Stages of Development

Although formation of the tooth is a continuous process, as a matter of convenience tooth morphogenesis has been divided into three stages: (1) bud, (2) cap, and (3) bell (Fig. 12-3).¹⁸⁶ In the bud stage the epithelial cells of the dental lamina proliferate and produce a budlike projection into the adjacent ectomesenchyme. The cap stage is reached when the cells of the dental lamina have proliferated to form a concavity with a caplike appearance. The outer cells of the cap are cuboidal and constitute the outer enamel epithelium. The cells on the inner or concave aspect of the cap are somewhat elongated and represent the inner enamel epithelium. Between the outer and inner epithelia is a network of cells termed the stellate reticulum because of the branched reticular arrangement of the cellular elements. The rim of the enamel organ (i.e., where the outer and inner enamel epithelia are joined) is termed the cervical loop. As the cells forming the loop continue to proliferate, there is further invagination of the enamel organ into the mesenchyme. During this time, the organ assumes a bell shape, and tooth development then enters the bell stage (Fig. 12-4). During the bell stage, the ectomesenchyme of the dental papilla becomes partially enclosed by the invaginating epithelium.

FIG. 12-3 Diagrammatic representation of (A), the bud, (B), the cap, and (C) the bell stages of tooth development.

FIG. 12-4 Bell stage of tooth development showing the outer enamel epithelium (OEE), stellate reticulum (SR), inner enamel epithelium (IEE), dental papilla (DP), cervical loop (CL), successional lamina (SL), and dental sac (DS).

The condensed ectomesenchyme surrounding the enamel organ and dental papilla complex forms the dental sac and ultimately develops into the periodontal ligament (see Fig. 12-4). During the bell stage, the primary dental lamina degenerates and is invaded and replaced by mesenchymal tissue. In this way the epithelial connection between the enamel organ and the oral epithelium is severed. The free end of the dental lamina associated with each of the primary teeth continues to grow and forms the successional (secondary) dental lamina. It is from this structure that the tooth germ of the succedaneous tooth arises. As the maxillary and mandibular processes increase in size, the permanent first molar arises from posterior extensions of the dental lamina. After birth, the second and third molar primordia appear as the dental laminae proliferate into the underlying mesenchyme.

The morphogenesis (shape) of the crown is controlled by specialized signaling centers called epithelial enamel knots (EK). The enamel knots are transient clusters of nonproliferative epithelial cells located in the dental epithelium.^159,215,353 They express members of molecules belonging to key conserved fibroblast growth factor (Fgf), bone morphogenetic protein (Bmp), Hedgehog (Hh), and Wnt families that are well known to be essential for embryo and organ formation.³³² The signaling activity of the enamel knot and expressed molecules such as Fgf4 and Bmp4 regulate cellular processes of the surrounding epithelial and mesenchymal cells, leading to altered proliferation and expression of genes essential for tooth formation, such as transcription factors Mxs1 and Pax9.^172,354 The first appearing primary enamel knot (PEK) is present at the tip of the epithelial bud at the bud stage.¹⁵⁹ At the cap stage, the PEK is located in the middle part of the anterior-posterior axis of the enamel organ.^159,353 The secondary enamel knots (SEK), which appear later at the site of cusp tips, regulate the formation and number of the cusps of the multicusped teeth.¹⁵⁹ The recently found tertiary enamel knot (TEK) in turn prevents enamel formation by the underlying ameloblasts and thus controls the formation of the enamel-free areas in animals having these areas, such as mice.²¹⁵

Development of the Tooth Nerve Supply

Peripheral innervation serves significant roles in pulp biology. The initial innervation of the tooth originates from the sensory trigeminal ganglia. The nerve fibers innervate specific sites in the adult tooth, and as a consequence, the development of dental innervation is not a random event but instead is a strictly spatiotemporally controlled process that is tightly linked to the advancing tooth morphogenesis and dental cell differentiation^173,209 (Fig. 12-5). In the well-analyzed mouse mandibular molar, the molar nerve²⁰⁹ from the inferior alveolar nerve branch reaches the tooth at the early bud stage. The nerve follows a narrow mesenchymal pathway under the tooth germ, where it divides into the buccal and lingual branches.^173,209,243 During the cap and bell stages, nerve fibers establish a nerve plexus beneath the primary apical foramina. Nerve fibers wait here before they are allowed to enter the dental papilla. This takes place only after the crown shape is being defined by both dentin and enamel appearance in the crown.^237,238 The nerve fibers enter the papilla at both the mesial and distal ends, which are the sites of the future mesial and distal root of the molar tooth germ. The nerve fibers innervate the subodontoblastic region, the coronal odontoblast layer, and predentin layer with some fibers terminating in the dentin. The nerve fibers of the nerve plexus also contribute to the innervation of the periodontium.¹⁴⁶ Pioneer sympathetic innervation of the dental pulp follows the sensory one.²³⁷

FIG. 12-5 Growth of the sensory trigeminal axons to the dental target areas during tooth formation. The schematic illustration is based on studies on the localization of nerve fibers in the developing mouse molar (A-F).^173,237,238 Pioneer dental axons follow a restricted mesenchymal pathway (marked green in B) and grow around the developing tooth germ, termed the mesenchymal dental follicle target field (green in C-F). Nerve fibers penetrate into the dental pulp (p) right after the onset of enamel formation (E) and innervate the major pulp-dentin border target area (marked green in F). a, ameloblasts; b, buccal; cm, condensed dental mesenchyme; cp, coronal pulp; d, dentin; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; dp, dental papilla; e, enamel; ian, inferior alveolar nerve; l, lingual; o, odontobaster; p, dental pulp; pe, periodontium; rp, radicular pulp; snp, subodontoblastic nerve plexus; nerve fibers are marked in black.

(Modified from Kenntunen P, et al: Regularing av utvikling av tannens form og sensorisk nerveforsying er samordent. Nor Tannlaegeforen Tid 119:162–166, 2009.)

Molecular Control of Trigeminal Axon Navigation

There is increasing evidence that the navigation of the growing dental axons involves concerted, redundant signaling activity by molecules of different families. The molecules exerting positive attractive effect on axon guidance or survival are expressed in the mesenchymal pathway or target areas around and within the tooth germ.²¹⁴ The molecules include neurotrophic factors, which promote survival, differentiation, and maintenance of neurons in developing and adult vertebrate nervous systems. NGF (nerve growth factor), which is essential for dental pulp nerve supply, and GDNF (glial cell line-derived neurotrophic factor) are expressed in the major target area of the developing tooth, namely the dental follicle and pulp-dentin border target areas. In addition, many other molecular families, such as laminins, neuregulin/ErbB, Ephs and Ncam are implicated in the development of the tooth nerve supply.^100,214

Repulsive axon guidance molecules in turn, are produced in the areas next to the axon pathways. They control axon navigation by preventing nerve fibers from entering areas that should be avoided. The secreted chemorepellant, semaphorin3A (Sema3A), which is expressed in mesenchymal restriction areas,¹⁷³ controls dental trigeminal axon fasciculation as well as the timing and patterning of tooth innervation.¹⁷³ In addition, Sema3A appears to control the site of the nerve fiber penetration to the dental pulp (i.e., through the future roots).²¹⁶ Because Sema3A expression in mesenchymal restriction areas is controlled by signaling from the dental epithelium, this indicates that tooth innervation development, like the formation of the tooth germ proper, is controlled by local tissue interactions. Of note, Fgfr2b, which is essential to tooth morphogenesis by regulating epithelial proliferation and function of the enamel knot, also controls Sema3A expression and therefore dental axon navigation. Thus, Fgf2b acts as a molecule that integrates tooth morphogenesis and dental trigeminal axon patterning.¹⁹⁷

Differentiation of Odontoblasts

Differentiation of epithelial and mesenchymal cells into ameloblasts and odontoblasts, respectively, occurs during the bell stage of tooth development. This differentiation starts in the region where the cusp tip will develop and proceeds towards the cervical loop. The preameloblasts lining the dental papilla induce differentiation of odontoblasts. Consequently, odontoblast-secreted dentin matrix induces enamel secretion by ameloblasts.

During the bell stage of development, there is still mitotic activity among the relatively immature cells of the inner enamel epithelium in the region of the cervical loop. As they commence to mature into ameloblasts, mitotic activity ceases, and the cells elongate and display the characteristics of active protein synthesis (i.e., an abundance of rough endoplasmic reticulum [RER], a well-developed Golgi complex, and numerous mitochondria).

As the ameloblasts undergo differentiation, changes are taking place across the basement membrane in the adjacent dental papilla because of a coordinated exchange of growth factors and other soluble mediators. Before differentiation of odontoblasts, the dental papilla consists of sparsely distributed polymorphic mesenchymal stem cells with wide intercellular spaces (Fig. 12-6). With the onset of differentiation, a single layer of cells, the presumptive odontoblasts (i.e., preodontoblasts) align themselves along the basement membrane separating the inner enamel epithelium from the dental papilla. These cells stop dividing and elongate into short columnar cells with basally situated nuclei (Fig. 12-7). Several cytoplasmic projections from each of these cells extend toward the basal lamina. At this stage, the preodontoblasts are still relatively undifferentiated.

FIG. 12-6 Bell stage shows preodontoblasts (PO) aligned along the basement membrane (BM) separating the inner enamel epithelium (IEE) from the dental papilla (DP).

FIG. 12-7 Diagrammatic representation of the stages of odontoblast differentiation.

As the odontoblasts continue to differentiate, they become progressively more elongated and take on the ultrastructural characteristics of protein-secreting cells. Cytoplasmic processes from these cells extend through the DBM toward the basal lamina, and more and more collagen fibrils appear within the ECM. The first formed collagen fibers pass between the preodontoblasts and extend toward the basal lamina to form large, fan-shaped bundles 100 to 200 nm in diameter, often referred to as von Korff fibers. These fibers stain with silver stains and are associated with a high content of proteoglycans. Some smaller collagen fibers, approximately 50 nm in diameter, also pass between the odontoblasts and are thought to arise in the dental papilla subjacent to the odontoblasts.²⁹

A study on collagen gene expression during rat molar tooth development found both types I and III collagen messenger ribonucleic acid (mRNA) in developing odontoblasts.⁷⁵ Levels of type I collagen mRNA increased with the progression of odontoblast differentiation, whereas type III collagen gene expression decreased as dentinogenesis proceeded. Both type I and type III collagen mRNA were detected in dental pulp mesenchymal cells.

Dentinogenesis first occurs in the developing tooth at sites where the cusp tips or incisal edge will be formed. It is in this region that odontoblasts first reach full maturity and become tall columnar cells, at times attaining a height of about 50 µm (see Fig. 12-7). The width of these cells remains fairly constant at approximately 7 µm. Production of the initial dentin matrix involves the formation, organization, and maturation of collagen fibrils and proteoglycans. As predentin is formed, the odontoblasts commence to move toward the central pulp, depositing predentin at a rate of approximately 4 to 8 µm per day in their wake during early tooth development. Within predentin, a process from each odontoblast becomes accentuated and remains to form the odontoblast process. The dentinal tubules are formed around these cellular processes. Similar to tooth initiation and morphogenesis, dentinogenesis is controlled by epithelial-mesenchymal interactions that are mediated by a combination of EMC and signaling molecules of different families such as BMP and Tgf-β1.¹⁹⁷

Root Development

Root development commences after the completion of enamel formation, when the cervical loop does not exist. The stellate reticulum and stratum intermedium cells disappear, and the inner and outer enamel epithelium in the distal part of the cervical loop forms Hertwig’s epithelial root sheath (HERS), composed of two cell layers, the inner and outer enamel epithelium (Fig. 12-8). HERS determines the size and shape of the root or roots of the tooth. As in the formation of the crown, the cells of the inner enamel epithelium induce the adjacent mesenchymal stem cells in the pulp to differentiate into preodontoblasts and odontoblasts. As soon as the layer of dentin matrix mineralizes, gaps appear within the HERS-derived Malassez epithelium, allowing mesenchymal cells from the dental sac to move into contact with the newly formed dentin. These mesenchymal stem cells thereafter differentiate into cementoblasts and deposit cementum matrix on the root dentin. Some cells of the Malassez epithelium persist within the periodontal ligament and are known as the epithelial cell rests of Malassez. These clusters of epithelial cells surrounded by basement membrane appear as a meshlike network around the root. Although the number of these rests gradually decreases with age, it has been shown that at least some of them retain the ability to undergo cell division³⁴⁷ (Fig. 12-9). In later life, if a chronic inflammatory lesion develops within the periapical tissues as a result of pulp disease, proliferation of the epithelial rests may produce a periapical (radicular) cyst.

FIG. 12-8 Root development showing dental pulp (DP), dental sac (DS), and epithelial root sheath (ERS).

FIG. 12-9 Autoradiograph of an epithelial rest cell showing ³H-thymidine labeling of the nucleus, indicating that the cell is preparing to divide.

(From Trowbridge HO, Shibata F: Mitotic activity in epithelial rests of Malassez. Periodontics 5:109, 1967.)

Accessory Canals in the Root

Occasionally during formation of the root sheath, a break develops in the continuity of the sheath, producing a small gap. When this occurs, dentinogenesis does not take place adjacent to the defect. The result is a small “accessory” canal between the dental sac and the pulp. An accessory canal can become established anywhere along the root, creating a periodontal-endodontic pathway of communication and a possible portal of entry into the pulp if the periodontal tissues lose their integrity. In periodontal disease, the development of a periodontal pocket may expose an accessory canal and allow microorganisms or their metabolic products to gain access to the pulp. Conversely, in the case of teeth with infected, necrotic pulps, the inability to cleanse, shape, and obturate these canals could result in periodontitis (see also Chapter 18).

Primary, Secondary, and Tertiary Dentin

Primary dentin is produced during tooth development until the teeth have erupted into the oral cavity. It is classified as orthodentin, the tubular form of dentin lacking of cells found in the teeth of all dentate mammals.²⁰⁴ The primary dentin is secreted at a relatively high rate and constitutes the major part of the dentin in the tooth. It is regular in structure and contains dentin tubules that form an S-shaped primary curvature as a result of the directional movement of odontoblasts, which have a cell process that extends from a broad peripheral border towards a narrow, centrally located cell layer. After tooth eruption, the odontoblasts continue to lay down dentin but slightly change their direction, which contributes to bending of the dentinal tubules. It is referred to as secondary dentin and is synthesized at a much lower rate and is less regular in structure than primary dentin. Secondary dentin is deposited during the rest of the tooth’s life. Unlike primary and secondary dentin, tertiary dentin, or reparative or reactionary dentin, is deposited as a result of a pathologic process such caries or occlusal abrasion. Tertiary dentin has been suggested to be secreted by original odontoblasts or in case of their death, by newly differentiated replacement odontoblasts originating from nearby mesenchymal stem cells. The function of the tertiary dentin is to protect the pulp from noxious influences. Tertiary dentin is disorganized in structure compared to primary and secondary dentin.

Mantle Dentin

The first layer of the primary dentin to be deposited is mantle dentin. It is produced by odontoblasts that are not yet fully differentiated. In the adult tooth, mantle dentin is the oldest dentin and is produced adjacent to the enamel in the crown. Odontoblasts first produce the collagen components, which are organized in a collagen fibril network. Electron microscopic analyses have shown that odontoblasts produce small membrane-closed matrix vesicles where precipitation of minerals starts.³³ After the rupture of the matrix vesicles, the crystals spread within the collagen matrix and are assumed to act as starting points for the growth of the apatite crystals. In addition to the crown, mantle dentin is also found in the root between cementum and Tomes granular layer. However, this is a controversial finding, and opposing data suggest that a hyaline layer or enameloid is found in the root instead of mantle dentin.

Later during the mineralization of the circumpulpal dentin, the matrix vesicles are no longer detected.²⁰⁵ Mantle dentin can be recognized by the characteristic thick, fan-shaped collagen fibers deposited immediately subjacent to the basal lamina in histologic sections. These fibers run roughly perpendicular to the DEJ. Spaces between the fibers are occupied by smaller collagen fibrils lying more or less parallel with the DEJ or CDJ. Mantle dentin is only 150 µm thick and is slightly less mineralized (about 4% less mineral) than underlying dentin¹³⁹ and is therefore softer.³⁶⁹ The mineralization of the mantle dentin may differ from mineralization of the rest of the dentin additionally, since dentinphosphoprotein (DPP), which regulates crystal growth in the circumpulpal dentin, does not appear to be present in mantle dentin.²⁴⁶

Circumpulpal Dentin

Circumpulpal dentin is formed after the layer of mantle dentin has been deposited, and it constitutes the major part of primary and secondary dentin. The odontoblasts first deposit the organic matrix, which is composed mainly of collagen fibrils approximately 500 nm in diameter, oriented at right angles to the long axis of the dentinal tubules. These collagen fibrils are closely packed together and form an interwoven network that will be embedded by the subsequent mineral crystals. Calcium is actively transported from the blood vessels by the odontoblasts into the collagen fibril template/network. There is evidence that mineralization of the dentin is under control of dentinphosphoprotein (DPP). DPP is able to bind calcium, since it is highly anionic. Conformational changes of DPP enable it to bind large numbers of calcium ions, which allows the formation and growth of mineral crystals in the collagen matrix network. By regulating the release and concentration of DPP, the odontoblasts may regulate the initiation and speed of dentin mineralization. Hydroxyapatite crystals are deposited on the surface and within the fibrils and continue to grow as mineralization proceeds, resulting in an increased mineral content of the dentin. The circumpulpal dentin is mineralized through calcospherites in the mineralization front between predentin and mineralizing dentin. The mineral crystals are organized in a radiating pattern in calcospherites, the size of which may vary in size and shape in different regions of dentin. As the calcospherites enlarge, they fuse with adjacent calcospherites until the dentin matrix is completely mineralized.

Predentin

Predentin is a 15- to 20-µm unmineralized organic matrix layer of dentin situated between the odontoblast layer and the mineralized dentin.²⁰⁴ Its prime protein constituents include type I and type II collagens.⁴² Noncollagenous elements consist of several proteoglycans (i.e., dermatan sulfate, heparan sulfate, hyaluronate, keratin sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate),^40,117 glycoproteins, glycosaminoglycans (GAGs),⁸³ Gla proteins, and dentin phosphoprotein (DPP)⁴² DPP is a highly phosphorylated protein that is exclusively found in bone and dentin.²⁹⁰ In dentin, DPP is produced by the odontoblasts and transported to the mineralization front. It is thought to bind to calcium and play a role in mineralization.^109,359 Growth factors such as TGF-β, insulin-like growth factor, platelet-derived growth factor, and angiogenic growth factor have also been identified in dentin.²⁹⁴ The presence of growth factors in dentin may have profound clinical significance because resorption of dentin conceivably releases these growth factors into pulp, where they alter biologic functions of this tissue. Dentinal resorption may stimulate formation of tertiary dentin.

Dentinal Tubules

A characteristic of human dentin is the presence of tubules that occupy from 1% (superficial dentin) to 30% (deep dentin) of the volume of intact dentin.^107,277 The diameter of tubules vary from 1 µ to 2.5 µm and traverse the entire thickness of dentin from the DEJ or CDJ to the pulp. They are slightly tapered, with the wider portion situated toward the pulp. This tapering is the result of the progressive formation of up to 1 µm of peritubular dentin, which leads to a continuous decrease in the diameter of the lumen of the tubules to a final diameter of about 1 mm.¹⁰⁷ No peritubular dentin is present when the tubules reach the predentin and pulp chamber.

In coronal dentin, the tubules have a gentle S shape as they extend from the DEJ to the pulp. The S-shaped curvature is presumably a result of the crowding of odontoblasts as they migrate toward the center of the pulp. As they approach the pulp, the tubules converge because the surface of the pulp chamber has a much smaller area than the surface of dentin along the DEJ. This results in a progressive increase in dentin permeability (discussed later).^275-277279

The number and diameter of the tubules has been determined and varies depending on the location in the tooth (Table 12-1).¹⁰⁷ The number and diameter of dentinal tubules are similar among rats, cats, dogs, monkeys, and humans, demonstrating a remarkable consistency in mammalian orthodentin.⁴

TABLE 12-1 Mean Number and Diameter per Square Millimeter of Dentinal Tubules at Various Distances from the Pulp in Human Teeth

Lateral microtubules containing branches of the main odontoblastic processes have been demonstrated by other researchers,^167,236 who suggested that they form pathways for the movement of materials between the main processes and the more distant matrix. It is also possible that the direction of the branches influences the orientation of the collagen fibrils in the intertubular dentin.

Near the DEJ, the dentinal tubules ramify into one or more terminal branches²³⁶ (Fig. 12-10). This occurs because during the initial stage of dentinogenesis, the differentiating odontoblasts extended several small cytoplasmic processes toward the DEJ, but as the odontoblasts migrated, their processes converged into one major process (see Fig. 12-7).

FIG. 12-10 Ground section of a tooth demonstrating branching of the dentinal tubules near the dentin and enamel junction (DEJ). This branching may account for the increased clinical sensitivity at the DEJ.

Intertubular Dentin

Intertubular dentin is located between the dentin tubules and constitutes the bulk of dentin (Fig. 12-11). Its organic matrix consists mainly of collagen fibrils having diameters of 50 to 100 nm. These fibrils are oriented approximately at right angles to the dentinal tubules. They are well mineralized and provide tensile strength to dentin.¹⁸⁴ Intertubular dentin is found between the DEJ and the mineralization front.²⁰⁴

FIG. 12-11 Diagram illustrating peritubular and intertubular dentin.

(From Trowbridge HO: Dentin today: research reveals new information about the complexity of dentin. Dentistry 2:22–29, 1982.)

Intratubular Dentin

Dentin lining the inner walls of tubules is termed intratubular or peritubular dentin (see Fig. 12-11). Intratubular dentin represents a specialized form of orthodentin not found in all mammals. The matrix of peritubular dentin differs from that of intertubular dentin by having relatively fewer collagen fibrils and a higher proportion of sulfated proteoglycans and mineral. Because of its lower content of collagen,^183,374 peritubular dentin is harder than intertubular dentin and therefore is more quickly dissolved in acid than intertubular dentin. By preferentially removing peritubular dentin, acid-etching agents used during dental restorative procedures and ethylenediaminetetraacetic acid (EDTA) used in endodontic treatment enlarge the openings of the dentinal tubules, making the dentin more permeable. The absence of intrafibrillar mineral in patients with dentinogenesis imperfecta type II may be responsible for their softer dentin.¹⁸⁵

Dentinal Sclerosis

Partial or complete obturation of dentinal tubules may occur as a result of aging or develop in response to persistent stimuli such as attrition of the tooth surface or dental caries. When tubules become filled with mineral deposits, the dentin is termed sclerotic. Dentinal sclerosis is easily recognized in histologic ground sections because of its translucency (due to homogeneity of the dentin). Studies using dyes, solvents, and radioactive ions have shown that sclerosis results in decreased permeability of dentin,^92,276,328 so by limiting the diffusion of noxious substances through the dentin, dentinal sclerosis helps shield the pulp from irritation.

One form of dentinal sclerosis is thought to represent an acceleration of intratubular dentin formation. This form appears to be a physiologic process, and in the apical third of the root it develops as a function of age.^323,328 Dentinal tubules can also become blocked by the precipitation of hydroxyapatite and whitlockite crystals within the tubules. This type occurs in the translucent zone of carious dentin and in attrited dentin and has been termed pathologic sclerosis.^263,328,371

Interglobular Dentin

Interglobular dentin refers to dentin that contains organic matrix that is unmineralized because the growing calcospherites failed to coalesce. This occurs most often in the circumpulpal dentin close to the mantle dentin in the crown and close to the Tomes granular layer in the root. In certain dental anomalies (e.g., vitamin D–resistant rickets, hypophosphatasia), large areas of interglobular dentin are a characteristic feature (Fig. 12-12).¹⁰⁸

FIG. 12-12 Section showing interglobular dentin (ID) in a deciduous incisor from a 3-year-old boy with childhood hypophosphatasia.

Dentinal Fluid

Free fluid occupies 1% of superficial dentin but about 22% of the total volume of deep dentin (i.e., near the pulp).²⁷⁷ This fluid is an ultrafiltrate of blood from the pulp capillaries, and its composition not surprisingly resembles plasma in many respects.⁶¹ Using calcium ion–specific electrodes, the ionized calcium concentration of dentinal fluid in predentin was two to three times higher than plasma.²¹² Although it contains plasma proteins, their concentration is only about 10% that of plasma.²¹⁹ The outward fluid flows between the odontoblasts through the dentinal tubules and is blocked peripherally by enamel on the crown and cementum on the root. It has been shown that the tissue pressure of the pulp is approximately 14 cm H₂O (10.3 mm Hg).⁶⁷ Consequently, a pressure gradient exists between the pulp and the oral cavity that accounts for the slow outward flow of fluid if any dentin becomes exposed. Exposure of the tubules by tooth fracture or during cavity preparation often results in the outward movement of fluid to the exposed dentin surface in the form of tiny droplets.¹⁵⁷ Dehydrating the surface of the dentin with compressed air, dry heat, or the application of absorbent paper can accelerate this outward movement of fluid.^37-39274 Rapid flow of fluid through the tubules is thought to be a cause of dentin sensitivity.²³⁰ The slow outward movement of dentinal fluid is not sufficient to activate the mechanoreceptor nerves responsible for dentin sensitivity. This slow outward fluid flow, about 0.02 ml/sec/mm², must increase to 1 to 1.5 ml/sec/mm² before these nerves begin to discharge.²³⁰

Bacterial products or other contaminants may be introduced into the dentinal fluid as a result of dental caries, restorative procedures, or growth of bacterial biofilms beneath restorations.^37,234,284 Dentinal fluid may thus serve as a sink from which injurious agents can diffuse into the pulp, producing an inflammatory response. Conversely, dentinal fluid may serve as a vehicle for the egress of bacteria from a necrotic pulp into the periradicular tissue. This has particular significance in the apical portion of the root canal system in the development or maintenance of apical periodontitis because bacteria are the key etiologic factor (see Chapter 15). Fortunately, the density of dentinal tubules is much lower in the apical portion of the root compared to the cervical portion, and this anatomic feature may contribute to the success of nonsurgical root canal treatment. In addition, endodontic surgical techniques have been modified to reduce exposure of dentinal tubules during apical root resection by reducing the angle of root resection; as the angle of resection approaches 90 degrees to the long axis of the root, a corresponding decrease is seen in dentinal tubules exposed to the periapical tissue (see Chapter 21 for details).

Dentin Permeability

The permeability of dentin has been well characterized.^{92,97,274-277,279,282} Dentinal tubules are the major channels for diffusion of material across dentin. Because fluid permeation is proportional to tubule diameter and number, dentin permeability increases as the tubules converge on the pulp (Fig. 12-13). The total tubular surface near the DEJ is approximately 1% of the total surface area of dentin,^274,277 whereas close to the pulp chamber, the total tubular surface may be nearly 45%. From a clinical standpoint, it should be recognized that dentin beneath a deep cavity preparation is much more permeable than dentin underlying a shallow cavity when the formation of sclerotic or reparative dentin is negligible.

FIG. 12-13 Diagram illustrating the difference in size and density of tubules in the dentinal floor between a shallow (A) and a deep (B) cavity preparation, in coronal dentin versus (C) root dentin.

(Redrawn from Trowbridge HO: Dentin today: research reveals new information about the complexity of dentin. Dentistry 22:22–29, 1982.)

One study⁹⁷ found that the permeability of radicular dentin is much lower than that of coronal dentin. This was attributed to a decrease in the density of the dentinal tubules from approximately 42,000/mm² in cervical dentin to about 8000/mm² in radicular dentin. These investigators found that fluid movement through outer radicular dentin was only approximately 2% that of coronal dentin. The low permeability of outer radicular dentin should make it relatively impermeable to toxic substances such as bacterial products emanating from plaque.

Factors modifying dentin permeability include the presence of odontoblast processes in the tubules and the sheathlike lamina limitans that lines the tubules. Collagen fibers have also been observed in most tubules.⁷¹ The functional or physiologic diameter of the tubules is only about 5% to 10% of the actual anatomic diameter (i.e., the diameter seen in microscopic sections).²³³ This is small enough to trap and remove bacteria from dentinal fluid,²³⁴ permitting sterile fluid to enter the pulp chamber.

In dental caries, an inflammatory reaction develops in the pulp long before the pulp actually becomes infected with micororganisms.³⁴⁵ This indicates that bacterial byproducts reach the pulp in advance of the bacteria themselves,^19,284 and the early inflammatory response is elicited by the accumulation of bacterial antigens or byproducts into the pulp, not by the bacteria themselves. Dentinal sclerosis (i.e., reparative dentin formation) beneath a carious lesion reduces permeability³²⁸ by obstructing the tubules, thus decreasing the delivery of irritants into the pulp.

The cutting of dentin during cavity preparation or root canal therapy produces a microcrystalline debris that coats the dentin and clogs the orifices of the dentinal tubules. This layer of debris is termed the smear layer. Because of the small size of the particles, the smear layer is capable of physically preventing bacteria from penetrating dentin.^234,244 Removal of the debris by acid etching or EDTA greatly increases the permeability of the dentin by widening the orifices of the tubules. Consequently, the incidence of pulpal inflammation may be increased significantly if cavities are treated with an acid cleanser unless a cavity liner, base, or dentin-bonding agent is used. Continued pulpal protection will then depend on the durability of the bond.

When the dentin surface of vital and nonvital teeth was exposed to the oral environment for 150 days, bacterial invasion of dentinal tubules occurred more rapidly in the nonvital teeth.²⁴⁴ Presumably this was due to the resistance offered by the outward movement of dentinal fluid and the presence of odontoblast processes in the tubules of vital teeth.^280,361,362 Importantly, it has been shown that antibodies or other antimicrobial components are present within the dentinal fluid of teeth with vital pulps.¹²⁵

The Pulp-Dentin Complex

The dental pulp and dentin function as a unit, and the odontoblasts represent a crucial element in this system. The odontoblasts are located in the periphery of the pulp tissue, with extensions into the inner part of dentin. Dentin would not exist unless produced by odontoblasts, and the dental pulp is dependent on the protection provided by the dentin and enamel. Likewise, integrated dynamics of the pulp-dentin complex imply that impacts on dentin may affect the pulpal components, and that disturbances in the dental pulp will in turn affect the quantity and quality of the dentin produced.

Odontoblast Layer

The outermost stratum of cells of the healthy pulp is the odontoblast layer (Figs. 12-15 and 12-16). This layer is located immediately subjacent to the predentin. The odontoblast processes, however, pass on through the predentin into the inner part of dentin. Consequently, the odontoblast layer is actually composed of the cell bodies of odontoblasts. In addition, capillaries, nerve fibers, and dendritic cells may be found among the odontoblasts.

FIG. 12-15 Morphologic zones of the mature pulp.

FIG. 12-16 Diagrammatic representation of the odontoblast layer and subodontoblastic region of the pulp.

In the coronal portion of a young pulp that is actively secreting collagen, the odontoblasts assume a tall columnar form.⁶³ The odontoblasts vary in height; consequently, their nuclei are not all at the same level and are aligned in a staggered array, often described as a palisade appearance. This organization makes the layers appear to be three to five cells in thickness even though there is only one actual layer of odontoblasts. Between adjacent odontoblasts there are small intercellular spaces approximately 30 to 40 nm in width. Odontoblast cell bodies are connected by tight and gap junctional complexes.^30,63,149 Gap junctions are formed by connexin proteins¹¹⁰ that permit cell-to-cell passage of signal molecules.

The odontoblast layer in the coronal pulp contains more cells per unit area than in the radicular pulp.²²⁴ Whereas the odontoblasts of the mature coronal pulp are usually columnar, those in the midportion of the radicular pulp are more cuboidal (Fig. 12-17).⁶³ Near the apical foramen, the odontoblasts appear as a squamous layer of flattened cells. Because fewer dentinal tubules per unit area are present in the root than in the crown of the tooth, the odontoblast cell bodies are less crowded and are able to spread out laterally.²²⁴ During maturation and aging, there is a continued ongoing crowding in the odontoblast layer, particularly in the coronal pulp, due to narrowing of the pulp space. Apoptosis of odontoblasts seems to adjust for this limited space during development.²³⁵

FIG. 12-17 Low columnar odontoblasts of the radicular pulp. The cell-rich zone is inconspicuous.

There are a series of specialized cell-to-cell junctions (i.e., junctional complexes), including desmosomes (i.e., zonula adherens), gap junctions (i.e., nexuses), and tight junctions (i.e., zonula occludens) that connect adjacent odontoblasts. Spot desmosomes located in the apical part of odontoblast cell bodies mechanically join odontoblasts together. The numerous gap junctions provide permeable pathways through which signal molecules can pass between cells (Fig. 12-18) to synchronize secretory activity that produces relatively uniform predentin layers (see Fig. 12-16). These junctions are most numerous during the formation of primary dentin. Gap junctions and desmosomes have also been observed joining odontoblasts to the processes of fibroblasts in the subodontoblastic area. Tight junctions are found mainly in the apical part of odontoblasts in young teeth. These structures consist of linear ridges and grooves that close off the intercellular space. However, tracer studies suggest direct passage of small elements from subodontoblastic capillaries to predentin and dentin between the odontoblasts.³³⁰ It appears that tight junctions determine the permeability of the odontoblast layer when dentin is covered by enamel or cementum by restricting the passage of molecules, ions, and fluid between the extracellular compartments of the pulp and predentin.³⁰ During cavity preparation, these junctions are disrupted, thereby increasing dentin permeability.^350,351

FIG. 12-18 A, Electron micrograph of a mouse molar odontoblast demonstrating gap junctions (arrows), nucleus (N), mitochondria (M), Golgi complex (G), and rough endoplasmic reticulum (RER). B, High magnification of a section fixed and stained with lanthanum nitrate to demonstrate a typical gap junction.

(Courtesy Dr. Charles F. Cox, School of Dentistry, University of Alabama.)

Cell-Poor Zone

Immediately subjacent to the odontoblast layer in the coronal pulp, there is often a narrow zone approximately 40 µm in width that is relatively free of cells (see Fig. 12-15) and hence is called the cell-free layer of Weil. It is traversed by blood capillaries, unmyelinated nerve fibers, and the slender cytoplasmic processes of fibroblasts (see Fig. 12-16). The presence or absence of the cell-poor zone depends on the functional status of the pulp.⁶³ It may not be apparent in young pulps, where dentin forms rapidly, or in older pulps, where reparative dentin is being produced.

Cell-Rich Zone

In the subodontoblastic area, there is a stratum containing a relatively high proportion of fibroblasts compared with the more central region of the pulp (see Fig. 12-15). It is much more prominent in the coronal pulp than in the radicular pulp. Besides fibroblasts, the cell-rich zone may include a variable number of immune cells like macrophages and dendritic cells, but also undifferentiated mesenchymal stem cells.

On the basis of evidence obtained in rat molar teeth, it has been suggested¹¹⁹ that the cell-rich zone forms as a result of peripheral migration of cells populating the central regions of the pulp, commencing at about the time of tooth eruption. Migration of immunocompetent cells out of and into the cell-rich zone has been demonstrated as a result of antigenic challenge.³⁸⁸ Although cell division within the cell-rich zone is rare in normal pulps, the death of the odontoblasts triggers a great increase in the rate of mitosis. Because odontoblasts are postmitotic cells, irreversibly injured odontoblasts are replaced by cells that migrate from the cell-rich zone onto the inner surface of the dentin.⁹⁶ This mitotic activity is probably the first step in the formation of a new odontoblast layer.^{73,240-242,320} Studies implicate stem cells as a source for these replacement odontoblasts.³²⁶

Pulp Proper

The pulp proper is the central mass of the pulp (see Fig. 12-15). It consists of loose connective tissue and contains the larger blood vessels and nerves. The most prominent cell in this zone is the fibroblast.

Odontoblast

Because odontoblasts are responsible for dentinogenesis, both during tooth development and aging, the odontoblast is the most characteristic and specialized cell of the dentin-pulp complex. During dentinogenesis, the odontoblasts form dentin and the dentinal tubules, and their presence within the tubules makes dentin a living responsive tissue.

Dentinogenesis, osteogenesis, and cementogenesis are in many respects quite similar, and odontoblasts, osteoblasts, and cementoblasts have many characteristics in common. Each of these cells produces a matrix composed of collagen fibrils, noncollagenous proteins, and proteoglycans that are capable of undergoing mineralization. The ultrastructural characteristics of odontoblasts, osteoblasts, and cementoblasts are likewise similar in that each exhibits a highly ordered RER, a prominent Golgi complex, secretory granules, and numerous mitochondria. In addition, these cells are rich in RNA, and their nuclei contain one or more prominent nucleoli. These are the general characteristics of protein-secreting cells.

The most significant differences among odontoblasts, osteoblasts, and cementoblasts are their morphologic characteristics and the anatomic relationship between the cells and the mineralized structures they produce. Whereas osteoblasts and cementoblasts are polygonal to cuboidal in form, the fully developed odontoblast of the coronal pulp is a tall columnar cell.^63,224 In bone and cementum, some of the osteoblasts and cementoblasts become entrapped in the matrix as osteocytes or cementocytes, respectively. The odontoblast, on the other hand, leaves behind a cellular process to form the dentinal tubule, and the cell body resides outside the mineralized tissue. Lateral branches between the major odontoblast processes interconnect^167,236 through canaliculi, just as osteocytes and cementocytes are linked together through the canaliculi in bone and cementum. This provides a pathway for intercellular communication and circulation of fluid and metabolites through the mineralized matrix.

The ultrastructural features of the odontoblast have been the subject of numerous investigations. The cell body of the active odontoblast has a large nucleus that may contain up to four nucleoli (Fig. 12-19). The nucleus is situated at the basal end of the cell and is contained within a nuclear envelope. A well-developed Golgi complex, centrally located in the supranuclear cytoplasm, consists of an assembly of smooth-walled vesicles and cisternae. Numerous mitochondria are evenly distributed throughout the cell body. RER is particularly prominent, consisting of closely stacked cisternae forming parallel arrays that are dispersed diffusely within the cytoplasm. Numerous ribosomes closely associated with the membranes of the cisternae mark the sites of protein synthesis. Within the lumen of the cisternae, filamentous material (probably representing newly synthesized protein) can be observed.

FIG. 12-19 Diagram of a fully differentiated odontoblast. RER, Rough endoplasmic reticulum.

The odontoblast appears to synthesize mainly type I collagen,^196,375 although small amounts of type V collagen have been found in the ECM. In addition to proteoglycans^40,82,117 and collagen,^196,202 the odontoblast secretes dentin sialoprotein⁴² and phosphophoryn,^42,70 a highly phosphorylated phosphoprotein involved in extracellular mineralization.^42,75 Phosphophoryn is unique to dentin and is not found in any other mesenchymal cell types.⁷⁵ The odontoblast also secretes both acid phosphatase and alkaline phosphatase. The latter enzyme is closely linked to mineralization, but the precise role of alkaline phosphatase in dentinogenesis is not completely understood. Acid phosphatase, a lysosomal enzyme, may be involved in digesting material that has been resorbed from predentin matrix.⁸⁵

In contrast to the active odontoblast, the resting or inactive odontoblast has a decreased number of organelles and may become progressively shorter.^63,224 These changes can begin with the completion of root development and eruption when dentin production shifts from primary to secondary dentin.

Odontoblast Process

A dentinal tubule forms around each of the major odontoblastic processes. The odontoblast process occupies most of the space within the tubule and coordinates the formation of peritubular dentin.

Microtubules and microfilaments are the principal ultrastructural components of the odontoblast process and its lateral branches.^106,150 Microtubules extend from the cell body out into the process.^106,151 These straight structures follow a course that is parallel with the long axis of the cell and impart the impression of rigidity. Although their precise role is unknown, theories as to their functional significance suggest that they may be involved in cytoplasmic extension, transport of materials, or the provision of a structural framework. Occasionally, mitochondria can be found in the process where it passes through the predentin.

The plasma membrane of the odontoblast process closely approximates the wall of the dentinal tubule. Localized constrictions in the process occasionally produce relatively large spaces between the tubule wall and the process. Such spaces may contain collagen fibrils and fine granular material that presumably represents ground substance. The peritubular dentin matrix within the tubule is lined by an electron-dense limiting membrane called the lamina limitans.^244,333,372 A narrow space separates the limiting membrane from the plasma membrane of the odontoblast process, except for the areas where the process is constricted.

In restoring a tooth, the removal of enamel and dentin often disrupts odontoblasts.* It would be of considerable clinical importance to establish the extent of the odontoblast processes in human teeth, because with this knowledge, the clinician would be in a better position to estimate the impact of the restorative procedure on the underlying odontoblasts. However, the extent to which the process penetrates into dentin has been a matter of considerable controversy. It has long been thought that the process is present throughout the full thickness of dentin. Although ultrastructural studies using transmission electron microscopy have described the process as being limited to the inner third of the dentin,^{51,107,333,372} it should be noted that this could possibly be the result of shrinkage occurring during fixation and dehydration. Other studies employing scanning electron microscopy have described the process extending further into the tubule, often as far as the DEJ,^{121,168,318,381} but it has been suggested that what has been observed in scanning electron micrographs is actually the lamina limitans.^333,334,372

In an attempt to resolve this issue, monoclonal antibodies directed against microtubules were used to demonstrate tubulin in the microtubules of the process. Immunoreactivity was observed throughout the dentinal tubule, suggesting that the process extends throughout the entire thickness of dentin.³¹⁸ However, a study employing confocal microscopy found that odontoblast processes in rat molars do not extend to the outer dentin or DEJ, except during the early stages of tooth development.⁵¹ It is likely that the walls of tubules contain many proteins originally derived from odontoblasts that no longer remain at that site. Because dentin matrix does not turn over, these antigens remain fixed in place. From a clinical perspective, it is important to remember that these processes in the tubules represent appendages from living odontoblasts in the pulp, which explains why the dentin must be considered a vital tissue, the destruction of which will affect the pulp.

The odontoblast is considered to be a fixed postmitotic cell in that once it has fully differentiated, it apparently cannot undergo further cell division. If this is indeed the case, the lifespan of the odontoblast coincides with the lifespan of the viable pulp. However, its metabolic activity can be dynamically altered (described under the heading, Pulpal Repair).

Relationship of Odontoblast Structure to Secretory Function

Studies using radiolabeled chemicals have shed a great deal of light on the functional significance of the cytoplasmic organelles of the active odontoblast.^375,376 In experimental animals, intraperitoneal injection of a collagen precursor (e.g., ³H-proline) is followed by autoradiographic labeling of the odontoblasts and predentin matrix³⁷⁵ (Fig. 12-20). Rapid incorporation of the isotope in the RER soon leads to labeling of the Golgi complex in the area where the procollagen is packed and concentrated into secretory vesicles. Radiolabeled vesicles can then be followed along their migration pathway until they reach the base of the odontoblast process. Here they fuse with the cell membrane and release their tropocollagen molecules into the predentin matrix by the process of exocytosis.

FIG. 12-20 Autoradiograph demonstrating odontoblasts and predentin in a developing rat molar 1 hour after intraperitoneal injection of ³H-proline.

It is now known that collagen fibrils precipitate from a solution of secreted tropocollagen, and that the aggregation of fibrils occurs on the outer surface of the odontoblast plasma membrane. Fibrils are released into the predentin and increase in thickness as they approach the mineralized matrix. Whereas fibrils at the base of the odontoblast process are approximately 15 nm in diameter, fibrils in the region of the calcification front have attained a diameter of about 50 nm.

Similar tracer studies³⁷⁶ have elucidated the pathway of synthesis, transport, and secretion of the predentin proteoglycans. The protein moiety of these molecules is synthesized by the RER of the odontoblast, whereas sulfation and addition of the GAG moieties to the protein molecules take place in the Golgi complex. Secretory vesicles then transport the proteoglycans to the base of the odontoblast process, where they are secreted into the predentin matrix. Proteoglycans, principally chondroitin sulfate, accumulate near the calcification front. The role of the proteoglycans is speculative, but mounting evidence suggests that they act as inhibitors of calcification by binding calcium. It appears that just before calcification, the proteoglycans are removed, probably by lysosomal enzymes secreted by the odontoblasts.⁸³

Pulp Fibroblast

Fibroblasts are the most numerous cells of the pulp. They appear to be tissue-specific cells capable of giving rise to cells that are committed to differentiation (e.g., odontoblast-like cells) if given the proper signal. These cells synthesize types I and III collagen, as well as proteoglycans and GAGs. Thus they produce and maintain the matrix proteins of the ECM. Because they are also able to phagocytose and digest collagen, fibroblasts are responsible for collagen turnover in the pulp.

Although distributed throughout the pulp, fibroblasts are particularly abundant in the cell-rich zone. The early differentiating fibroblasts are polygonal and appear to be widely separated and evenly distributed within the ground substance. Cell-to-cell contacts are established between the multiple processes that extend out from each of the cells. Many of these contacts take the form of gap junctions that provide for electronic coupling or chemical signaling from one cell to another. In terms of ultrastructure, the organelles of the immature fibroblasts are generally in a rudimentary stage of development, with an inconspicuous Golgi complex, numerous free ribosomes, and sparse RER. As they mature, the cells become stellate in form, and the Golgi complex enlarges, the RER proliferates, secretory vesicles appear, and the fibroblasts take on the characteristic appearance of protein-secreting cells. In addition, collagen fibrils accumulate along the outer surface of the cell body. With an increase in the number of blood vessels, nerves, and collagen fibers, there is a relative decrease in the number of fibroblasts in the pulp.

Many fibroblasts of the pulp are characterized by being relatively undifferentiated. A more modern term for undifferentiated cells is stem cells. Many pulpal cells do seem to remain in a relatively undifferentiated modality (compared with fibroblasts of most other connective tissues¹²⁸). This perception has been supported by the observation of large numbers of reticulin-like fibers in the pulp. Reticulin fibers have an affinity for silver stains and are similar to the argyrophilic fibers of the pulp. However, in a careful review, it appears that actual reticulin fibers may not be present in the pulp; instead the previously described fibers are actually argyrophilic collagen fibers.¹⁷ The fibers apparently acquire a GAG sheath, and it is this sheath that is impregnated by silver stains. In the young pulp, the nonargyrophilic collagen fibers are sparse, but they progressively increase in number as the pulp ages.

Many experimental models have been developed to study wound healing in the pulp, particularly dentinal bridge formation after pulp exposure or pulpotomy. One study⁹⁶ demonstrated that mitotic activity preceding the differentiation of replacement odontoblasts appears to occur primarily among perivascular fibroblasts.

Pulpal fibroblasts seem to take active part in signaling pathways in the dental pulp. For example, fibroblast growth and synthesis is stimulated by neuropeptides; in turn, fibroblasts produce NGF and proinflammatory cytokines during inflammation.^32,380,382 NGF plays an important role not only in development but also in regulating neuronal and possibly odontoblast responses to injury via activation of similar neurotrophin receptors expressed on both cell types (see also Plasticity of Intradental Nerves Fibers in this chapter).³⁸⁰

Macrophage

Macrophages are monocytes that have left the bloodstream, entered the tissues, and differentiated into various subpopulations. The different subpopulations can be studied by their antigenic properties in immunohistochemical studies. Many are found in close proximity to blood vessels. A major subpopulation of macrophages is quite active in endocytosis and phagocytosis (Fig. 12-21). Because of their mobility and phagocytic activity, they are able to act as scavengers, removing extravasated red blood cells, dead cells, and foreign bodies from the tissue. Ingested material is destroyed by the action of lysosomal enzymes. Another subset of macrophages participates in immune reactions by processing antigen and presenting it to memory T cells.²⁶⁵ The processed antigen is bound to class II major histocompatibility complex (MHC) molecules on the macrophage, where it can interact with specific receptors present on naïve or memory T cells.¹²⁴ Such interactions are essential for T cell–dependent immunity. Similar to fibroblasts, macrophages take an active part in the signaling pathways in the pulp. When activated by the appropriate inflammatory stimuli, macrophages are capable of producing a large variety of soluble factors, including interleukin 1, tumor necrosis factor, growth factors, and other cytokines. A recent study has shown that a subset of macrophages express lymphatic markers, indicating a link between macrophages and lymphatic function and development.²⁰

FIG. 12-21 Immunoelectron micrograph of an HLA-DR+ matured macrophage (M) in the human pulp, showing a phagosome (P). Ly, Lymphocyte.

Dendritic Cell

Dendritic cells are accessory cells of the immune system. Similar cells are found in the epidermis and mucous membranes, where they are called Langerhans’ cells.^164,264 Dendritic cells are primarily found in lymphoid tissues, but they are also widely distributed in connective tissues, including the pulp³⁰³ (Fig. 12-22). These cells are termed antigen-presenting cells and are characterized by dendritic cytoplasmic processes and the presence of class II MHC complexes on their cell surface (Fig. 12-23). In the normal pulp, they are mostly located in the periphery of the coronal pulp close to the predentin but migrate centrally in the pulp after antigenic challenge.³⁸⁸ They are known to play a central role in the induction of T cell–dependent immunity. Like antigen-presenting macrophages, dendritic cells engulf protein antigens and then present an assembly of peptide fragments of the antigens and MHC class II molecules. It is this assembly that T cells can recognize. Then the assembly binds to a T-cell receptor and T-cell activation occurs (Fig. 12-24). Fig. 12-25 shows a cell-to-cell contact between a dendritic-like cell and a lymphocyte.

FIG. 12-22 Class II antigen-expressing dendritic cells in the pulp and dentin border zone in normal human pulp, as demonstrated by immunocytochemistry. D, Dentin; OB, odontoblastic layer.

FIG. 12-23 Immunoelectron micrograph of a dendritic-like cell (DC) in the human pulp, showing a dendritic profile with a relatively small amount of lysosomal structures.

FIG. 12-24 Function of MHC class II molecule-expressing cells. They act as antigen-presenting cells that are essential for the induction of helper T cell–dependent immune responses.

FIG. 12-25 Immunoelectron micrograph of a cell resembling a dendritic cell and a lymphocyte. They show cell-to-cell contact.

Lymphocyte

Hahn et al.¹²⁴ reported finding T lymphocytes in normal pulps from human teeth. T8 (suppressor) lymphocytes were the predominant T-lymphocyte subset present in these samples. Lymphocytes have also been observed in the pulps of impacted teeth.¹⁹¹ The presence of macrophages, dendritic cells, and T lymphocytes indicates that the pulp is well equipped with cells required for the initiation of immune responses.^164,303 B lymphocytes are scarcely found in the normal pulp.

Mast Cell

Mast cells are widely distributed in connective tissues, where they occur in small groups in relation to blood vessels. Mast cells are seldom found in the normal pulp tissue, although they are routinely found in chronically inflamed pulps.³⁰³ The mast cell has been the subject of considerable attention because of its dramatic role in inflammatory reactions. The granules of mast cells contain heparin, an anticoagulant, and histamine, an important inflammatory mediator, as well as many other chemical factors.

Hyaluronan

Another major structural component of the interstitial matrix is hyaluronan. It is an unbranched, random-coil molecule from repeating nonsulfated disaccharide units and is found in the interstitium as free molecules or bound to cells, possibly via the connection to fibronectin.¹⁹⁴ Its large molecular weight together with its protein structure accounts for its unique properties. It has a high viscosity even at low concentration, exhibits exclusion properties, and has a strong affinity for water.

Hyaluronan is one of several types of GAGs in the pulp.^203,221 The hyaluronan receptor-1 is expressed on lymphatic vessels and also on immune cells in the dental pulp.²⁰ Hyaluronan is removed from the tissue by the lymphatics and metabolized in the lymph nodes⁹⁸ and by endothelial cells in the liver.^126,285

Elastic Fibers

Elastic fibers comprise an elastin core and a surrounding microfibrillar network and provide elasticity to the tissue.²⁷² The amount of elastin in the interstitial matrix in most tissue is small. There is no evidence for elastic fibers in the matrix in the pulp.^126,285

The Inflamed Interstitium

Hyaluronidases, and chondroitin sulfatases of lysosomal and bacterial origin are examples of the hydrolytic enzymes that can attack components of the interstitium. During infection and inflammation, the physical properties of the pulp tissue may then be altered due to production of such degrading enzymes.^133,302 In addition to their own damaging effect, they may also pave the way for the deleterious effects of bacterial toxins, increasing the magnitude of the damage.¹⁶⁶

The pathways of inflammation and infection are strongly influenced by the particular composition of the interstitium in every tissue and its degradation by either host or microbial enzymes.

Tissue Injury and Deafferentation

When a peripheral nerve is cut or crushed, an interruption of the afferent input to the CNS occurs, which is called deafferentation. It would be logical to assume that the result of deafferentation would be anesthesia of the formerly innervated area, but occasionally other symptoms may occur, which may surprisingly include pain. Following nerve injury, a dramatic shift in transcription of neuropeptides, receptors, and sodium channels has been documented. The bidirectional contact between the nerve cell and the peripheral target tissue is lost, and the neurons change into a state of either regeneration or neuronal cell death. The impact on neurons in the trigeminal ganglion is dependent on the injury site. A peripheral injury has less effect than a more centrally located one. However, even a small pulp exposure induces neuronal changes, both in the trigeminal ganglion and at the second-order neuronal level in the brainstem.^46,367 Because each single-rooted tooth contains about 2000 nerve fibers,^160,161 extirpation of the pulp is shown to cause both neurochemical and degenerative changes of their cell bodies in the gasserian (trigeminal) ganglion.^120,174,315 The central projection of these nerves to the spinal nucleus of the trigeminal nerve are also affected,³⁴³ and there is evidence for transsynaptic changes^314,315 that are reflected in the sensory cortex. Even larger responses would be expected from tooth extraction, where both periodontal ligament and pulpal innervation are destroyed.

When an axon is severed peripherally, a complete degeneration of the cell bodies may not always occur.³⁶⁸ Attempted regeneration by axonal sprouting may result in altered expression of various receptors, resulting in sensitivity to norepinephrine (via increased adrenergic receptor activity)²⁶¹ or acetylcholine (via increased cholinergic receptor activity),⁷² sensitizing sensory neurons to autonomic activity. In addition, dorsal horn neurons, deprived of their normal sensory input, may begin to respond to other nearby afferents. Thus normal inhibitory influences are reduced, and a widening of the sensory receptive field is produced, which can produce central sensitization (see Chapter 19). Phantom tooth pain is another term often used synonymously with pain following deafferentation. Different reports suggest an incidence of persistent pain following pulpectomy to be in the range of 3% to 6%.^222,360

Following tissue injury or tissue inflammation, extensive changes occur in the gene expression of sensory ganglion nerves and, by way of transsynaptic mechanisms, in their central projections.^{47,253,254,315} One example is the upregulation of inducible gene transcription factors such as c-fos⁵³ and different subsets of sodium channels.¹¹⁴ This is thought to result in alterations in threshold properties and the size of receptor fields. C-fos is not normally expressed in neurons in the brainstem, but chronic pulpitis causes prolonged increases in c-fos expression is some brainstem neurons.

If such changes also occur in humans, they may help to explain why certain patients may complain of vague, poorly described pain for months following endodontic treatment. If their pulpitis caused sprouting of periapical nerves,^52-54182 these nerves may have been due to the transport of peripheral signaling molecules to the cell body by way of retrograde axoplasmic flow.^45,54 This could induce changes in the expression of many genes, resulting in central senstization^8,44 that may require many months to correct.³²⁷ Reports of nerve sprouting in human inflamed pulps have been confirmed in different studies.^296,297,303 Such reactions might contribute to increases in dentin sensitivity as well as expansion of receptive fields.^253,254,256 Sprouting of sympathetic fibers has also been reported,¹³⁵ but the timing appears to be different. The functional implications and how this is related to pain mechanisms are unknown, but it has been suggested that these reactions are involved in healing and nociception following pulpal inflammation.^111,135,136

Pulp Testing

The electric pulp tester delivers a current sufficient to overcome the resistance of enamel and dentin and stimulate the sensory A fibers at the dentin-pulp border zone. Smaller C fibers of the pulp do not respond to the conventional pulp tester because significantly more current is needed to stimulate them.²⁵² Bender et al.¹⁸ found that in anterior teeth, the optimal placement site of the electrode is the incisal edge, since the response threshold is lowest at that location and increases as the electrode is moved toward the cervical region of the tooth.

Cold tests using carbon dioxide (CO₂) snow or liquid refrigerants and heat tests employing heated gutta-percha or hot water activate hydrodynamic forces within the dentinal tubules, which in turn excite the intradental A fibers. C fibers are generally not activated by these tests unless they produce injury to the pulp. It has been shown that cold tests do not injure the pulp.¹⁰⁴ Heat tests have a greater potential to produce injury, but if the tests are used properly, injury is not likely.

Sensitivity of Dentin

The mechanisms underlying dentin sensitivity have been a subject of interest in recent years. How are stimuli relayed from the peripheral dentin to the sensory terminals located in the region of the dentin-pulp border zone? Converging evidence indicates that movement of fluid in the dentinal tubules is the basic event in the arousal of dentinal pain.^{38,39,249,361,363} It now appears that pain-producing stimuli, such as heat, cold, air blasts, and probing with the tip of an explorer, have in common the ability to displace fluid in the tubules.^38,230 This is referred to as the hydrodynamic mechanism of dentin sensitivity. The hydrodynamic theory suggests that dentinal pain associated with stimulation of a sensitive tooth ultimately involves mechanotransduction. Recently, classical mechanotransducers have been recognized on pulpal afferents, providing a mechanistic support to this theory.¹³⁸ Thus fluid movement in the dentinal tubules is translated into electric signals by receptors located in the axon terminals innervating dentinal tubules. Indeed, there is a positive correlation between rate of fluid flow in the tubules and discharge evoked in intradental nerves,^229,230,363 with outward fluid movements producing a much stronger nerve response than inward movements.²³⁰

In experiments on humans, brief application of heat or cold to the outer surface of premolar teeth evoked a painful response before the heat or cold could have produced temperature changes capable of activating sensory receptors in the underlying pulp.^250,346 The evoked pain was of short duration: 1 or 2 seconds. The thermal diffusivity of dentin is relatively low; yet the response of the tooth to thermal stimulation is rapid, often less than 1 second. Evidence suggests that thermal stimulation of the tooth results in a rapid movement of fluid into the dentinal tubules. This results in activation of the sensory nerve terminal in the underlying pulp. Presumably, heat expands the fluid within the tubules faster than it expands dentin, causing the fluid to flow toward the pulp, whereas cold causes the fluid to contract more rapidly than dentin, producing an outward flow. It is speculated that the rapid movement of fluid across the cell membrane of the axon terminal activates a mechanosensitive receptor, similar to how fluid movement activates hair cells in the cochlea of the ear. All axon terminals have membrane channels through which charged ions pass, and this initial receptor current, if sufficient, can trigger voltage gated sodium channels to depolarize the cell, leading to a barrage of impulses to the brain. Some ion channels are activated by voltage, some by chemicals, and some by mechanical pressure.^230,249,253 In the case of pulpal nerve fibers that are activated by hydrodynamic forces, pressure would be transduced, gating mechanosensitive ion channels.

The dentinal tubule is a capillary tube with an exceedingly small diameter.*¹⁰⁷ The physical properties of capillarity are significant because fluid force increases as the diameter is reduced. If fluid is removed from the outer end of exposed dentinal tubules by dehydrating the dentinal surface with an air blast or absorbent paper, capillary forces produce a rapid outward movement of fluid in the tubule (Fig. 12-37). According to Brännström,³⁸ desiccation of dentin can theoretically cause dentinal fluid to flow outward at a rate of 2 to 3 mm/sec. In addition to air blasts, dehydrating solutions containing hyperosmotic concentrations of sucrose or calcium chloride can produce pain if applied to exposed dentin.

FIG. 12-37 Diagram illustrating movement of fluid in the dentinal tubules resulting from the dehydrating effect of a blast of air from an air syringe.

Investigators have shown that it is the A fibers rather than the C fibers that are activated by hydrodynamic stimuli (e.g., heat, cold, air blasts) applied to exposed dentin.^251,256 However, if heat is applied long enough to increase the temperature of the dentin-pulp border by several degrees Celsius, then C fibers may respond, particularly if the heat produces injury. It seems that the A fibers are mainly activated by a rapid displacement of the tubular contents.²⁴⁹ Slow heating of the tooth produced no response until the temperature reached 111° F (43.8° C), at which time C fibers were activated, presumably because of heat-induced injury to the pulp. These C fibers are called polymodal nociceptors because they contain numerous receptors that confer the ability to detect and respond to many different types of stimuli.^253,254 Capsaicin, the pungent active ingredient in hot peppers, is known to simulate C fibers and a subset of A-delta fibers.¹⁵⁴ Capsaicin activates transient receptor potential, subtype vanilloid 1, (TRPV1).⁵⁹ The TRPV1 receptor is expressed primarily on a major subclass of nociceptors and responds to heat (>110° F [>43° C]), certain inflammatory mediators, and acid (pH < 6). Thus TRPV1 has been considered a molecular integrator of polymodal noxious stimuli.²⁶⁰ The ability of the TRPV1 antagonist, capsazepine, to inhibit acid-, heat-, and capsaicin-activated trigeminal neurons²⁰⁶ has led to the development of new drugs (i.e., TRPV1 antagonists) for the treatment of pulpal pain. Eugenol is known to activate and ultimately desensitize TRPV1, and this may explain the anodyne action of zinc oxide eugenol temporary restorations.³⁸⁴

It has also been shown that pain-producing stimuli are more readily transmitted from the dentin surface when the exposed tubule apertures are open^148,162 and the fluid within the tubules is free to flow outward.^162,230,363 For example, acid treatment of exposed dentin to remove the smear layer opens the tubule orifices and makes the dentin much more responsive to stimuli such as air blasts and probing.^148,254

Perhaps the most difficult phenomenon to explain is dentinal pain associated with light probing of dentin. Even light pressure of an explorer tip can produce strong forces.* These forces have been shown to mechanically compress dentin and close open tubule orifices with a smear layer that causes sufficient displacement of fluid to excite the sensory receptors in the underlying pulp (Fig. 12-38).^55,56 Considering the density of the tubules in which hydrodynamic forces would be generated by probing, thousands of nerve endings would be simultaneously stimulated when a dental explorer is scratched across dentin.

FIG. 12-38 Scanning electron micrograph of a shallow groove (between white arrowheads) created in polished dentin by a dental explorer tine under a force of 30 g (30 cN). Note the partial occlusion of the tubules by smeared matrix.

(From Camps J, Salomon JP, Meerbeek BV, et al: Dentin deformation after scratching with clinically relevant forces. Arch Oral Biol 48:527, 2003.)

Another example of the effect of strong hydraulic forces that are created within the dentinal tubules is the phenomenon of odontoblast displacement. In this reaction, the nuclei and cell bodies of odontoblasts are displaced upward in the dentinal tubules, presumably by a rapid movement of fluid in the tubules produced when exposed dentin is desiccated, as with the use of an air syringe or cavity-drying agents (Fig. 12-39).^76,192 Such cellular displacement results in the destruction of odontoblasts, because cells thus affected soon undergo autolysis and disappear from the tubules. Displaced odontoblasts may eventually be replaced by stem cells that migrate from the cell-rich zone of the pulp, as discussed later in the chapter.

FIG. 12-39 Odontoblasts (arrows) displaced upward into the dentinal tubules.

The hydrodynamic theory can also be applied to an understanding of the mechanism responsible for hypersensitive dentin.^37,39 There is controversy regarding whether exposed dentin is simply sensitive or becomes truly hypersensitive.^253,254,256 Growing evidence indicates that new sodium channels responsible for activating nerves are expressed in nerve tissue exposed to inflammation.^114,129,293 An increase in the density of sodium channels or their sensitivity may contribute to dentinal hypersensitivity. Hypersensitive dentin is also associated with the exposure of dentin normally covered by cementum or enamel. The thin layer of cementum is frequently lost as gingival recession exposes cementum to the oral environment. Cementum is subsequently worn away by brushing, flossing, or using toothpicks. Once exposed, the dentin may respond to the same stimuli to which any exposed dentin surface responds (e.g., mechanical pressure, dehydrating agents). Although the dentin may at first be very sensitive, within a few weeks the sensitivity usually subsides. This desensitization is thought to occur as a result of gradual occlusion of the tubules by mineral deposits, thus reducing hydrodynamic forces. In addition, deposition of reparative dentin over the pulpal ends of the exposed tubules probably also reduces sensitivity, because reparative dentin is less innervated by sensory nerve fibers.⁴⁹ However, some hypersensitive dentin does not spontaneously desensitize, so hypersensitivity may be caused by either inflammatory changes in the pulp or mechanical changes in the patency of dentinal tubules.

Currently the treatment of hypersensitive teeth is directed toward reducing the functional diameter of the dentinal tubules to limit fluid movement. Four possible treatment modalities^271,348,365 can accomplish this objective:

Dentin sensitivity can be modified by laser irradiation, but clinicians must be concerned about its effect on the pulp.^326,336

Neuropeptides

Of immense importance in pulp biology is the presence of neuropeptides in pulpal nerves.^45,48,52,327 Pulpal nerve fibers contain neuropeptides such as calcitonin gene–related peptide (CGRP) (see Figs. 12-31, 12-33, 12-34),⁴⁴ substance P (SP),^253,268 neuropeptide Y (see Fig. 12-29), neurokinin A (NKA),¹⁴ and VIP.^213,365 In rat molars, the largest group of intradental sensory fibers contains CGRP. Some of these fibers also contain other peptides, such as SP and NKA.^45,47 Release of these peptides can be triggered by numerous stimuli, including tissue injury,¹⁴ complement activation, antigen-antibody reactions,²⁶⁴ or antidromic stimulation of the inferior alveolar nerve.^268,269 Once released, vasoactive peptides produce vascular changes that are similar to those evoked by histamine and bradykinin (i.e., vasodilation).²⁶⁹ In addition to their neurovascular properties, SP and CGRP contribute to inflammation and promote wound healing.^32,344 The release of CGRP can be modified by sympathetic agonists and antagonists,^111,130 offering the promise of using such agonists to treat dental pain. This latter point is important because sympathetic agonists are used by clinicians every day—the vasoconstrictors present in local anesthetic solutions may have direct effects on inhibiting dental nerve activity. Local anesthetic reduction in pain may be due to the actions of both the local anesthetic and the vasoconstrictor on the nerves.^129,130 In cats, capsaicin acutely activates and chronically blocks the TRPV1-expressing classes of C and A-delta nociceptors in the pulp.¹⁵⁴ In addition, the chronic application of capsaicin ointment to skin has been shown to relieve pain in patients, suggesting that clinical trials evaluating chronic application of capsaicin for treating pulpal or periradicular pain may be of value.

Antidromic stimulation of nerves (i.e., towards the peripheral terminals) simply means that the afferent barrage goes in the opposite direction of the orthograde stimulation (towards the CNS). Normally, sensory nerves are stimulated at their peripheral terminations, and their action potentials then travel toward the brain. In antidromic nerve stimulation, the sensory nerve is usually cut, then the peripheral end of the nerve is electrically stimulated. This causes an action potential to travel backward toward the periphery, which causes release of neuropeptides in the pulp.²⁶⁸ All branches of the nerve also depolarize and release neuropeptides (the so-called axon reflex).³⁰⁶

It has been reported^230,268 that mechanical stimulation of dentin produces vasodilation within the pulp, presumably by causing the release of neuropeptides from intradental sensory fibers.²⁶⁸ Electric stimulation of the tooth has a similar effect.¹⁴² The pulpal concentrations of CGRP, SP, and NKA are elevated in painful human teeth over healthy control teeth extracted for orthodontic treatment.¹⁴ These peptides are also elevated in pulps beneath advancing carious lesions.^295,296

Plasticity of Intradental Nerve Fibers

It has become apparent that the innervation of the tooth is a dynamic situation in which the number, size, and cytochemistry of nerve fibers can change because of aging,^99,207,327 tooth injury,^45,47,48,52 and dental caries.²⁹⁶ For example, in rats, nerve fibers sprout into inflamed tissue surrounding sites of pulpal injury, and the content of CGRP and SP increases in these sprouting fibers.^{45,47,48,52,54} When inflammation subsides, the number of sprouts decreases. Fig. 12-40 compares the normal distribution of CGRP-immunoreactive sensory fibers in an adult rat molar with those beneath a shallow cavity preparation. The innervational pattern in normal and inflamed teeth is governed by neuronal growth factors. Neurotrophic and target-derived factors regulate neuronal structure, survival, and function and are important for the maintenance of neuronal phenotype characteristics. During development, all dental fibers appear to require nerve growth factor (NGF) and express its receptor, TrkA, at some stages,²²⁷ whereas in adult teeth the large trigeminal neurons are potentially dependent only on dental pulp–derived, glial cell line–derived neurotrophic factor (GDNF); the smaller trigeminal neurons remain dependent on NGF.^190,289 This suggests that GDNF may function as a neurotrophic factor for the subset of larger nerves in the tooth, which apparently mediate mechanosensitive stimuli, whereas NGF is suggested to support neurons responsible for nociception.²⁴³ NGF is the most extensively investigated among the trophic factors.¹⁹⁸ Binding of target-derived NGF is dependent on specific TrkA receptors located on the axonal surface, with subsequent internalization and transport to the cell body, where the effects are mediated.

FIG. 12-40 A, Normal distribution of calcitonin gene–related peptide (CGRP)-immunoreactive sensory fibers in adult rat molar. Nerve fibers typically are unbranched in the root (R), avoid interradicular dentin (ir), and form many branches in coronal pulp (C) and dentin (D). Nerve distribution is often asymmetric, with endings concentrated near the most columnar odontoblasts (in this case on the left side of the crown). When reparative dentin (rd) forms, it alters conditions so that dentinal innervation is reduced. B, Shallow class I cavity preparation on the cervical root of a rat molar was made 4 days earlier. Primary odontoblast (O) layer survived, and many new CGRP-immunoreactive terminal branches spread beneath and into the injured pulp and dentin. Terminal arbor can be seen branching (arrowhead) from a larger axon and growing into the injury site. Scale bar: 0.1 mm. A, ×75. B, ×45.

(From Byers MR: Dynamic plasticity of dental sensory nerve structure and cytochemistry. Arch Oral Biol 39[suppl]:13S, 1994.)

Regulation of neural changes during inflammation seems to be a function of NGF expression.^50,54 NGF receptors are found on intradental sensory fibers and Schwann cells.⁴⁷ Evidence indicates that NGF is synthesized by fibroblasts in the coronal subodontoblastic zone (i.e., cell-rich zone), particularly in the tip of the pulp horn.⁴⁷ Maximal sprouting of CGRP- and SP-containing nerve fibers corresponds to areas of the pulp where there is increased production of NGF.⁵⁴ Fig. 12-41 shows the expression of NGF-mRNA in a pulp horn subjacent to cavity preparation.

FIG. 12-41 NGF-mRNA is upregulated in the mesial pulp horn 6 hours after cavity preparation.

(From Pimental E: Handbook of growth factors, vol 3, London, Taylor & Francis, 1994).

It has been suggested that neuroimmune interactions take place in the dental pulp, since a coordinated increase of pulpal nerves and immune cells has been demonstrated.^164,303 In addition, recruitment of immunocompetent cells has been demonstrated in the dental pulp after electrical tooth stimulation.^66,103 Similar responses have been seen in periapical bone in rats with radicular pulpitis.¹⁸² Neurogenic inflammation is generally thought to enhance healing, because denervated teeth show poorer healing following pulp exposures than innervated teeth.^45,53