining the mineralizing front and running obliquely to the surface. These striae end on the surface of the enamel as perikymata, except for the first-formed striae overlying the cusps of the tooth. In teeth mineralizing before birth, an exaggerated stria, the neonatal line, is present representing the enamel formed during the general disturbance in metabolism occurring over the few days following birth.

Transition stage

The young enamel that is deposited initially is high in water and protein content, low in mineral content, and porous. The process that converts it to fully mineralized enamel is termed maturation. Enamel maturation is carried out by the same ameloblast cells that secreted the primary matrix, but in a very changed form. The period during which the ameloblasts change from a secretory to a maturation form is the transition stage. During this phase, enamel secretion stops and some of the matrix is removed. A reduction in height of the ameloblasts signals the onset of the transition. The number of ameloblasts is reduced by as much as 50% by apoptosis (programmed cell death). In those ameloblasts that remain, the organelles associated with protein synthesis (e.g. the rough endoplasmic reticulum and the Golgi apparatus) are reduced by autophagocytosis. The amount of stellate reticulum is reduced so that blood vessels invaginating the external enamel epithelium come to lie close to the proximal end (base) of the ameloblasts.

Maturation stage

Once the entire thickness of the enamel has formed, it is structurally complete and possesses the morphological features seen in mature enamel. Newly formed enamel consists of about 65% water, 20% organic material and 15% calcium hydroxyapatite crystallites by weight. The stage during which enamel changes from its lightly calcified and organic-rich state into its final highly mineralized and organic poor state is termed the maturation stage. In addition to a quantitative loss of organic matrix from 30% to 1%, there is also a qualitative change; young enamel protein is comprised of approximately 90% amelogenins and 10% non-amelogenins, whereas in adult enamel 90% of the protein is comprised of non-amelogenins and 10% of amelogenin proteins. During maturation, enamel crystallites increase in width and thickness at the expense of water and organic matrix. Calcium and phosphate ions move through the ameloblasts and into the maturing enamel, while water and degraded enamel proteins pass in the opposite direction. During this process, the average thickness of the crystallites increases from about 1.5 nm to about 25 nm. The degradation of the enamel matrix, probably by serine proteases released from the enamel organ, seems to precede mineral gain. Indeed, at the initial stage of maturation, the space caused by matrix loss is occupied by water, with the enamel becoming more porous.
To reflect the change in function from the secretory stage to the maturation stage, the ameloblasts undergo morphological changes. Having a reduced height and a great reduction in the amount of organelles associated with protein synthesis, the distal end of the cell shows numerous infoldings, forming a striated border. The ameloblast in this form is described as ruffle-ended. This morphology alternates with that of the smooth-ended ameloblast, in which the striated border is absent. Modulation between the two forms appears to occur between five and seven times during maturation. This modulation may indicate alternation between resorptive phases, during which water and organic components are removed, and secretory phases, when mineral ions are added to the maturing enamel. Maturation takes a considerable period of time and, like enamel formation, proceeds from the tips of the cusps down towards the cervical margins (and fissures).
Not all areas within enamel will achieve full mineralization. Near the enamel–dentine junction, hypomineralized regions with a prismatic appearance are found as enamel tufts. Other faults give rise to enamel lamellae.

Enamel matrix

At this point, it is necessary to consider the composition and functions of the developing enamel matrix in more detail.

Amelogenin

Approximately 20% of young, developing enamel is almost all proteinaceous. The majority of the developing enamel organic matrix we amelogenins, secreted by the ameloblasts. This protein is rich in proline and glutamine, and is 178 amino acids in length with a hydrophobic core and protein–protein interaction domain. Within the enamel matrix, it self-assembles into spherical nanospheres that lie between the hydroxyapatite crystals, acting as spacers that allow the crystals to grow as enamel matures. It has a hydrophilic, mineral-binding domain, binding tightly to the enamel crystals through the C-terminal, which is cleaved shortly after secretion. This is the key to regulating crystal growth, as these nanospheres promote growth of the hydroxyapatite crystals, preventing premature crystal–crystal fusion. Amelogenin is degraded by proteolytic enzymes as enamel matures.

Non-amelogenins

The remaining proteins forming a small (10%) component of the organic matrix are grouped together as non-amelogenins. These include enamelin, tuftelin and ameloblastin:
• Enamelin is the largest enamel protein and is an acidic glycoprotein. This means that it has a high affinity for binding hydroxyapatite. It is rapidly processed after secretion and may interact with enamel crystals and be involved with nucleation, although evidence of this is limited.
• Tuft protein is highly anionic and can be found within the enamel tufts at the dentine–enamel junction.
• Ameloblastin is a tooth-specific protein expressed by cells of the inner enamel epithelium, in ameloblasts and transiently in odontoblasts during tooth development. Ameloblastin cleavage products lacking the C-terminus have been found to accumulate at the prism boundary throughout the enamel layer. Its localization suggests a possible role in regulating mineralization.

Proteinases

Proteinases are also expressed within the developing enamel matrix. Enamel matrix contains metalloproteinases during early enamel development and serine proteinases during the late stages of enamel formation. Their roles are involved with proteolytic processing of the enamel proteins and, as such, drive enamel maturation by degrading those proteins which inhibit mineral deposition. Such proteinases include enamelysin (MMP-20) and enamel matrix serine proteinase-1 (EMSP-1):
• Enamelysin is secreted primarily during the secretory and transition stages of enamel development. It is secreted from the secretory face of the Tomes process directly into the enamel matrix. The 45 kDa active MMP cleaves amelogenin at the majority of sites observed in vivo.
• EMSP-1 is a trypsin-like serine proteinase and is expressed in the pulp tissue as well as enamel. Activity of this enzyme is markedly increased during the transition stage of enamel development and remains at a high level throughout maturation. Its optimal activity is at pH 5.7, which is the pH present within the enamel matrix during maturation.

Nucleation

Nucleation is the mechanism whereby a hydroxyapatite crystal is seeded onto the organic matrix, allowing it to grow at the expense of calcium and phosphate surrounding it. In enamel, the process of heterogeneous nucleation, also known as epitactic nucleation (epitaxis), occurs. This is defined as the growth of one crystalline substance on a different solid surface having similar lattice spacings, the organic matrix.

Biomineralization of enamel

Biomineralization of enamel takes place in a tissue-specific micro-environment. The size, morphology and stability of the formed crystals are determined by the degree of supersaturation of calcium and phosphate in the fluid phase, and are influenced by the presence of a large number of regulators (matrix proteins). Calcium reaches the matrix through the enamel organ by intercellular and transcellular pathways. Active transport systems, using carrier proteins in cell membranes, may be involved, and calcium may also flow through concentration gradients from blood plasma to enamel matrix. First-formed enamel is poorly organized, with random crystal sizes and morphology. Initial crystals grow by fusion of nucleation sites but, once a prismatic structure takes shape, growth is by increased length, not width, and controlled by amelogenin nanospheres.
Nanospheres control growth by acting as spacers between the crystals, providing space for new crystal deposition and inhibiting crystal fusion. There is good correlation between the size of the nanospheres and spacing of the enamel crystallites, suggesting that the width of the nanospheres controls the final thickness of the enamel crystals. In the maturation phase, the matrix proteins have a reduced role to play, as most organic material has been degraded and lost. Matrix proteins are removed long before crystal growth ends. Such degraded matrix proteins may accumulate in the extracellular space around the ameloblast cells where they may inhibit cell activity and so control or limit the thickness of enamel deposition.

Post-maturation stage

Once maturation of the enamel is complete, the ameloblasts undergo further changes in morphology associated with changes in function, which can be considered as the post-maturation stage. The cells become flattened and a thin, amorphous layer of protein, the primary enamel cuticle, separates the cells from the surface enamel. This cuticle can be considered as a basal lamina and the distal, flattened end of the ameloblast are linked to it by hemidesmosomes. Together with the shrunken remnants of the enamel organ, the ameloblast layer forms the reduced enamel epithelium. During eruption, this reduced enamel epithelium protects the enamel surface from the possible addition of a surface layer of cementum as it erupts through the adjacent connective tissue. The primary enamel cuticle, together with the remnants of the enamel organ (reduced enamel epithelium), form Nasmyth’s membrane. Once the tooth has erupted into the oral cavity, the surface layer shows a further slight increase in mineralization through interaction with saliva.
On eruption, the reduced enamel epithelium undergoes yet another transformation as it is converted into the junctional epit/>

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Jan 5, 2015 | Posted by in General Dentistry | Comments Off on 12. Dental tissues. I
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