Chapter 13. Dental tissues. II
Dentine/pulp complex: structure, composition, development and oral pain
Structure and composition of dentine161
Dentine development (dentinogenesis)165
Organization and composition of the dental pulp 169
Blood supply 170
Lymphatic vessels 170
Nerve supply 170
Age-related changes 173
Self-assessment: questions (Structure and composition of dentine) 174
Self-assessment: answers (Structure and composition of dentine) 177
Self-assessment: questions (Dentine development) 181
Self-assessment: answers (Dentine development) 183
Self-assessment: questions (Dental pulp) 186
Self-assessment: answers (Dental pulp) 187
Structure and composition of dentine
Dentine is the mineralized connective tissue that forms the bulk of the tooth. It surrounds and protects the dental pulp. In the crown it is covered by enamel, in the root by cementum. Unlike enamel, dentine is sensitive and is formed throughout life, giving rise to secondary dentine. Though the odontoblasts that form the tissue have processes that lie in tubules within the dentine, the cell bodies lie at the periphery of the pulp, constituting a dentine/pulp complex. Being a living tissue, dentine can react to trauma by forming tertiary dentine.
• know the composition and main structural features of dentine and be able to contrast these with enamel
• appreciate the different zones in dentine and understand the changes that take place in its structure with age. As with other dental tissues, you should be able to relate structure to function
• know the basis of dentine sensitivity
• understand how dentine reacts to trauma and how it bonds to restorative materials, as such knowledge has clinical relevance.
Dentine is the mineralized connective tissue that forms the bulk of the tooth. In the crown it is covered by enamel, in the root by cementum. It surrounds and protects the dental pulp. Unlike enamel, dentine is formed throughout life and is sensitive so that it is able to react to stimuli.
Dentine is pale yellow in colour. As enamel is semitranslucent, it is the underlying dentine that imparts the slight yellowish colour on the crown. Dentine is softer than enamel but harder than cementum. The combination of its organic matrix and mineral composition gives it both strength and a degree of flexibility. Dentine is traversed by a system of very narrow tubules that render it permeable. However, this permeability is reduced by the development of peritubular dentine.
The chemical composition of dentine by weight is approximately 70% inorganic, 20% organic and 10% water. The inorganic component is in the form of (impure) calcium hydroxyapatite crystallites that give it its hardness. The crystallites are smaller than enamel, having a width of about 35 nm and a thickness of about 10 nm. The organic component of dentine consists of collagen fibrils embedded in an amorphous ground substance.
Collagen gives dentine its strength. The fibrils comprise over 90% of the organic matrix and are mainly type I collagen (with very small traces of type III and type V); they have a mean diameter of approximately-100 nm.
Concerning its structure and formation, type I collagen has:
• a triple helix of two α-1 and one α-2 chains
• intracellular formation of procollagen (triple helix formation/secretion)
• propeptides removed in processing of procollagen to tropocollagen
• fibril formation with alignment of tropocollagen molecules, guided by non-collagenous proteins, e.g. decorin
• pyridinoline cross-link formation between lysine and hydroxylysine residues; this increases as predentine is modified, allowing for mineralization of dentine.
The collagen fibril orientation, along with the presence of proteoglycans, can be said to be the scaffold on which mineral can be deposited, and collagen itself is a good nucleator of hydroxyapatite. Hydroxyapatite crystal deposition occurs at the gap zone within collagen fibrils of dentine.
Comprising only about 10% of the organic matrix, there are a large number of non-collagenous proteins in dentine. Although their functions are poorly understood, they are concerned with collagen formation and mineralization, and with odontoblast cell function and adhesion. The non-collagenous proteins include the proteoglycans decorin and biglycan, with the glycosaminoglycan side-chains of chondroitin-4-sulphate and chondroitin-6-sulphate. Also contained within this non-collagenous matrix are specific dentine phosphoproteins, acidic proteins, including Gla-protein, osteopontin, osteonectin, dentine and bone sialoproteins, and numerous growth factors such as TGF-β1, BMP-2, BMP-4, BMP-6, BMP-7, insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF).
Acidic proteins such as γ-carboxyglutamate-containing proteins (Gla-proteins) are found in dentine in low amounts. It is not known what their function is, but their acidic nature allows them to bind strongly to hydroxyapatite crystallites, and therefore they may play some role in directing or guiding mineral deposition as predentine matures through to dentine. The other acidic proteins, osteopontin and osteonectin, may also be important in directing mineralization, and these are classically associated with bone. Dentine matrix protein is another acidic phosphorylated protein found in dentine and bone, and may also be involved in regulating nucleation of mineral crystals.
The two dentine phosphoproteins, dentine sialoprotein (DSP) and dentine phosphoprotein (DPP) are post-transcriptionally cleaved from the same gene product from the dentine sialophosphoprotein (DSPP) gene. The two proteins may have similar, but slightly different roles:
• DPP is secreted at the mineralizing front and is not present in predentine. It is highly phosphorylated (85–90%) and highly acidic due to the high aspartic acid residues and very high phosphate content. DPP has a very high affinity for calcium and hydroxyapatite surfaces and may function in mineral deposition due to its acidic nature and high calcium ion-binding properties.
• DSP is also acidic, with a high carbohydrate content (sialic acid 10%), and is phosphorylated. It is present in odontoblasts, predentine and dentine, and is considered to be tooth-specific.
Dentine contains a cocktail of bioactive proteins called growth factors. Such growth factors are sequestered into the dentine during dentinogenesis and are thought to be held within the mineralized matrix, bound to proteoglycans or latency-associated peptides. Such factors include TGF-β1, BMP-2, 4 and 7, IGF and the angiogenic growth factor VEGF. Such factors have been shown to be important in regulating dentinogenesis and tooth development, and it is possible that release of these factors during trauma or disease (and possibly cavity lining) directs the tertiary dentinogenic response which the pulp-dentine complex exhibits.
The basic repeatable unit in dentine is the dentinal tubule. This runs through the dentine and is widest at the pulp surface, with a diameter of about 3 μm.
Intertubular and circumpulpal dentine
Initially, the tubule contains an odontoblast process surrounded by the mineralized intertubular dentine. The dentine tubules follow a curved, sigmoid course (primary curvature) that is most prominent at the sides of the crown. In the root and beneath the cusps, the tubules run a straighter course. Along the length of each primary curvature, minor undulations of the tubule constitute the secondary curvatures. Occasionally, adjacent secondary curvatures may coincide, giving rise to a feature called a contour line (of Owen). The dentinal tubules branch along their course, this being particularly evident at the enamel–dentine junction. In the root, just beneath the cementum, the terminal branching is exaggerated and the dentinal tubules also appear to loop. These features are thought by some to be responsible for the appearance of the granular layer (of Tomes) seen in ground sections. In the bulk of the dentine (circumpulpal dentine), the collagen fibrils of the mineralized matrix are aligned parallel to the enamel–dentine junction and transverse to the dentinal tubules.
Peritubular (intratubular) dentine
Soon after the dentinal tubules have been formed, another type of dentine is deposited on the walls of the tubule, narrowing the size of the lumen. This explains why the dentinal tubule is narrower (about 1 μm) in the outer part of the dentine. This type of dentine is known as peritubular (intratubular) dentine and may eventually lead to the complete obliteration of the tubule. Peritubular dentine differs from intertubular dentine in lacking a collagenous fibrous matrix and may be considered as representing mineralized ground substance. It is hypermineralized when compared with intertubular dentine, being 10–15% more mineralized; although containing hydroxyapatite crystals, other forms of calcium phosphate may be encountered. Peritubular dentine is present in unerupted teeth. In demineralized sections, the peritubular dentine is completely lost, as it lacks a residual collagenous framework, and the tubule is restored to its original dimensions.
Associated with physiological ageing, especially in root dentine, the dentinal tubules become completely occluded by peritubular dentine formation. The contents of the tubule acquire the same refractive index as the intertubular dentine. When a ground section of a root is placed in water (which has a refractive index different from that of dentine), regions blocked by peritubular dentine will appear translucent (‘translucent dentine’), while regions with patent tubules will fill with water and appear opaque. Translucent dentine forms initially near the root apex and extends both cervically and apically with age. There is a good correlation between the amount of translucent dentine and the age of the patient, and this feature is used in forensic odontology to help age teeth.
Contents of dentinal tubules
The contents of dentinal tubules may include the odontoblast process, afferent nerve terminals and processes from antigen-presenting cells. The tubule is also bathed in ‘dentinal’ fluid. Movement of this fluid is thought to be involved in dentine sensitivity.
The odontoblast process contains vesicles, microtubules and intermediate filaments. The main organelles associated with protein synthesis (e.g. rough endoplasmic reticulum Golgi material) are present in the odontoblast cell body but do not extend into the process. The extent of the odontoblast process inside the dentinal tubule has not been established with certainty due to considerable technical difficulties. Evidence suggests that, with age and with the formation of peritubular dentine, it is limited to the inner third of dentine. A thin, proteinaceous membrane termed the lamina limitans may be seen lining the wall of the dentinal tubule and its presence may give rise to the erroneous impression of an odontoblast process.
Sensory nerve terminals
Sensory nerve terminals may be seen adjacent to the odontoblast processes. They are limited mainly to the dentine of the crown beneath the cusps (where they may be found in 50–80% of the tubules) and project up to 200 μm from the pulp. Fewer tubules are innervated in the midcoronal dentinal regions and fewer than 5% of tubules are innervated in the cervical and root dentine. The axon is narrower than the odontoblast process and often contains mitochondria; there is no evidence of any specialized contacts between the nerve and the odontoblast process.
Regional variations in dentine structure and composition
The structure and composition of mineralized dentine varies in different parts of the tissue and one can distinguish the following zones: mantle dentine, interglobular dentine, granular layer, hyaline layer and predentine.
The outermost thin (about 20 μm) layer of dentine in the crown is termed mantle dentine. It differs from the bulk of the circumpulpal dentine in that its collagen fibrils are largely oriented perpendicular to the enamel–dentine junction rather than parallel to it, and is generally 5% less mineralized than the rest of the circumpulpal dentine. It contains no dentine phosphoprotein and the dentinal tubules show considerable branching in this region. During the initial mineralization of dentine, mantle dentine exhibits the presence of matrix vesicles. The mantle dentine lies adjacent to the three-dimensional, scalloped architecture of the enamel–dentine junction and some odontoblast processes initially extend into the enamel and give rise to enamel spindles.
Much of dentine is deposited as calcospherites, with the hydroxyapatite crystallites arranged radially. These normally fuse to form a uniformly calcified tissue. However, in some areas, usually beneath the mantle layer in the crown, the fusion may be incomplete, giving rise to uncalcified, interglobular dentine.
In ground sections, the outermost layer of root dentine is a clear hyaline layer, whose precise origin is in dispute. This narrow band (up to 20 μm wide) appears to be non-tubular and therefore structureless. The hyaline layer may serve to bond cementum to dentine and may be of considerable clinical significance when considering periodontal regeneration.
Granular layer (of Tomes)
Immediately beneath the hyaline layer when viewed in ground sections is a narrow, dark zone called the granular layer (of Tomes).
As it is formed throughout life, the inner surface of dentine adjacent to the pulp is lined by an unmineralized zone of dentine matrix called predentine. In demineralized sections, predentine stains differently to that of the matrix of the rest of the (mineralized) dentine matrix. This reflects a difference in the composition of its matrix. Predertine contains a type I collagen which provides an organic scaffold for eventual mineralization. Non-collagenous proteins (such as decorin and biglycan) direct matrix organization and prevent premature mineralization, and this matrix is modified by proteolytic enzymes prior to mineral deposition at the mineralization front. Such remodelling includes removal of inhibitors to mineral nucleation and the processing of proteins for new functions. The presence of matrix metalloproteinases indicates that proteolytic processing occurs as predentine is modified. The mineralizing front may show a globular or linear outline, reflecting two different mineralization processes. The width of the predentine can vary from 10 to 40 μm, depending on the rate at which dentine is being deposited.
Like enamel, dentine has regularly spaced incremental lines. There are two types: short-period and long-period markings. They have been attributed to:
• circadian fluctuations in acid–base balance that affect both the mineral content and the refractive index of forming hard tissues
• changes in collagen fibril orientation.
Short-period markings (von Ebner lines) may be seen as alternating dark and light bands, each pair reflecting the diurnal rhythm of dentine formation and lying approximately 3 μm apart. The coarser, long-period lines (Andresen lines) are approximately 20 μm apart. Between each long-period line there are 6–10 pairs of short-period lines. The cause of this periodicity is unknown. A similar periodicity exists in enamel, making it likely that a common mechanism exists. As with enamel, an exaggerated line, the neonatal line, can be seen in the dentine of teeth mineralizing at birth.
Age-related and posteruptive changes
Once the tooth is erupted and fully formed, dentine can undergo a number of changes that either are related to age or occur as a response to a stimulus applied to the tooth, such as caries or attrition.
Secondary dentine is the term given to the dentine that starts to form once the root is complete, about 3 years after the tooth erupts. Its structure is very similar to that of primary dentine and it may be difficult to distinguish between the two. However, primary and secondary dentine are often delineated as a result of a change in direction of the dentinal tubules with coincidence of secondary curvatures, producing a contour line (of Owen). The increased crowding of odontoblasts as secondary dentine formation continues throughout life, together with the slower rate of deposition, make secondary dentine a little less regular than primary dentine and the incremental markings somewhat closer together. The overall result is the smaller pulp chambers and narrower root canals in the teeth of older patients.
With more severe stimuli, such as attrition and dental caries, the dental pulp may be induced to produce a less regular form of dentine known as tertiary dentine. It may have a tubular structure or it may be relatively atubular.
Reactionary dentine refers to the tertiary dentine forming in response to an insult in which, although some damage has been sustained, the existing odontoblasts upregulate their synthetic and secretory functions and continue to form dentine.
Reparative dentine relates to the tertiary dentine forming after a stimulus in which the original odontoblasts in the associated region have been destroyed and new calcified tissue (reparative dentine) has been formed by newly differentiated cells (referred to as ‘odontoblast-like’ cells). This matrix is rapidly deposited and has a dysplastic, irregular structure. No DPP is present and high levels of bone-associated proteins can be identified. This reparative dentine has also been referred to as osteodentine.
In addition to infilling with peritubular dentine as a physiological response to ageing, dentinal tubules commonly fill in as a response to an external stimulus such as surrounding a slowly advancing carious lesion. This type of dentine is termed sclerotic dentine and, like translucent dentine, will present as areas of dentine that lack structure and appear transparent. Little is known about the precipitated material, but it appears to differ from peritubular dentine and may not be formed by the odontoblast.
If the primary odontoblasts are killed by an external stimulus, or retract before peritubular dentine occludes the tubules, empty tubules will be left. They may be sealed at their pulpal end by tertiary dentine. When ground sections are routinely prepared and mounted, the mounting medium may not enter these sealed-off tubules and they will remain air-filled. Viewed through a microscope, transmitted light will be totally internally reflected; these tubules will appear dark and are termed ‘dead tracts’.
One of the most important clinical aspects of the dentine/pulp complex relates to the intense pain that can be generated from this site when given appropriate stimuli. Stimuli applied to dentine can be transmitted across the dentine to fire nerves at the periphery of the dental pulp. Three main hypotheses have been put forward to account for dentine sensitivity:
• One view states that stimuli are transmitted directly via nerves in dentine. However, arguing against this view is the relative scarcity of nerves and their apparent absence in the outer parts of dentine. In addition, the application of local anaesthetics to the surface of dentine does not abolish the sensitivity.
• A second hypothesis states that stimuli are transmitted via odontoblast processes. However, there is no physiological evidence to date that indicates that the odontoblast process is analogous to a nerve fibre and can similarly conduct impulses pulpwards (i.e. it has a low membrane potential). Furthermore, like nerves in dentine, the process may not extend far into dentine; nor is the application of substances designed to prevent transmission of such impulses effective. In addition, odontoblasts have not been shown to be synaptically connected to nerve fibres.
• The third and most plausible hypothesis to explain the transmission of sensory stimuli suggests that all effective stimuli applied to dentine cause fluid movement through the dentinal tubules, and that this movement is sufficient to depolarize nerve endings in the inner parts of tubules, at the pulp–predentine junction and in the subodontoblastic neural plexus. Movement in either direction would mechanically distort the terminals. This is discussed further on pages 172–173.
Dentine development (dentinogenesis)
Essential epithelial/mesenchymal interactions occur in the developing tooth germ to enable dentine formation to commence. Dentine formation, like bone and cementum, is a two-phase process, commencing with the secretion of an extracellular connective tissue matrix and its subsequent mineralization. It continues throughout life. The tissue that is produced shows regional variations in structure and well-defined age changes.
• understand the process whereby dentine is formed and be able to compare and contrast it with the formation of enamel
• be able to relate the structures seen in a ground section of dentine with the development of the tissue
• be able to appreciate how age changes relate to the clinical situation.
Dentine formation begins when the tooth germ has reached the bell stage of development. In the enamel organ, the internal enamel epithelium starts to differentiate but, as yet, no enamel has been deposited. The dentine-forming cells, the odontoblasts, differentiate from the peripheral cells of the underlying dental papilla and are of neural crest origin.
Dentine formation begins, as with enamel, in the region of the cusp (or incisal margin) of the tooth and gradually extends down the slopes of the crown to the cervical margin. This represents coronal dentine. Within the dentinal tubules, peritubular dentine formation soon begins to narrow the size of the lumen. Following crown completion, root dentine then forms as the epithelial root sheath (of Hertwig) extends apically and odontoblasts differentiate from the adjacent dental papilla. Dentinogenesis continues and the thickness of dentine increases steadily until, at about the time root completion occurs, it slows down to give rise to secondary dentine.
Differentiation of odontoblasts
The differentiation of odontoblasts is the result of epithelial/mesenchymal interactions (see page 115), requiring the involvement of many signalling agents, transcription factors and growth factors (e.g. Ssh (sonic hedgehog), BMPs, fibroblast growth factors (FGFs) and TGFs). At the early bell stage of development, the cells of the dental papilla are relatively undifferentiated. As the internal enamel epithelial cells elongate to become pre-ameloblasts, the adjacent cells of the dental papilla start to differentiate. They elongate and the nucleus comes to lie in the basal part of the cell (that furthest from the internal enamel epithelium). The Golgi material becomes pronounced and the rough endoplasmic reticulum increases in size. Cell-to-cell junctions, particularly between odontoblasts but also linking odontoblasts to subodontoblastic cells, increase in number. The odontoblasts migrate pulpwards, each trailing a number of small cell processes, one of which predominates to form the odontoblast process of the cell.
From the distal end of the cell, the differentiating odontoblast secretes the initial organic matrix, the predentine of mantle dentine that surrounds the odontoblast process. This matrix is composed of small collagen fibrils (nearly all type I) embedded in its characteristic ground substance. The cell may now be referred to as an odontoblast. The collagen fibrils in mantle dentine are oriented perpendicular to the basal lamina (the site of the future enamel–dentine junction). The basal lamina soon breaks down. As the predentine thickens, it matures sufficiently to allow its matrix to mineralize.
Mineralization of mantle dentine
Mineralization of mantle dentine is thought to be initiated by matrix vesicles. These membrane-bound organelles (30–200 nm) are budded off from the odontoblast. They contain a variety of enzymes (including alkaline phosphatase) and other molecules that lead to the formation of the first mineral crystals of hydroxyapatite within the vesicles. The crystals then break out of the vesicles and subsequent mineralization of the remainder of the dentine occurs without the presence of matrix vesicles. Similar matrix vesicles have been implicated in the initial mineralization of bone and calcified cartilage.
Once the initial thin layer of mantle dentine has formed, the fully differentiated odontoblasts continue retreating pulpwards, trailing out an odontoblast process around which the odontoblast continues to secrete the predentine associated with circumpulpal dentine. Compared with that of mantle dentine, the collagen fibrils are now oriented parallel to the enamel–dentine junction. When the predentine reaches a thickness of about 10–20 μm, it attains a state of maturity that will allow it to mineralize. At the mineralizing front, degradation of some molecules in predentine may occur, while other molecules may be added (e.g. dentine phosphophoryn) after being transported along the odontoblast process, thereby bypassing the bulk of the predentine layer. As the biochemistry of predentine will differ when compared with that of the organic matrix associated with mineralized dentine, predentine will stain differently. The main path of the retreating odontoblasts will give rise to the outline of the primary curvatures, whilst minor undulations in the odontoblast process will give rise to the secondary curvatures.
The outline of the mineralizing front indicates that two distinct patterns of mineralization can occur (and in the absence of matrix vesicles): a linear or a spherical (calcospheritic) pattern. In calcospherites, the crystallites are arranged in a radial pattern and, despite complete mineralization of dentine, this pattern can still be discerned using polarized light. Failure of calcospherites to fuse may result in the appearance of interglobular dentine, representing small regions of unmineralized matrix.
Biochemical aspects of dentine mineralization
Concerning biochemical aspects of dentine mineralization, collagen fibril formation proceeds, with alignment of tropocollagen molecules being guided by non-collagenous proteins such as decorin. Pyridinoline cross-link formation between lysine and hydroxylysine residues increases as predentine is modified, allowing for mineralization to dentine. The collagen fibril orientation, along with the presence of proteoglycans, can be said to be the scaffold on which mineral can be deposited, and collagen itself is a good nucleator of hydroxyapatite. Hydroxyapatite crystals are initially deposited at the gap zone within collagen fibrils of mineralized dentine.
Two pools of proteoglycans are synthesized as dentine is formed. One pool is found in predentine and the other pool is found in dentine. This reflects different roles that these molecules may play in dentine formation.
Decorin and biglycan, with dermatan sulphate glycosaminoglycan side-chains, and versican are synthesized in predentine. These forms of the molecules are also predominant in soft connective tissues. They are found bound to collagen fibres in the gap zone and are involved with initial matrix formation, collagen fibril formation and prevention of premature mineralization. Versican has a high proportion of GAG chains attached to core protein and has also been implicated in the inhibition of mineralization. All of these proteoglycans are removed or remodelled as predentine matures.
At the mineralization front, a second pool of decorin (and biglycan) with chondroitin sulphate glycosaminoglycan side-chains is secreted. These proteoglycans, (together with acidic proteins) may be transported intracellularly along the odontoblast process, bypassing most of the predentine layer. Levels of these proteoglycans are lower than those found in predentine; they are distributed in mineralized connective tissues interfibrilly and are associated with the gap zones of the collagen fibrils. This second pool may be associated with directing mineral deposition, as the chondroitin sulphate may be involved in transport of Ca 2+ and HPO 42− to the gap zones in the collagen fibrils.
Acidic proteins such, as γ-carboxyglutamate-containing proteins (Gla-proteins), are found in dentine in low amounts. It is not known what their function is, but their acidic nature allows them to bind strongly to hydroxyapatite crystallites and therefore they may play some role in directing or guiding mineral deposition as predentine matures through to dentine. The other acidic proteins, osteopontin and osteonectin, may also be important in directing mineralization and these are classically associated with bone. Dentine matrix protein is another acidic phosphorylated protein found in dentine and bone, and may also be involved in regulating nucleation of mineral crystals.
The two dentine phosphoproteins, dentine sialoprotein (DSP) and dentine phosphoprotein (DPP), are post-transcriptionally cleaved from the same gene product from the dentine sialophosphoprotein (DSPP) gene. The two proteins may have similar, but slightly different roles: DPP:
• is secreted at the mineralizing front and is not present in predentine
• is highly phosphorylated (85–90%)
• is highly acidic due to the high aspartic acid residues and very high phosphate content
• has a very high affinity for calcium hydroxyapatite surfaces
• may function in mineral deposition due to its acidic nature and high calcium ion-binding properties.
• is acidic
• has a high carbohydrate content (sialic acid 10%)
• is phosphorylated
• is present in odontoblasts, predentine and dentine
• is considered to be tooth-specific.
Dentine is considered to be a bioactive matrix, as it contains a cocktail of bioactive proteins called growth factors. Such growth factors are sequestered into the dentine during dentinogenesis and are thought to be held within the mineralized matrix, bound to proteoglycans or latency-associated peptides. Such factors include TGF-β1, BMP-2, 4 and 7, IGF and the angiogenic growth factor VEGF. Such factors have been shown to be important in regulating dentinogenesis and tooth development, and it is possible that release of these factors during trauma or disease (and possibly cavity lining) directs the tertiary dentinogenic response which the pulp/dentine complex exhibits.
Dentinogenesis in the root
The basic process of dentinogenesis in the root does not differ fundamentally from that occurring in the crown. However, differences are evident in the earliest stages.
Epithelial root sheath
Following crown formation, the external enamel and internal enamel epithelia proliferate apically as the epithelial root sheath to map out the shape of the root. Internal to the root sheath lies the dental papilla, while the dental follicle is situated extenally. As in the crown, epithelial/mesenchymal interactions induce the peripheral cells of the dental papilla to differentiate into odontoblasts, which commence laying down the initial root dentine. Unlike dentinogenesis in the crown, the cells of the epithelial root sheath do not enlarge or become columnar, and do not differentiate into ameloblasts. Instead, they remain cuboidal. As the odontoblasts of the root migrate pulpwards, they do not initially trail behind a process and the resulting matrix contains some organic material derived from the odontoblasts (though with fewer collagen fibrils), as well as some enamel-related proteins secreted by the epithelial root sheath cells. This epithelial contribution is reflected in the structure of the internal enamel epithelial cells, which possess some of the intracellular organelles associated with protein synthesis and secretion (e.g. Golgi material and endoplasmic reticulum). The subsequent fate of the epithelial root sheath is considered further in relation to cementum formation.
This thin, initial, organic predentine layer in root dentine will mineralize to form the hyaline layer, which is continuous in the crown with the mantle layer. Unusually, mineralization begins a few microns in from the surface and continues pulpwards, allowing these outer few microns to undergo delayed mineralization outwards and provide a firm union with the initial collagen fibrils of the cementum.
Following the formation of the hyaline layer, the migrating odontoblasts trail behind their odontoblast processes. Initially these branch, loop and appear dilated and, when the dentine matrix around them becomes mineralized, give rise to the granular layer immediately beneath the hyaline layer. After this, the main odontoblast process is formed in a relatively straight horizontal plane as the remaining circumpulpal dentine of the root is formed.
Once the crown has erupted into the mouth and root completion has occurred, dentine continues to form throughout life, but at a slower rate. This dentine is termed secondary dentine. As the pulp volume decreases with continuing dentine deposition, odontoblasts overlap and form a pseudostratified layer, mainly in the crown.
Peritubular dentine is slowly deposited on the wall of the dentinal tubule, commencing soon after the formation of the dentinal tubule. It differs from intertubular dentine in containing more mineral and in lacking collagen fibres, its organic matrix consisting of non-collagenous proteins such as glycoproteins, proteoglycans and lipids. The inorganic component of peritubular dentine differs somewhat to that of intertubular dentine. Peritubular dentine formation may be under the control of the odontoblast and may incorporate plasma proteins that have diffused along the cell membrane.
Peritubular dentine will slowly narrow the diameter of the lumen of the dentinal tubule, initially about 3 μm in diameter. The lumen may be completely occluded by peritubular dentine and this happens consistently in the apical third of the root, giving rise to the appearance of translucent dentine. The amount of translucent dentine correlates very well with age.
The rate of dentine formation varies, producing incremental lines. There are both short-term (diurnal) and long-period rhythms:
• A diurnal rhythm of formation produces short-period lines approximately 4 μm apart (von Ebner lines), resulting from slight differences in composition/orientation of the dentine matrix.
• Long-period lines (Andresen lines), approximately 20 μm apart, are also evident, suggesting a longer rhythm (of unknown aetiology) of about 7 days.
An exaggerated line, the neonatal line, is present in all teeth mineralizing at birth and represents the dentine formed during the first few days after birth with its disturbed nutrition.
Tertiary dentine is the tissue that is laid down in response to a stimulus, such as severe attrition or dental caries:
• If the stimulus is mild and the original odontoblasts remain alive, a tubular form of tertiary dentine termed reactionary dentine will be formed.
• If, however, the stimulus is more severe and sufficient to destroy the original odontoblasts, new odontoblast-like cells will differentiate from stem cells within the pulp and lay down another form of tertiary dentine called reparative dentine, which is more irregular and atubular.
Development of the pulp
Once the bud-stage enamel organ invaginates to become the cap stage, the cells and matrix within the invagination are recognized as the dental papilla. During growth of the tooth germ, although relatively undifferentiated, the dental papilla interacts with the overlying epithelium (epithelial/mesenchymal interactions) to drive morphogenesis and histogenesis. The cells of the dental papilla, including its neural crest cells, are densely packed, rapidly dividing and separated by relatively little extracellular matrix.
As the enamel organ surrounding the dental papilla enlarges and enters the bell stage, the cells within the dental papilla undergo cytodifferentiation into a peripheral layer of odontoblasts and a central mass of fibroblasts. As the pulp develops, the cytoplasmic component of these central cells expands and synthetic organelles appear. The organic matrix of the pulp is released into the extracellular space and forms fine collagen fibres that are embedded in an amorphous ground substance. In the early stages of pulpal development, the ground substance has a high glycosaminoglycan content and a low quantity of hyaluronan. This balance is reversed in the mature tooth. Once the odontoblasts have begun to lay down dentine, the dental papilla becomes, by convention, the dental pulp. A proportion of cells present in the dental pulp remain as stem cells, retaining the potential to differentiate into other cells in later life, especially the odontoblast-like cells associated with the formation of tertiary dentine.
Once the full length of the root is established, the development of the dental pulp can be considered complete, although dentine deposition will continue throughout life. A cell-rich zone may be evident beneath the odontoblast layer at the time of eruption, probably the result of the migration of more central cells rather than by local cell division. Between the cell-rich zone and the odontoblast layer, a cell-free zone may become apparent at the time of eruption. However, this zone may be a fixation artefact. With age, as the odontoblasts migrate pulpally, the odontoblast layer appears pseudostratified.
Vascularization of the developing pulp starts during the early bell stage, with small branches from the principal vascular trunks of the jaws entering the base of the papilla. Of these small pioneer vessels, a few become the principal pulpal vessels, enlarge and run through the pulp towards the cuspal regions, where they give off numerous branches to form the subodontoblast plexus. The vascularity of the odontoblast layer increases as dentine is progressively laid down.
Nerves do not enter the dental pulp until dentinogenesis is well under way. The first fibres to enter the developing pulp become located close to the blood vessels. These nerves, although anatomically part of the sensory nervous system, probably play an important role via axon reflexes in controlling blood flow. The autonomic, sympathetic innervation follows later. Although a large number of nerves are found in the developing pulp, the formation of the subodontoblastic nerve plexus (of Raschkow) is not established until root formation is complete.
The dental pulp is the soft connective tissue occupying the core of the tooth that is responsible for nourishing and maintaining the dentine. Its peripheral cells form the odontoblast layer, from which the odontoblast processes pass into the dentine tubules. Being almost totally surrounded by mineralized tissue gives rise to a number of specialized features. As it forms dentine throughout life, the pulp undergoes a number of important age changes. Unusually for a soft connective tissue, it has a very rich blood and nerve supply. Its extreme sensitivity gives it profound clinical significance.
• know the composition and structure of the dental pulp, including all cell types present
• be able to compare the dental pulp with other soft connective tissues and be aware of specializations that may relate to its position, being surrounded by dentine
• be able to describe the blood vessels and nerves of the pulp and understand the physiology of pain
• appreciate the age changes that occur in the dental pulp and how these may relate to the clinical situation.
Organization and composition of the dental pulp
The dental pulp is a loose, soft, connective tissue derived from the dental papilla and is responsible for the formation and maintenance of dentine. It is contained within the pulp chamber and root canals of the tooth. At the apical constriction of the root canal it becomes continuous with the periodontal ligament. After a tooth has erupted into the oral cavity, the pulp forms secondary dentine slowly, but regularly, throughout life so that its size diminishes. It is able to respond to strong stimuli (such as caries, trauma, tooth movement and restorative procedures) by producing tertiary dentine. Some of the more specialized properties of the dental pulp relate to its situation, being almost completely encased in, and protected by, dentine.
As with other connective tissues, the dental pulp is made up of a combination of cells embedded in an extracellular matrix of fibres in a semifluid gel. It has a very rich blood and nerve supply. It contains 75% by weight of water and 25% organic material. The pulp is a dynamic functional matrix which has an important role in controlling the activity of the cells within it. The complex framework of collagen and associated proteoglycans and glycoproteins creates a scaffold to stabilize the structure of the pulp; however, the nature of the organic molecules within it allow it to influence cell migration, proliferation, adhesion, differentiation and function.
The collagen of the dental pulp is a combination of types I (60%) and III (40%). The collagen fibrils are about 50 nm in diameter and form thin fibres irregularly scattered throughout the tissue. A small amount of type IV collagen is present in the basement membrane of blood vessels.
GAGs and proteoglycans
The young, immature pulp has large quantities of chondroitin sulphate, with dermatan sulphate being found in much smaller amounts. GAGs are hydrophilic and form gels that fill most of the extracellular space. They swell when hydrated which may explain the high fluid pressure within the pulp, but will also contribute to the mechanical support. Hyaluronan is found unbound to protein and is thought to facilitate cell migration through the matrix. In the older, mature pulp, the ratios are reversed, with 60% of the GAG content being hyaluronan, 20% dermatan sulphate and only about 12% chondroitin sulphate (the remainder consisting of heparin sulphate).
Among the proteoglycans within the pulp are versican, syndecan and decorin, whilst among the glycoproteins are fibronectin and tenascin. The diverse nature and role of the proteoglycans found within the pulp influence its bioactivity by binding and conferring protection to growth factors, and acting as adhesion molecules to influence cell behaviour.
The pulp contains all the usual cells expected for a soft connective tissue (i.e. fibroblasts, defence cells and stem cells). However, also present at the periphery are the cell bodies of odontoblasts, a cell type unique for this tissue.
Odontoblasts are the cells responsible for the formation of dentine. They originate from neural crest (ectomesenchyme) cells. The fully differentiated odontoblast is a polarized columnar cell 50 μm long and 5 μm in width, with a very long cell process that extends into a dentine tubule. The cell has numerous smaller processes which link it to adjacent odontoblasts and adjacent pulp cells. The nucleus lies in the basal (pulpal) half of the cell, with the other organelles involved in dentine synthesis (e.g. rough endoplasmic reticulum, Golgi material and mitochondria) above it in the distal part of the cell. Initially forming a single layer of cells, odontoblasts in the crown retreat pulpwards so that, in a mature tooth, the odontoblasts overlap and give the false appearance of multiple layers (‘pseudostratification’). In the root, they are commonly more cuboidal and maintain the appearance of a single layer.
Odontoblasts have numerous cell junctions on their cell membranes namely, macula adherens junctions (desmosomes), tight junctions and gap junctions. These allow for cell signalling, maintaining the integrity of the cell layer and governing its degree of permeability. The odontoblast cell layer would appear to provide a controlled barrier between the pulp and the dentine.
During their life cycle, the activity of the odontoblasts is affected by a number of signalling and growth factors. In mature odontoblasts, these include factors such as transforming growth factor (TGF)-β and bone morphogenetic protein. As mature odontoblasts express numerous membrane receptors for the TGF-β family, it has been suggested that this growth factor may be of importance in the initiation of tertiary dentine. Although some have implied that the odontoblast could act as a sensory receptor, passing on afferent information from the outer dentine to nerve fibres in the peripheral pulp, there is no evidence of specialized junctions between nerves and odontoblasts.
Beneath the odontoblast layer in the crown, a layer relatively free of cell bodies may be seen once the tooth has erupted. This has been termed the cell-free zone (although it may represent a preparation artefact). Beneath the cell-free zone there may be a zone where there appears to be an increase in the number of cell bodies, the cell-rich zone.
Beneath the odontoblast layer, the most common cell type within the dental pulp is the fibroblast; these form a loose network throughout the tissue. As elsewhere, their morphology is highly variable. Their main function is associated with the development and maintenance of the extracellular matrix that provides the framework and support for the odontoblasts and for the neurovascular elements of the pulp. As the turnover of this matrix does not appear to be particularly rapid, pulpal fibroblasts show only moderate amounts of associated intracellular organelles such as endoplasmic reticulum, Golgi material or mitochondria.
A population of stem/progenitor cells must be present within the pulp to replace pulpal fibroblasts. There must also be a population of such progenitor cells that can, in response to a severe challenge, also produce tertiary dentine. Whether these two populations represent the same stem cell is not known. However, the subject has profound clinical implications. It is to be remembered that, for odontoblasts to differentiate initially during tooth development, signalling from epithelial cells is a prerequisite. However, epithelial cells are not present within the adult dental pulp during the formation of tertiary dentine. Therefore, during repair, another analogous method must take control. As dentine contains a number of growth factors which are known to be involved in the differentiation of odontoblasts during development, it is now thought that these are released during trauma to the dentine and are critical in signalling to the undifferentiated mesenchymal cells beneath the odontoblast layer and around blood vessels to differentiate into a new generation of odontoblast-like cells.
The typical defence cells (e.g. lymphocytes, macrophages and mast cells) exist in the healthy dental pulp. In addition, dendritic antigen-presenting cells are also an important component of the normal dental pulp. They are at least 50 μm long and have a number of branching processes. They are present particularly at the periphery of the dental pulp and around nerves and blood vessels. Some dendritic processes extend into the dentinal tubules. The cells initiate a primary immune response and migrate, with trapped antigen, to regional lymph nodes, inducing T-lymphocyte division and differentiation there.
The pulp has a rich blood supply. Arterioles and venules enter and leave the dental pulp via the apical foramina. The largest of the arterioles are approximately 150 μm in diameter. They run longitudinally through the root canals, within which they send off side branches to the periphery. The vessels divide and branch profusely once they are within the coronal pulp and form a rich capillary plexus beneath the odontoblast layer. Capillary loops extend towards the dentine and pass between the odontoblasts and the predentine. Returning vessels connect to venules. The capillary n/>