Читать книгу Oral Cells and Tissues - Philias R. Garant - Страница 9

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Dentin is deposited by odontoblasts that develop ectomesenchymal cells of the dental papilla on contact with the basal lamina formed by the inner enamel epithelium.

Differentiation of Odontoblasts

Odontoblast precursors migrate into the developing jaw from the neural crest as part of a large population of ectomesenchymal cells that participate in the formation of many parts of the face and oral cavity. During the cap stage of tooth formation, the preodontoblasts concentrate adjacent to the inner enamel epithelium (IEE) of the enamel organ. Preodontoblasts exit the cell cycle and differentiate before the preameloblasts of the IEE have stopped dividing.¹^,²

Contact with the IEE basement membrane and/or with other associated extracellular material of epithelial origin has long been held to be a requirement for initial odontoblastic differentiation.³ Recent experiments suggest that a fibronectin-rich substratum is a key requirement.⁴ Early studies implicating the importance of the basement membrane in odontoblast differentiation were reviewed by Ruch¹^,² and Ruch et al.⁵

Aperiodic fibrils are key structures regulating the differentiation of odontoblasts. They are deposited first at the tip of the future cusp, and then apically toward the cervical border of the developing tooth. Shortly after the first aperiodic fibrils form, the preodontoblasts bind to them through leading-edge cytoplasmic processes (see Fig 1-8).³^,⁶^,⁷ As leading-edge contacts increase in number, the preodontoblasts are immobilized across the basal lamina from the cells of the IEE. Polarity toward the basal lamina is established at this time.⁸^,⁹ Odontoblast differentiation in organ culture fails when the basement membrane is removed by prior incubation in trypsin.³

Electron microscopy reveals that aperiodic fibrils, about 15 nm wide and 1.0 to 2.0 μm long, are attached to the basal lamina beneath the IEE. Fluorescent antibodies to collagen types I, III, IV, and VI, tenascin, proteoglycan, and fibronectin bind to basement membrane molecular components in the same location, suggesting that the aperiodic fibrils may consist of one or more of these matrix proteins.¹⁰^–¹⁶ Similar patches of extracellular matrix have been observed adjacent to the plasma membrane of preodontoblasts.¹²^,¹⁷

Fibronectin receptors (165-kDa protein) are present in the leading-edge plasma membrane of preodontoblasts during differentiation and stabilization. Adherence of a cell surface 165-kDa fibronectin receptor appears to stabilize cytoskeletal elements, promote preodontoblast polarization, and trigger other cytoplasmic processes associated with differentiation.¹⁸^,¹⁹ Attachment to fibronectin leads to its uptake and removal at the leading edge of the differentiating odontoblast.

Transforming growth factor β1 (TGF-β1), a growth factor that binds to fibronectin, is a well-known inhibitor of cell proliferation and a promoter of odontoblast differentiation and matrix synthesis. Thus, one important function of fibronectin may be to serve as a reservoir for growth factors that cause preodontoblasts to exit the cell cycle and to undergo differentiation. The importance of fibronectin in dentinogenesis is underscored by the observation that cells of the dental papilla can differentiate into odontoblast-like cells when grown in contact with a supporting surface that is rich in fibronectin and other soluble dentin matrix components.⁴^,²⁰

Odontoblasts sequentially express several members of the TGF-β superfamily of growth factors and their receptors.²¹ During normal development, TGF-β1 is expressed in the IEE before and during odontoblast polarization. Differentiated odontoblasts express receptors for TGF-β1 and secrete TGF-β1 into the dentin matrix.²² Loss-of-function mutations in the Tgf-β1 gene in mice cause dentin and pulpal pathoses.²³ Evidence accumulated over nearly 2 decades suggest that spatial and temporal interactions between cell surface receptors and extracellular matrix molecules and growth factors, such as fibronectin and TGF-β1, provide the necessary information to coordinate odontoblast differentiation.

It has been suggested that the entry of calcium ions might act as a signal for mediating restructuring of the cytoskeleton during the establishment of odontoblast shape and polarity toward the IEE. Cell membrane ligand-gated calcium channels have been localized to the apical pole of the preodontoblasts (nearest the basement membrane).²⁴

In addition to the potential signaling effects of calcium, fibronectin, and TGF-β, there is evidence to suggest that enamel matrix proteins may serve a similar instructional role during odontoblast differentiation. The expression of enamel proteins in the IEE begins before the cells have acquired the secretory ameloblast phenotype. Electron microscopic studies have identified the presence of enamel matrix protein across the basal lamina in close contact with the apical pole of the developing odontoblasts.¹⁶^,²⁵ The enamel proteins, identified by antiamelogenin antibodies, are endocytosed in coated vesicles at the odontoblast cell surface.¹⁶^,²⁶ The potential instructive role, if any, for these enamel proteins in regulating odontoblast development is unclear.

Secretion of Dentin Matrix

Subsequent to odontoblast differentiation, the basal lamina is degraded. Application of in situ hybridization techniques has shown that preameloblasts and preodontoblasts express matrix metalloproteinase 2, an enzyme that degrades collagen IV and fibronectin, coincident with the removal of the basal lamina.¹⁴ Evidence from electron microscopy suggests that the preameloblasts of the IEE phagocytose the partially degraded basal lamina.

After the breakup of the basal lamina, heterotypic cell-to-cell contacts form between cell processes of the newly differentiated odontoblasts and the distal ends of the preameloblasts. Although it was speculated that such contacts might allow the transmission of informational messages needed for differentiation, there has never been any evidence presented that functional gap junctional contacts exist between these two cell types.

In contrast, stable gap junctions and macula adherens–type junctions develop between adjacent odontoblasts during aggregation (see chapter 1).⁸^,²⁷^–³⁰ Coordination of dentin matrix secretion may require communication across gap junctions, permitting ions and small metabolites to cross from odontoblast to odontoblast. Soon after alignment of the odontoblasts, a junctional complex consisting of fascia adherens and fascia occludens forms in the distolateral cell membranes. The fascia adherens is associated with a highly developed terminal web of cytoplasmic filaments.⁸ The tight junctions of the fascia occludens do not form a zonula occludens.²⁷

Concomitant with the onset of dentin matrix secretion, odontoblasts grow in length and develop large amounts of rough endoplasmic reticulum (RER). A prominent Golgi complex develops in the supranuclear cytoplasm facing the IEE. In addition to increased expression of messenger ribonucleic acid (mRNA) for collagen type I, developing odontoblasts also express mRNA for osteocalcin, dentin phosphophoryn, and high levels of alkaline phosphatase.³¹^,³² As synthesis of type I collagen increases, the expression of type III collagen decreases in odontoblasts. Dentin matrix contains type I collagen and a variety of glycoproteins and glycosaminoglycans.³³^,³⁴

The earliest layer of dentin to form is called mantle dentin (Fig 2-1). The collagen fibers of the mantle dentin are thicker than those that form later in circumpulpal dentin. In coronal dentin, the collagen fibers of mantle dentin are polymerized perpendicular to the dentinoenamel junction, while the fibers of the circumpulpal dentin form approximately parallel to the dentinoenamel junction.

Fig 2-1 Components of dentin. The outermost layer of dentin is the mantle dentin (MD). It is deposited during the early stage of odontoblast development. With the appearance of the odontoblastic process, the major portion of dentin, the circumpulpal dentin (CD), is deposited. It consists mainly of intertubular dentin (ITD) and narrow bands of peritubular dentin (PTD) surrounding the dentinal tubule (DT). (E) Enamel; (D) dentin; (P) pulp.

As dentin matrix is deposited, the odontoblast cell body is pushed backward away from the dentin surface. A single dominant cytoplasmic process, the odontoblastic process, forms during the retreat of the cell. It remains embedded in the dentin, undergoing elongation as more dentin matrix is deposited (Fig 2-2). With the appearance of the odontoblastic process, formation of circumpulpal dentin begins.

Fig 2-2 Mature secretory odontoblast. (D) Dentin; (N) nucleus; (PD) predentin matrix; (FA) fascia adherens; (FO) fascia occludens; (RER) rough endoplasmic reticulum; (TW) terminal web; (Fb) fibroblast.

Structure of Mature Secretory Odontoblasts

Fully differentiated odontoblasts are tall columnar cells, 50 to 60 μm in length, characterized by a highly polarized distribution of cytoplasmic organelles (Figs 2-2 and 2-3).⁸^,³⁵^,³⁶ For descriptive purposes, it is convenient to divide the odontoblast into two parts, the cell body and the odontoblastic process. The terminal web of cytoplasmic filaments, associated with fascia adherens junctions, provides a line of demarcation between the cell body and the odontoblastic process (see Fig 2-2). Mature odontoblasts are aligned as a single layer of columnar cells, but when crowded, as in the pulp horns or in the most incisal portion of the rodent incisor, odontoblasts assume a pseudostratified organization (see Fig 2-3).

Fig 2-3 Cross section of a rat incisor, illustrating mature secretory odontoblasts. (BV) Blood vessels; (CR zone) cell-rich region of the pulp containing numerous fibroblasts; (D) dentin; (OP) odontoblastic process; (PD) predentin. (Epon section [1 μm] stained with toluidine blue; original magnification × 260.)

Odontoblasts are joined and attached at their distal extremities by well-developed terminal webs and associated fascia adherens junctions (see Fig 2-2).⁸ Physical evidence of the strength of this bond is provided by the fact that the odontoblastic layer can be isolated relatively intact after demineralization and digestion of the dentin matrix. When observed macroscopically and histologically, the terminal web apparatus appears to form a continuous membranous structure. Early histologists called it the pulpodentinal membrane. This zone of attachment prevents the entrapment of odontoblasts in the predentin matrix and ensures that the developing surface of dentin remains relatively flat.

Although physiologic evidence suggests that a paracellular barrier to calcium exists at the distal end of the cell, no zonula occludens junction is present. Morphologic studies have revealed only a partial (fascia) occludens junction at that site. Gap junctions are formed between adjacent odontoblasts and between odontoblasts and the fibroblasts of the subodontoblast-rich zone.²⁹^,³⁷^,³⁸

The narrow intercellular spaces between adjacent odontoblasts contain collagen fibers, aperiodic microfibrils, proteoglycans, and fibronectin.¹⁵^,³⁹^–⁴³ These intercellular fibers (von Korff fibers) follow a spiral pathway through the interodontoblastic space, passing into the predentin between adjacent odontoblasts at interruptions of the fascia occludens and fascia adherens junctions.

During odontoblast differentiation, the RER and the Golgi complex undergo hypertrophy in preparation for protein secretion. The nucleus is restricted to the pulpal end of the cell body and is characterized by an abundant euchromatic matrix, prominent nucleoli, and many nuclear pores (see Figs 2-2 and 2-3). The RER is the major cytoplasmic organelle within active odontoblasts. Parallel cisterns of RER occupy the supranuclear cytoplasm, the borders of the Golgi complex, and the cytoplasm proximal to the terminal web (see Fig 2-2).⁸^,⁴⁴^–⁴⁶ Mitochondria are dispersed throughout the cell body.

The Golgi complex, containing aggregates of smooth-walled vesicles and cisterns, occupies a central location (see Fig 2-2).⁶^,⁴⁵^–⁴⁷ Each stack of Golgi cisterns displays morphologic and functional polarity, with a forming face (the convex surface) and a mature face (the concave surface). The forming face develops from, and is continuously replenished by, fusion of small intermediate (transport) vesicles originating from the RER. Presecretory granules containing type I procollagen, glycoproteins, and glycosaminoglycans develop from the cisterns of the mature face of the Golgi apparatus.³⁵^,³⁶^,⁴⁸ Phosphophoryns appear to be packaged in small, narrow vesicles.⁴⁹ The complex cytoplasmic machinery operating in the Golgi complex for targeting secretory proteins to their appropriate final destination is briefly discussed later in the chapter, in the “Basic Science Correlation” section.

After their release from Golgi cisterns, the presecretory granules of the dentin matrix undergo condensation to form smaller secretory granules, approximately 300 nm long and 30 nm wide.⁸^,³⁵ The long axis of the secretory granule is roughly equal to the length of a type I procollagen molecule (about 280 nm long). The diameter of the granule is wide enough to contain many procollagen molecules, packaged side by side.

An essential component of the secretory machinery of the odontoblasts is its network of microtubules.⁵⁰ Interference with the assembly of microtubules prevents the migration of secretory granules from the Golgi complex to the secretory pole of the odontoblast.⁵¹^–⁵³ The cytosolic motor-protein kinesin, using adenosine triphosphate (ATP) as an energy source, interacts with microtubules and the membranes of secretory granules to propel the secretory granules in an anterograde direction toward the secretory pole of the cell. Similar interactions between microtubules and cytoplasmic motor-proteins are involved in maintaining the organization of the Golgi complex and the polarized distribution of cytoplasmic organelles. Lysosomes and acid phosphatase are also present in mature odontoblasts, especially prominent in the distal portion of the cell body near the predentin.⁵⁴^,⁵⁵

During formation of primary dentin, the internal perimeter of the pulp becomes smaller, forcing the odontoblasts into a pseudostratified organization. With further deposition of secondary dentin, some odontoblasts undergo programmed cell death. It has been reported that half of the odontoblasts in human premolar teeth are lost over 4 years.⁵⁶

Dentin matrix is deposited in incremental amounts in a daily (circadian) biologic rhythm. These microscopic increments are visible in dentin as stripes running parallel to the mineralization front. In human dentin, the daily increment is about 4 μm wide. Additional periodicity occurs at roughly 5-day intervals, producing the lines of Von Ebner, spaced about 20 μm apart. Circadian rhythms may contain further oscillations, which produce ultradian increments. In dentin from rodent incisors, three ultradian lines are spaced about 8 μm apart within the wider 20-μm circadian incremental lines.⁵⁷

Various explanations have been put forth to explain these rhythmic patterns of matrix deposition. Feeding and/or sleeping patterns were originally suggested to be the most likely causes of variation in secretory function. Fluctuating levels of hormones and growth factors regulated by central neural activity are the probable cause of these patterns.

Mature odontoblasts express parathyroid hormone receptors. Parathyroid hormone has an anabolic effect on odontoblasts, increasing the level of cyclic adenosine monophosphate and alkaline phosphatase.⁵⁸

Composition of the Dentin Matrix

The organic matrix of dentin contains collagen, non-collagenous proteins (proteoglycans, phosphophoryns, and glycoproteins), phospholipids, and growth factors.

Collagen

Type I collagen is the major protein of dentin matrix. Lesser amounts of types III, V, and VI collagen are also found in dentin matrix.

Electron microscopic autoradiographic studies with tritiated proline and immunocytochemical studies have shown that the procollagen of dentin matrix is secreted mainly from the predentinal segment of the odontoblastic process (Figs 2-4 to 2-6).³⁵^,⁴⁹ Tritiated proline–labeled granules accumulate in the distal part of the cell body and are discharged by a process of merocrine exocytosis. A smaller number of labeled secretory granules are present in more distal parts of the process beyond the predentin. Presumably they are secreted at sites distal to the mineralization front.

Fig 2-4 Interaction of odontoblast secretory products in predentin, dentin, and the mineralization front. Phosphate ions in phosphophoryns sequester calcium and initiate the growth of hydroxyapatite crystals. The linkage of phosphophoryns and collagen leads to deposition of mineral along the collagen fibrils. A portion of the proteoglycans are degraded and removed from the predentin before mineralization of the collagenous matrix. Growth factors (bone morphogenetic protein 2 [BMP-2] and transforming growth factor β [TGF-β]) are retained in the matrix. (NCPs) Noncollagenous proteins. (Adapted from Veis.²²⁶)

Following exocytosis of procollagen into the extracellular space, neutral proteinases remove the terminal amino and carboxy propeptides of the procollagen molecules, permitting collagen molecules to assemble into 64-nm banded fibrils of the predentin and dentin matrix (see chapter 6). Predentin matrix remains unmineralized for several days following its secretion. Typically, a layer of unmineralized predentin, approximately 10 to 20 μm thick, separates mineralized dentin from the cell body of the odontoblast (see Figs 2-2 and 2-3). A widened predentin layer is usually a sign of abnormal mineral metabolism and/or matrix mineralization.

Fibronectin is also found in association with collagen fibrils in the predentin. Tissue inhibitor of matrix metalloproteinase 1, another secretory product of odontoblasts, is found in high concentration in predentin.⁵⁹

Figs 2-5a to 2-5d Light microscopic autoradiographs of the utilization of tritiated proline (reflecting collagen synthesis) by odontoblasts at various time periods after intravenous injection. (Original magnification × 500.)

Fig 2-5a At 10 minutes, the label is at the cell periphery. (PD) Predentin; (O) odontoblast.

Fig 2-5b At 20 minutes, the Golgi complex is heavily labeled. (arrowheads) Odontoblastic process; (*) approximate location of Golgi; (D) dentin.

Fig 2-5c At 30 minutes, the grains are mostly over the odontoblastic process (arrowheads).

Fig 2-5d At 2 hours, most of the radioactivity is now in the predentin.

Fig 2-6a Scanning electron microscopic view of the surface of predentin after the pulp and odontoblasts have been stripped away. The oval depressions represent the spaces (dentinal tubules) previously occupied by the odontoblastic processes. Note the uniform diameter (about 3 μm) of the collagen fibrils and their orientation around and perpendicular to the long axis of the dentinal tubule. (Adapted from Tabata et al⁴³ with permission from John Wiley & Sons. Original magnification × 18,000.)

Fig 2-6b Higher magnification of the wall of the tubule in the predentin. No lamina limitans is present in predentin. Note the compact and woven arrangement of the collagen fibrils. These fibrils constitute the collagenous component of the intertubular dentin. (Adapted from Tabata et al⁴³ with permission from John Wiley & Sons. Original magnification × 31,000.)

Noncollagenous proteins

Odontoblasts secrete noncollagenous proteins consisting of proteoglycans, phosphophoryns, and glycoproteins (see Fig 2-4). Electron microscopic autoradiographic localization of sulfate 35 and tritiated fucose have shown that the proteoglycan and glycosaminoglycan components of the matrix are concentrated at the mineralization front.²⁹^,³⁸^,⁴⁸^,⁶⁰^,⁶¹

Biochemical and immunohistochemical studies indicate that there are specific differences in proteoglycan composition between predentin and dentin.⁶²^,⁶³ Because proteoglycans interact with collagen during fibril formation, a function of predentin proteoglycans might be to regulate the size and orientation of dentin collagen fibers. It has also been suggested that predentin proteoglycans might control the timing and site of mineralization, either by sequestering calcium or by shielding potential mineral nucleation sites in the matrix. Evidence that some proteoglycans are degraded near the mineralization front by proteoglycanases and metalloproteinases supports the idea that some proteoglycans may indeed inhibit mineralization of the dentin matrix.⁶⁴^,⁶⁵

Decorin, a chondroitin–dermatan sulfate proteoglycan with binding affinity for type I collagen, is found in dental pulp, odontoblasts, at the mineralization front, and along the mineralized walls of the dentinal tubules.⁶⁶ In contrast, decorin is conspicuously absent from predentin.

Porcine predentin matrix contains active neutral metalloproteinases (56- and 61-kDa gelatinases and 25-kDa proteoglycanase) capable of degrading proteoglycans at the mineralization front.⁶⁴^,⁶⁵ The activity of these enzymes is calcium dependent. A mechanism must exist to regulate the availability of calcium for enzyme activation and matrix mineralization at the mineralization front. Endocytosis of proteoglycan degradation products, and membrane retrieval by coated vesicles, occurs in the proximal part of the odontoblastic process.

Not all of the matrix proteoglycans are degraded prior to mineralization. Chondroitin-6-sulfate, chondroitin-4-sulfate, and hyaluronate, associated with core proteins, have been extracted from demineralized dentin. Five dentin proteoglycans, ranging in size from 100 to 400 kDa and rich in chondroitin-4-sulfate, have been partially characterized by Steinfort et al.⁶³

Three noncollagenous proteins, dentin phosphophoryn (DPP), dentin matrix protein 1 (DMP1), and dentin sialoprotein (DSP), all contained in dentin matrix, appear to be specifically associated with biomineralization. The role of noncollagenous proteins in dentin formation has been the subject of recent reviews.⁶⁷^,⁶⁸

Dentin phosphophoryn is the major noncollagenous component of dentin. Immunocytochemical studies indicate that phosphophoryns are localized in small granules distinct from larger collagen-containing secretory granules. The DPPs are secreted from the odontoblastic process at the mineralization front.⁴⁹ Mineral crystal nucleation is attributed to DPP, a protein rich in aspartic acid and serine residues. Biochemical studies indicate that the DPPs are covalently linked to specific sites on the collagen fibrils of dentin.⁶⁹^–⁷¹ The serine residues of DPPs are phosphorylated by casein kinase in the extracellular space prior to mineralization.⁷²^,⁷³ Because of their many phosphate groups, and their capacity to bind calcium, DPPs create a template for calcium and phosphate concentration, and thereby drive crystal nucleation (see Fig 2-4).⁷⁴^,⁷⁵

An acidic phosphoprotein, DMP1 is localized to mature odontoblasts, cementoblasts, and osteoblasts.⁷⁶^–⁷⁸ It is not expressed in the enamel organ and pulp. The precise role of DMP1 has yet to be identified. The gene coding for DMP1 has been localized to human chromosome 4q21, a region implicated in the autosomal-dominant form of dentinogenesis imperfecta type II.⁷⁹ Teeth affected by this disease are characterized by discolored and abnormally soft dentin and by fewer and irregular dentinal tubules.⁸⁰

Dentin sialoprotein is a sialic acid–rich glycoprotein that is expressed early in tooth development, prior to degradation of the basement membrane. The mRNA for DSP has been detected in preameloblasts and preodontoblasts, suggesting that it may have a signaling role during ameloblast and odontoblast differentiation.⁸¹^,⁸²

Both DSP and DPP are transcribed from a single mRNA, coded by a gene on human chromosome 4, and coexpressed during tooth development.⁸³^,⁸⁴ Both proteins are expressed in preodontoblasts and odontoblasts throughout dentin matrix production. Preameloblasts also express DSP and DPP until enamel secretion, at which point mRNA for DSP and DPP is no longer detected in ameloblasts.

Additional glycoproteins, such as osteocalcin and thrombospondin 1, are found in dentin. Osteocalcin, a glycoprotein rich in glutamic acid, is present in odontoblasts and dentin matrix.³¹^,⁸⁵ In bone, osteocalcin is a chemotactic factor for osteoclasts. Thrombospondin 1 is present in high levels within predentin, especially near the mineralization front.⁸⁶ Thrombospondin 1 mRNA is localized in odontoblasts but not in the cells of the dental pulp.

Phospholipids

Cytochemical and autoradiographic studies have demonstrated the presence of phospholipids in predentin and dentin matrix. Because they are closely associated with hydroxyapatite crystals at the mineralization front, it has been speculated that they may have a role in mineral nucleation.⁸⁷

Growth factors

Bone morphogenetic proteins and TGF-β have been isolated from demineralized dentin matrix.⁸⁸ It has been suggested that they may trigger the differentiation of new odontoblasts during the induction of reparative dentin. Transforming growth factor β may be retained in the dentin matrix because of its ability to bind to decorin proteoglycan. Odontoblasts express high levels of TGF-β and its receptor.²²^,⁸⁹ In addition, TGF-β promotes matrix production in most connective tissue cell types. Mutations in TGF-β genes lead to severe pulpal inflammation and attrition of occlusal surfaces.²³

Mineralization of Mantle and circumpulpal Dentin

Two mechanisms for initiating crystal nucleation are responsible for mineralization of the dentin matrix: matrix vesicles in mantle dentin and collagen-phosphophoryn complexes in circumpulpal dentin.

Matrix vesicles

Matrix vesicles in mantle dentin are similar to those first described in mineralizing cartilage.⁹⁰^,⁹¹ Matrix vesicles in mantle dentin are believed to bud from the tips of odontoblast cytoplasmic processes.

Matrix vesicles initiate mineralization by concentrating calcium and phosphate ions.⁹² Adenosine triphosphatase (ATPase) activity in matrix vesicle membranes may concentrate ions across the limiting membrane prior to nucleation.⁹³ As the ion concentration increases, hydroxyapatite crystallizes along the inner surface of the matrix vesicle membrane. Calcium-binding phospholipids in the limiting membrane may serve as templates for hydroxyapatite precipitation.

Elemental analysis of freeze-dried matrix vesicles in mantle predentin indicates that earliest mineral deposits appear as dotlike nuclei inside the vesicles.⁹⁴ Continued crystal growth leads to vesicle rupture, with release of hydroxyapatite crystals into the extracellular matrix. The newly formed crystals act as seeds for continued mineralization of mantle dentin matrix.

After mineralization is initiated, there appears to be no further need for matrix vesicles. Thus matrix vesicles do not participate in mineralization of late mantle dentin and circumpulpal dentin.⁹⁵ Matrix vesicles are also discussed in chapter 12.

Collagen-phosphophoryn complexes

Mineralization of circumpulpal dentin is initiated by phosphophoryns, independent of matrix vesicle activity.⁹⁶ Extracellular kinases have been identified in dental matrix.⁷³ These enzymes phosphorylate dentin phosphophoryn. Dentin phosphophoryns, linked to collagen fibrils, act as nucleators of hydroxyapatite crystals in late mantle dentin and circumpulpal dentin. The steric arrangement of negative charges on the phosphophoryns creates a template for hydroxyapatite deposition.⁹⁷

Morphologic and histochemical studies of the predentin-dentin border in teeth that have been preserved by rapid pressure freezing and freeze substitution to avoid exposure to aqueous solvents have revealed a 0.5- to 2.0-μm border zone within which mineral deposition occurs gradually.⁹⁸ In this zone, some proteoglycans are degraded and calcium is bound by phosphophoryn and perhaps by phospholipid as well.

At the mineralization front in the zone of initial mineralization, dotlike mineral nuclei are aligned parallel to and superimposed on collagen fibrils.⁹⁹^,¹⁰⁰ The mineral nuclei appear positioned over the hole regions of the collagen fibrils, suggesting that the DPPs are bound to collagen at those sites. Additional hydroxyapatite crystals have been located on the surface of the fibrils and in perifibrillar spaces. It has been suggested that dentin sialoproteins and proteoglycans act as nucleating agents for perifibrillar hydroxyapatite crystals. Collagen mineralization is discussed further in chapter 8.

Structure of the Odontoblastic Process and Dentinal Tubules

With progressive deposition of dentin matrix, the odontoblastic process lengthens and becomes embedded in mineralized tissue (Figs 2-2, 2-3, 2-6, and 2-7). The space occupied by the odontoblastic process is known as the dentinal tubule. The dentinal tubule extends from within the mantle dentin to the predentin.¹⁰¹^,¹⁰²

Odontoblastic process

The cytoplasm of the odontoblastic process contains a rich network of microtubules and microfilaments, both of which are oriented parallel to its long axis (see Fig 2-7).⁵⁰^,¹⁰³^–¹⁰⁵ The microtubules and microfilaments form a network that runs the length of the odontoblastic process. Microtubules provide a substratum for granule translocation (discussed in chapter 3). The microfilaments (actin) provide a contractile mechanism that might enable the odontoblastic process to retract toward the pulp. It has been suggested that the odontoblastic process is retracted toward the cell body when exposed to noxious stimuli. This is an interesting idea that should be explored in new research.

Figs 2-7a to 2-7c Electron micrographs of the odontoblastic process (OP) in longitudinal section (a) and cross section (b, c). The process contains a dense network of microtubules (Mts) and microfilaments, and is free of cytoplasmic organelles. Nerve endings (NE) can be found in close juxtaposition to the OP. (mD) Mineralized dentin; (PD) predentin; (dD) demineralized dentin. (Original magnification × 24,000 [a], × 48,000 [b], and × 30,000 [c].)

When the odontoblastic process is viewed in cross section, it is apparent that microtubules and secretory granules are associated in a consistent pattern. The microtubules are distributed evenly around the circumference, about 30 to 40 nm from the granule membrane. Most secretory granules are secreted in the predentin or at the mineralization front.

Some secretory granules, however, are found in the odontoblastic process beyond the mineralization front, suggesting that collagen, proteoglycans, or other constituents of the dentin matrix might be secreted from the distal regions of the process. Secretion of organic matrix from the distal parts of the process might be responsible for the formation of peritubular dentin and/or sclerosis of the tubules.

Numerous coated pits and coated vesicles indicative of membrane retrieval and receptor-mediated endocytosis are conspicuous elements of the odontoblastic process.¹⁰⁶

A sheath, rich in glycosaminoglycans, separates the process from the surface of the peritubular dentin (Figs 2-8 and 2-9).¹⁰⁷^,¹⁰⁸ This sheath is similar to the lamina limitans found around osteocyte cell processes.¹⁰⁹ Thin cytoplasmic side branches, arising from the main odontoblastic process, pierce through the sheath and extend toward adjacent odontoblastic processes.¹¹⁰^–¹¹² These small branches of the odontoblastic process contain only fine filaments.

Although it has been suggested that odontoblastic processes might communicate via side branches, there is no evidence that gap junctions are formed between adjacent side branches. If adjacent odontoblastic processes made gap junctional contacts via smaller side branches, their organization would be comparable to the canalicular cell processes of bone, whereby osteocytes intercommunicate through gap junctions.

Fig 2-8 Components of tubular dentin. The odontoblastic process (OP) occupies the dentinal tubule space (DTS) and is surrounded by the lamina limitans (LL). (PTD) Peritubular dentin; (ITD) intertubular dentin.

Fig 2-9 Odontoblastic process (OP) from middle region of the dentin viewed in longitudinal section. The lamina limitans (LL) is closely applied to the plasma membrane of the process. Demineralized peritubular matrix (PTM) abuts the LL. A secretory granule (SG) is present in the process. (Original magnification × 54,000.)

Many investigators who have tried to determine the true length of the odontoblastic process by transmission electron microscopy have concluded that the process does not extend beyond the middle of the dentin.¹⁰¹^,¹⁰³^,¹⁰⁴^,¹¹³ In contrast, most investigators who have examined fractured dentin surfaces by scanning electron microscopy have reported that the odontoblastic process extends out to the dentinoenamel junction.¹¹⁰^,¹¹⁴^–¹¹⁷ Dramatic scanning electron micrographs were obtained of dentin pretreated with hydrochloric acid to remove the mineral phase and with collagenase to remove the collagen fibrils of the organic matrix.¹¹⁷ A remarkable system of branching tubular structures, stretching from the predentin to the dentinoenamel junction, was revealed when these methods were used. The tubular structures were erroneously identified as odontoblastic processes.

The contrasting results obtained by routine transmission electron microscopy and scanning electron microscopy were resolved when it was shown that the structures presumed to represent odontoblastic processes could be removed by digestion with hyaluronidase.¹⁰⁷^,¹⁰⁸^,¹¹⁸ This was taken as proof that the tubular structures, erroneously identified as odontoblastic processes, were in fact organic sheaths (lamina limitans) located between the odontoblastic process and the peritubular dentin. The true length of the odontoblastic process in mature dentin remained to be established.

Another approach to this problem was the use of fluorescein-labeled antitubulin as a method for identifying odontoblastic processes. In those studies, it was found that antitubulin was localized along the entire length of the dentinal tubule, suggesting, once again, that the odontoblastic process extended all the way to the dentinoenamel junction.¹¹⁹ An alternate explanation for the positive fluorescence might be that, following retraction or degradation of the odontoblastic process, tubulin might bind to the lamina limitans or to the walls of the dentinal tubules in sufficient amounts to give positive staining.

Confocal microscopic studies of odontoblasts infused with a fluorescent dye have shown that the odontoblastic process in fully developed teeth does not extend to the outer dentin.¹²⁰^,¹²¹ The longest processes were found in coronal dentin.

Another explanation put forth to explain the difficulty encountered in establishing the true length of the odontoblastic process is that the process might contract toward the cell body in response to noxious stimuli, such as the fixatives used to preserve cells prior to routine electron microscopy. This idea received support in studies of teeth that had been frozen rapidly to immobilize cytoplasmic structures prior to chemical tissue fixation.¹²² When this approach was used, structures resembling odontoblastic processes were observed in dentin near the dentinoenamel junction. Additional research is needed to explore the interesting possibility that the odontoblastic process contracts in response to stimuli in its immediate environment.

Dentinal tubules

Dentinal tubules extend from the mantle dentin to the predentin, across the full thickness of circumpulpal dentin (see Fig 2-1). The distal end of the dentinal tubule branches extensively into fine secondary tubules that permeate the mantle dentin.¹²³^,¹²⁴ Small side branches extend from tubule to tubule in the circumpulpal dentin.¹¹¹^,¹²⁴ Dentinal tubules are wider toward the pulp and generally more concentrated in the region of the pulp horns.¹⁰²^,¹²⁵

Some dentinal tubules appear to be obliterated by nonmineralized collagen fibrils, while others are blocked with mineral.⁸^,¹²⁶^,¹²⁷ The physiology of dentin permeability is to a great degree a function of the patency of the tubules.¹⁰⁸^,¹²⁸^,¹²⁹ Dentinal tubules contain serum proteins, including fibrinogen, albumin, and immunoglobulins.¹³⁰^–¹³² These proteins are carried into the tubules in dentinal fluid, where they may become trapped in the lamina limitans or bound to the mineral phase of dentin. The flow of dentinal fluid increases following dental operative procedures and during pulpal inflammation.¹³³

Carious dentin contains higher levels of immunoglobulins. It has been suggested that the high affinity of antibody binding to hydroxyapatite may serve as a protective reservoir of antibacterial immunoglobulin.¹³⁴

The first description of tubular “canals” in dentin was made by Leeuwenhoek in the 17th century. He fabricated simple microscopes capable of magnifications of no more than × 100. With these rudimentary instruments, he amused his contemporaries by demonstrating microscopic canals in dentin, and animalcules (bacteria) in saliva. More than 300 years later, with microscopes capable of achieving magnifications of more than × 1,000,000, there are still new frontiers to explore in the structure of dentin.

Formation of Intertubular and Peritubular Dentin

The greater bulk of the mineralized circumpulpal dentin is called intertubular dentin (see Fig 2-1). It is formed by the mineralization of predentin. The matrix of intertubular dentin is rich in type I collagen fibrils. The uniform size and the arrangement of the collagen fibrils are best viewed in scanning electron micrographs (see Figs 2-6a and 2-6b).⁴³ Hydroxyapatite crystals, about 40 nm in length, are formed in and around the collagen fibrils of the intertubular dentin.

A second and minor component of circumpulpal dentin, the peritubular dentin, develops around the odontoblastic process (see Fig 2-1). The organic matrix of peritubular dentin is rich in glycosaminoglycans and relatively free of collagen fibrils. Bone sialoprotein and osteonectin have been localized in peritubular dentin. The hydroxyapatite crystals that develop in the peritubular dentin matrix are small in comparison to those that form in the intertubular dentin. Because of its small crystallites (large surface area) and the noncollagenous nature of its organic matrix, peritubular dentin is more susceptible to demineralization and degradation during the caries process.

Transport Across the Odontoblastic Layer

Over the course of several decades, there have been numerous contradictory reports on the transport of calcium and other substances into developing dentin. Some investigators have presented autoradiographic evidence of an intracellular (transcellular) pathway, while others have shown that ions move from pulp to dentin through a paracellular route between odontoblasts.

Increasing evidence supports a direct role for odontoblastic transport of ions into the predentin and dentin. The calcium ion activity in the fluid phase of predentin is two to three times higher than that in the pulp.¹³⁵ Current physiologic evidence suggests that an ionic gradient exists across the odontoblastic layer and that, under normal conditions, the paracellular pathway is closed. Under these conditions small molecules and ions would be forced to pass through the odontoblast cytoplasm to reach the dentin.

Recent studies have provided indirect support that calcium is transported into the dentin across the odontoblast cell membrane by a calcium ATPase pump, and by Na⁺-Ca⁺⁺ antiports.¹³⁶^,¹³⁷ L-type voltage–gated and agonist-gated calcium channels have been localized in odontoblasts by immunocytochemical methods.²⁴^,¹³⁷^,¹³⁸ Calcium channel inhibitors (nifedipine and neomycin) have been shown to decrease the entry of calcium into the odontoblasts, and eventually to reduce the level of calcium entry into the predentin. Secretory odontoblasts express high levels of inositol 1,4,5-triphosphate–regulated channel proteins. These channel proteins permit calcium flux from intracellular stores into the cytosol. Smutzer et al suggested that 1,4,5-triphosphate receptors might regulate calcium release for dentin mineralization.¹³⁹

The presence of ion concentration gradients across the odontoblastic layer appears to be at odds with the morphologic evidence that no zonula occludens is formed at the distal end of the odontoblast cell body. Furthermore, there is free diffusion of intravenously administered tracer molecules into predentin. Additional research is needed to identify the mechanisms responsible for generating and maintaining ion concentration gradients and for regulating solute movement across the odontoblast layer.

Innervation of Dentin and Mechanisms of Pain Sensation

Organization of the nerve supply

The nerve supply to the coronal pulp is especially rich, forming a subodontoblastic plexus of nerve fibers (plexus of Raschkow). Unmyelinated nerves from this plexus penetrate between the odontoblasts and enter the predentin. Although most unmyelinated nerves appear to terminate in the predentin, some enter the dentinal tubules. Nerve endings containing many small vesicles and mitochondria have been identified in close association with the odontoblast cell body and the odontoblastic process in dentin (Figs 2-7 and 2-10).¹⁴⁰^–¹⁴³ The majority of these nerves are afferent somatosensory pain fibers.

Nerve growth factor and its receptor have been localized in relatively high amounts in the odontoblastic and subodontoblastic layers of the coronal pulp. The presence of nerve growth factor may be responsible for concentrating nerve endings in the vicinity of the odontoblasts. Experimental evidence has shown that nerves grow toward concentrations of nerve growth factor.

Because the cell processes of fibroblasts and odontoblasts can be confused with unmyelinated nerve fibers, special staining techniques are needed to accurately distinguish nerve fibers and their terminals. Protein gene product 9.5, a neuron-specific protein, has been used to identify pulpal nerve fibers at both the light and electron microscopic level (see Figs 2-10a to 2-10d).¹⁴⁰^,¹⁴⁴ Fibers containing protein gene product were present in both radicular and coronal predentin. Nerve endings in the predentin have also been identified by immunocytochemistry for calbindin, a calcium-binding protein found in high concentrations in nerve cells.¹⁴⁵ Tracer experiments with tritiated proline injected into the brainstem nuclei of the trigeminal nerve have provided convincing proof of a rich supply of sensory nerve terminals in the predentin and dentinal tubules.¹⁴⁶

Figs 2-10a to 2-10d Nerve structure of a human premolar. (Human protein gene product [PGP] 9.5 antibody stain. Adapted from Maeda et al¹⁴⁰ with permission from Elsevier Science.)

Fig 2-10a Pulpodentinal region of human premolar stained with human PGP 9.5 antibody. A dense network of nerve endings can be seen in the predentin (PD). (D) Dentin; (P) pulp. (PGP 9.5 antibody stain. Original magnification × 70.)

Fig 2-10b Inset area in Fig 2-10a at higher magnification. Nerve terminals are beadlike axonal swellings (arrowheads). (Original magnification × 700.)

Fig 2-10c Electron immunocytochemistry. Nerve terminals (arrows) stained with PGP 9.5 antibody are juxtaposed to the odontoblastic processes (OP) in the predentin (PD). (Original magnification × 4,000.)

Fig 2-10d Electron immunocytochemistry. The nerve terminals contain mitochondria (M) and many smooth vesicles (arrows). Although the plasma membranes of the nerve terminals are in close apposition to the plasma membrane of the odontoblastic process (OP) (arrowheads), no evidence of a synaptic structure is present. (Original magnification × 12,000.)

Vasomotor nerves supply small arteries in the pulp, terminating in close apposition to arteriolar smooth muscle cells.¹⁴⁷ The nerve endings contain many dense-cored adrenergic synaptic vesicles.

Theories of dental pain

Although it is well established that most unmyelinated nerve endings in pulp and dentin are nociceptors, the exact mechanisms whereby noxious conditions are converted into dental pain stimuli have yet to be identified. The most widely held theory centers around fluid flow within dentinal tubules. The hydrodynamic theory of dental pain is based on the facts that fluid in the dentinal tubules is constrained by the rigid walls of the peritubular dentin and fluxes in temperature or in osmotic pressure produce rapid expansion and contraction of the dentinal fluid. The bulk flow of the dentinal fluid may distort nerve endings, thereby triggering nerve impulses. Experimental support for this theory is provided by the observation that pain occurs when heat or a substance that changes the osmotic pressure of the dentinal fluid is applied to cut dentin surfaces.

However, the hydrodynamic theory does not explain why some chemicals that do not alter osmotic pressure in dentin can still cause dental pain. Presumably these chemicals diffuse down their concentration gradients to act directly on nociceptive nerve endings in predentin. The hydrodynamic theory also fails to account for the rapidity of stimulus transduction, especially in relation to mechanoreceptor activity elicited from dentin (see chapter 9).

Despite the limitations of the hydrodynamic theory, the results of clinical and basic research on dentin sensitivity have shown that the patency of dentinal tubules is a significant factor in controlling the degree of stimulation of dentinal nerves.¹²⁹^,¹⁴⁸^–¹⁵⁰ Recent investigations have shown that the dentinal tubules are filled with a fibrous hydrogel.¹²⁹ Although the hydrogel may limit bulk fluid flow, it still permits the diffusion of solutes down their concentration gradient. Dentin sensitivity can be reduced by obliteration of the tubules, either by the physiologic formation of peritubular dentin and the intratubular deposition of collagen fibrils or by the clinical application of agents that cause mineral precipitation inside the tubules.

It has also been speculated that the odontoblast (and its process) might act as a transducer to convert noxious stimuli into nerve impulses. This concept is based on the notion that odontoblasts make gap junctions (electronic synapses) with adjacent nerves and that the flow of ions through these junctions, in response to changes in the odontoblasts, could lead to depolarization of somatosensory neurons. Patch-clamp recordings made on segments of plasma membrane from isolated odontoblasts have demonstrated potassium and chloride channels and a resting membrane potential of about –40 to –50 mV.¹⁵¹ It has been suggested that mechanosensitive ion channels could lead to changes in ion conductance across the plasma membrane in response to hydrodynamic forces exerted on the odontoblastic process.¹⁵² Although gap junctions between odontoblasts and nerves have been reported, there is still no proof that odontoblasts communicate electrically with nerve endings.

A subpopulation of cells cultured from human dental pulp have voltage-gated sodium channels and other properties associated with neuronal satellite cells.¹⁵³ Whether these pulp cells originate from the odontoblastic layer and whether or not they have a role in pulpal somatosensation remains to be established. Additional discussion of dentinal and pulpal sensory mechanisms is contained in chapter 10.

It has also been suggested that dentinal nerves may have an effector function on odontoblasts. Indirect evidence for an effector activity includes the fact that many dentinal nerves contain the neuropeptide, calcitonin gene–related peptide (CGRP).¹⁵⁴ Evidence of protein secretion from dentinal nerves has also been reported.¹⁴² Recent studies of the presence of several exocytosis regulatory proteins (synapsin and synaptogamin) in dentinal nerve endings adds more support that dentinal nerves have an effector function in addition to their somatosensory afferent actions.¹⁵⁵

Many of the nerve endings in the odontoblastic layer and predentin contain substance P and CGRP.¹⁴⁴^,¹⁵⁶^–¹⁵⁸ These neuropeptides may be involved in vascular dilation and neurogenic inflammation.¹⁵⁹^–¹⁶⁰ Indirect evidence supports the idea that the release of neuropeptides from dental sensory nerve fibers is important in the recruitment of immunocompetent cells to the dental pulp.¹⁶¹ Experimental studies also suggest that these neuropeptides may promote dentinogenesis.¹⁶²^,¹⁶³

The potential for development of neurogenic inflammation in pulp is supported by the demonstration that coronal and apical pulp contain CGRP-positive nerves in association with blood vessels and within the connective tissue stroma.¹⁵⁹^,¹⁶⁰^,¹⁶⁴ Some CGRP-positive nerve fibers are found in the subodontoblastic layer. Sprouting of CGRP-positive nerve endings occurs following dental injury.¹⁶⁵^,¹⁶⁶ The release of CGRP increases vascular permeability of pulpal blood vessels.¹⁶⁷ Recent studies have demonstrated excitatory amino acid receptors in bovine dental pulp.¹⁶⁸ Activation of excitatory amino acid receptors leads to the release of CGRP in pulp.

Supply of Blood to the Pulp

Blood vessels enter the tooth through the apical foramen and course coronally in the midregion of the radicular pulp. The largest arteries have a muscular coat of three to six layers of smooth-muscle cells. The outer adventitia is rather inconspicuous, because it blends gradually with the pulpal connective tissue. The endothelial cells that line the arteries often appear to bulge into the lumen. The accompanying veins have one to two layers of smooth-muscle cells and a wider lumen. As the major vessels course through the radicular pulp, they give off peripheral branches that arborize to form a rich capillary network associated with the odontoblastic layer.¹⁶⁹^,¹⁷⁰ The greatest degree of branching occurs in the coronal pulp, especially below the cusps.

The endothelial lining is of the continuous type, except for the fenestrated capillaries that are adjacent to the predentin. The cell-to-cell junctions of the endothelium are characterized by adherens junctions and overlapping cell processes. Numerous pynocytotic vesicles are present on the luminal and abluminal surfaces of the endothelium. The luminal surface of capillaries and venules contain many cytoplasmic processes. Spindle-shaped pericytes contain moderately high numbers of cytoplasmic filaments and are spaced apart as single cells in close contact with the basal lamina of the endothelial cells. The pericytes serve as stem cells capable of multipotential differentiation. Recent studies have shown that a population of dendritic cells is associated with the major pulpal blood vessels.¹⁷¹^,¹⁷² These cells are specialized for phagocytosis, processing, and presentation of antigens.

Blood is supplied to the odontoblastic layer by capillaries that are in close apposition to the odontoblast cell bodies and the predentin (Figs 2-11a to 2-11c).¹⁷³^–¹⁷⁵ Individual capillaries penetrate the intercellular spaces between the odontoblasts to form the predentinal capillary plexus. The predentinal capillary plexus reaches a peak of development coincident to the most active phase of dentin formation. Fenestrated endothelial linings have been reported in capillaries located close to the predentin.¹⁶⁹ The close proximity of thin-walled capillaries to the odontoblasts and the predentin suggests that there is a high requirement for oxygen, ions, and metabolites during the rapid phase of dentin formation. When dentin formation is completed, the predentinal capillary plexus is no longer present. At this stage, nutrients reach the odontoblasts from the subodontoblastic plexus.

Reparative dentinogenesis is preceded by angiogenesis. Several angiogenic growth factors (platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor) have been isolated from dentin matrix.¹⁷⁶ It has been proposed that angiogenic growth factors are released during dentin degradation, thereby stimulating the development of new blood vessels in the zone of injury.¹⁷⁶

Figs 2-11a to 2-11c Blood supply to the pulp. (Adapted from Ohshima and Yoshida¹⁶⁹ with permission from Springer-Verlag.) (a) Electron micrograph illustrating the presence of capillaries (CL) in the odontoblastic (OB) layer. (Original magnification × 3,000.) (b) Left inset area in Fig 2-11a at higher magnification. Capillaries (CL) are generally of the fenestrated type near the predentin (arrowheads). (Original magnification × 8,000.) (c) Right inset area in Fig 2-11a at higher magnification. Capillaries (CL) are generally of the continuous type nearer to the pulp. (Original magnification × 8,000.)

Cells and Extracellular Matrix of the Dental Pulp

The dental pulp is a connective tissue derived from the proliferation and differentiation of the cells of the dental papilla. In its developmental stage, the dental pulp contains a relatively high content of glycosaminoglycans and sparsely distributed, fine collagen fibrils (types I and III).¹⁷⁷^,¹⁷⁸ Initially, the network of collagen fibers is composed mostly of argyrophilic reticular fibers rich in type III collagen. As the pulp matures, the synthetic capacity of pulpal fibroblasts increases, and more collagen bundles of type I collagen are formed. Despite the increased amount of type I collagen, the mature pulp continues to have an unusually high content of type III collagen.¹⁷⁹ Although the numbers of collagen fibers continue to increase with age, the pulp maintains its appearance of a loose connective tissue. Collagen fibers are concentrated to form supporting elements for blood vessels and nerve trunks that course from the root apex to the coronal pulp chamber.

The pulp contains a relatively large concentration of glycosaminoglycans and proteoglycans.¹⁸⁰^,¹⁸¹ Versican, a chondroitin-6-sulfate–rich proteoglycan, has been detected in high concentrations in peripheral pulp.¹⁸² Fibroblasts are distributed evenly throughout the middle regions of the pulp and concentrated beneath the odontoblastic layer in the coronal pulp of erupted teeth to form a cell-rich zone. The cell-rich zone also contains numerous major histocompatibility complex–positive dendritic cells that have an increased capacity for capturing and processing antigens.¹⁷² These cells have numerous cell processes that make contact with odontoblasts and nerves.¹⁸³ Dendritic cells are part of the surveillance arm of the immune system (see chapter 13). Because the cell-rich zone makes its appearance after the tooth has erupted into the oral cavity and is limited in its extent to the coronal pulp (excluding the floor of the coronal pulp), it is believed to form as a defensive response to external stimulation.

Dental pulp cells respond to a variety of growth factors.¹⁸⁴ Deoxyribonucleic acid synthesis in human dental pulp cells is stimulated by basic fibroblast growth factor and platelet-derived growth factor and is inhibited by interleukin 1 β. Transforming growth factor β stimulates the synthesis of collagen and fibronectin in cultures of pulp cells.¹⁸⁴ Vitamin D stimulates pulp fibroblasts to express osteopontin, a phosphoprotein typically found in bone.¹⁸⁵

Basic Science Correlation: The Secretory Pathway

During the 1960s and 1970s, the ultrastructure of the RER-Golgi system was characterized, and the morphologic aspects of a secretory pathway were established. It is now known that proteins destined to be exported from the cell, or to lysosomes and endosomes, are synthesized in the rough endoplasmic reticulum and transported to the Golgi complex.¹⁸⁶ In the Golgi complex, proteins are posttranslationally modified, sorted, and packaged for further transport to their ultimate destination, whether it be a secretory granule, a primary lysosome, or the cell membrane.

No specific signal recognition event appears to be required for the transport of proteins from the RER to the Golgi apparatus. The only prerequisite is that the proteins undergo correct three-dimensional folding within the RER. Transport vesicles destined for the Golgi apparatus develop from smooth-membrane segments of the RER, called transitional elements.

The Golgi complex is subdivided into a cis-Golgi network, Golgi stacks, and a trans-Golgi network (Fig 2-12).¹⁸⁷ The cis-Golgi network acts as a quality control gate, preventing the transport of defective proteins through the Golgi complex to the cell surface and/or secretion into the extracellular space. The small percentage of RER-resident proteins that escape during the formation of transport vesicles are recognized in the cis-Golgi network by their lysine–aspartic acid–glutamic acid–leucine amino acid sequence and are returned to the RER in a retrograde vesicular pathway (see Fig 2-12).¹⁸⁸ Retrograde traffic also returns membrane lipids to the RER compartment. Transport from the RER to the Golgi apparatus requires microtubules. However, the retrograde pathway from the Golgi apparatus back to the RER does not depend on an intact microtubular network.

Fig 2-12 Secretory pathway from the rough endoplasmic reticulum (RER) to the cis-Golgi network (CGN), across the Golgi stacks, and into the trans-Golgi network (TGN), where proteins are directed to appropriate destinations. (arrows) Unidirectional anterograde vesicular transport. (dashed lines) Retrograde pathways used to retrieve membrane and proteins that have escaped from the RER. (Adapted from Rothman and Orci¹⁸⁷ with permission from MacMillan Publishers.)

Secretory and cell membrane proteins undergo successive compartment-specific reactions during their transit through the Golgi stacks. Glycosyltransferase and glycosidases contained in the Golgi cisternae sequentially decorate the peptide backbone of the protein by the addition of carbohydrate side chains. These posttranslational modifications involve the addition of oligosaccharides by nitrogen-linkage to asparagine, and/or oxygen-linkage at serine and threonine residues. Formation of oxygen-linked glycans involves a two-step process consisting of the addition of N-acetyl-galactosamine, followed by the addition of galactose and sialic acid (N-acetyl-neuraminic acid). Studies have shown that the addition of N-acetyl-galactosamine occurs in transitional elements of the RER, while the addition of galactose and sialic acid occurs in the most mature cisternae of the Golgi apparatus.

The two-way traffic of vesicles from the RER to the Golgi complex, and from the Golgi complex to the cell membrane, requires numerous regulatory mechanisms.¹⁸⁹ The complex machinery for sorting proteins and controlling vesicular traffic inside the cell began to be deciphered in the 1980s and was accelerated by the advent of newly discovered molecular biology techniques.¹⁹⁰ Cell biologists view the Golgi complex as a dynamic system of membrane-bound compartments whose function requires constant intercompartmental vesicular exchange. Movement of substances from the RER to the cis-Golgi network, between Golgi stacks, and from the trans-Golgi network to the final target membrane is carried out in small transport vesicles that bud from surfaces of the donor compartment.¹⁹¹^,¹⁹² A great deal of research is being focused on identifying the molecular nature of the sorting, docking, and fusion events needed for this operation.

The budding process requires the recruitment and attachment of specific coat proteins (coatomers) on the parent cisternal membrane to form a mechanochemical “patch” capable of deforming the membrane into a separate vesicle.¹⁹³^–¹⁹⁵ As the vesicle forms, it concentrates a microscopic sample of specific cargo proteins from the cisternal fluid. Coatomer recruitment requires ATP, Ca²⁺, guanosine triphosphate (GTP), and several cytosolic proteins.¹⁹⁶

In the first step of the process (Fig 2-13), a transmembrane protein in the donor membrane, guanine nucleotide–releasing protein (GNRP), interacts with a cytosolic GTP-binding protein called adenosine diphosphate ribosylation factor (ARF). In the cytosol, ARF is in its guanosine diphosphate (GDP)-bound state (ARF-GDP). When ARF-GDP interacts with GNRP, GDP is released and GTP is bound in its place. Subsequently, ARF-GTP undergoes conformational change, exposing a fatty acid chain that anchors ARF-GTP to the donor membrane.

Fig 2-13 Formation of a coatomer-coated membrane. The first step involves the insertion of guanine nucleotide–releasing protein (GNRP) into the membrane of the donor compartment. In the second step, GNRP reacts with adenosine diphosphate ribosylation factor (ARF), converting ARF–guanosine diphosphate (GDP) to ARF–guanosine triphosphate (GTP). Attached to the donor membrane, ARF-GTP is then able to bind coatomer proteins. As more coatomer proteins are bound to the site, a vesicle will start to form from the donor compartment by a budding process. Soluble N-ethylmaleimide–sensitive fusion attachment protein receptors (SNAREs) project from the surface of the transport vesicle (v-SNARE).

In step two, segments of the membrane covered by ARF-GTP favor the recruitment and attachment of coatomer proteins (see Fig 2-13). In mechanisms yet to be clarified, the coatomer-coated membrane is deformed and pinched off to form a coatomer-coated transport vesicle (Fig 2-14). Transport vesicles retain their coatomer coats until they begin docking to the appropriate target membrane.

Sorting products to their appropriate destinations requires specific signals to control the docking of transport vesicles with the correct target compartment. This is accomplished by transmembrane proteins that act as surface markers. The transmembrane interacting proteins are soluble N-ethylmaleimide–sensitive fusion attachment protein receptors (SNAREs). Terrian and White reviewed the evolution of SNARE proteins and their role in traffic regulation (see Fig 2-14).¹⁹⁷ Specific surface markers (t-SNARES) have been identified in the membranes of the RER, Golgi complex, endosomes, and plasma membrane.

Docking of the transport vesicle to the target membrane occurs when vesicle SNAREs (v-SNAREs) bind to their “target” membranes (t-SNAREs) (see Fig 2-14). Rab-GTP, a second type of monomeric guanosine triphosphatase, present in the vesicle membrane, functions as a monitor and stabilizer of the fit between the two types of SNARE molecules. A guanosine triphosphatase–activating protein in the target membrane causes ARF-GTP to hydrolyze GTP to GDP. In its GDP-binding state, ARF retracts its fatty acid anchor and detaches from the vesicle membrane. Simultaneously, the ARF-coatomer complex disassembles.

Fig 2-14 Formation, docking, and fusion of the transport vesicle. The coatomer-coated vesicle, with soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (v-SNARE) molecules exposed beyond the coatomer coating, is available for binding (step 2, docking reaction) to the appropriate target membrane SNARE (t-SNARE) molecules. During the docking reaction, adenosine diphosphate ribosylation factor–guanosine triphosphate is hydrolyzed to adenosine diphosphate ribosylation factor–guanosine diphosphate and disassociates from the vesicle membrane. Coatomer proteins are also released. The attachment of v-SNARE to t-SNARE is monitored and stabilized by a second type of guanosine triphosphate, a Rab-GTP molecule present in the donor vesicle membrane. Fusion of the transport vesicle membrane to the target (step 3) is induced by a protein complex that includes N-ethylmaleimide–sensitive fusion protein (NSF-P) and soluble NSF attachment proteins (SNAPs).

For membrane fusion to occur, special fusion proteins are required to displace water molecules and to overcome the electrostatic repulsive forces between the two closely juxtaposed lipid membranes.¹⁹⁸ The space between the adjacent membranes must be reduced to less than 1.5 nm. The v-SNARE to t-SNARE receptor-ligand docking reaction between the transport vesicle and its target membrane recruits fusion proteins to the site of attachment. N-ethylmaleimide– sensitive fusion (NSF) protein and soluble NSF attachment proteins (SNAPs) have been shown to carry out fusions in eukaryotic cells by interacting with SNAREs (see Fig 2-14). Conformational changes in the fusion protein, driven by ATP, destabilize the lipid membranes, leading to the formation of a fusion pore that rapidly expands to permit total fusion of the two membranes. Exactly how this machinery is assembled and how it functions during the fusion event is still speculative.

Application of this new knowledge of the regulation of cytoplasmic traffic must be applied to future studies of the odontoblastic Golgi complex to gain a clearer understanding of the secretion and retrieval of dentin matrix components.

From the trans-Golgi network, there are two basic pathways of secretion: the regulated pathway and the constitutive pathway.¹⁹⁹^,²⁰⁰ In the regulated pathway, secretory product is stored in vesicles or granules until secretion is triggered by an appropriate signal. Secretory granule formation involves condensation of the secretory product from larger condensing vacuoles (presecretory granules) considered to be part of the trans-Golgi network (see Fig 2-13).²⁰¹ Budding of membrane from the condensing vacuole continues, until a smaller and denser secretory granule is formed. In the constitutive pathway, products are exported immediately after they are packaged in the Golgi apparatus.

Regulated secretion requires an intact microtubular system to transport granules to a specific region of the cell surface, usually the apical end of the cell. Constitutive secretion does not appear to be dependent on microtubules and may occur from many regions of the cell surface.

Microtubules form a radiating network, extending from the centrosome outward to the cell periphery. This network provides a structural pathway for the translocation of secretory granules. The energy source for granule transport is derived from the hydrolysis of ATP. Enzymatic motor proteins (mechanochemical ATPases) associated with the granule membrane hydrolyse ATP molecules when activated by contact with the microtubules.

Cyclical attachment and detachment produces movement of the granule along the length of the microtubule. The mechanochemical ATPase responsible for anterograde movement of secretory granules is a member of the kinesin family of motor proteins. Substances that interfere with microtubule assembly, such as antimitotic (antispindle) agents, and substances that deplete ATP or stop its production produce abnormalities in deposition of dentin, enamel, and bone matrices.

Microtubule-directed transport delivers secretory granules to the periphery of the secretory pole of the cell. At that point, further transport toward the plasma membrane is dependent on myosin. The final approach and fusion is both nonmicrotubule and nonmyosin dependent.²⁰² Discussion of microtubules is continued in chapter 3.

A proposed pathway for secretion in the odontoblast, based on ultrastructural studies of the odontoblast and the current theories of Golgi organization, is outlined in Fig 2-15. Additional studies of odontoblast structure and function are needed to determine whether there are multiple forms of secretory granules in odontoblasts and to identify the signals that direct secretory granule discharge.

Fig 2-15 Odontoblast secretory pathway. Intermediate coated transport vesicles (CTV) bud from the rough endoplasmic reticulum (RER) and migrate to the cis-Golgi network (CGN), where they fuse with the outermost cisternae of the Golgi stack. Presecretory granules (PSG) form part of the trans-Golgi network (TGN). Concentration of the secretory product occurs by aggregation of proteins inside the PSGs and by the removal of fluid and membrane via budding of small vesicles. Vesicles containing membrane proteins and lipids are secreted in the constitutive pathway. Matrix secretory granules (SG) are transported via the regulated pathway, along microtubules, to the odontoblastic process.

Clinical Correlations

Secondary, tertiary (reactive), and reparative dentin

Odontoblasts are nondividing cells with a long life span. During tooth development, they produce primary dentin at the rate of about 4 to 8 μm per day. Once the crown is completed and the apical length of the root has been established, odontoblasts produce secondary dentin at 1 to 2 μm per day. Histologic studies of human teeth have shown that all teeth contain secondary dentin. It is deposited throughout life as long as the pulp remains vital.

Although there are no reliable data on the estimated longevity of individual odontoblasts, the continued slow deposition of secondary dentin suggests that odontoblasts are long-lived cells. Odontoblasts lose nearly half of their RER and Golgi profiles following formation of primary dentin.²⁰³ Biochemical studies indicate that, on completion of primary dentin, there is an 80% reduction in alkaline phosphatase activity at the predentin-odontoblast region, and a concomitant reduction of ATPase activity in the odontoblasts. This reduced metabolic function is consistent with a slow production of secondary dentin.

Secondary dentin is characterized by a regular arrangement of dentinal tubules, usually in direct continuity with those of the primary dentin. Micro-hardness measurements indicate that secondary dentin is about 30% to 40% softer than primary dentin. The biochemical and matrix factors responsible for the decrease in mineral content have not been identified.

Tertiary or reactive dentin is produced in response to nonlethal irritation of the odontoblasts. Once activated, either by a slowly progressing carious lesion, dental abrasion, or restorative dentistry procedure, odontoblasts resume deposition of dentin at rates that approach those measured for formation of primary dentin. Reactive dentin is deposited subjacent to the area of injury as a protective barrier for the pulp.

Reactive dentin does not have the well-organized histologic structure of primary or secondary dentin. The dentinal tubules are fewer and less likely to be neatly parallel to each other. A calciotraumatic line is commonly found to separate the secondary dentin from the reactive dentin. Excessive formation of reactive dentin in the root portion of the pulp can lead to varying degrees of pulp canal obliteration, a condition that complicates pulp canal therapy.

Recent studies of superficial carious lesions, where the zone of demineralization had not reached the dentinoenamel junction, demonstrated increased deposition of peritubular dentin and a decreased width of the predentin.²⁰⁴ With progression of the lesion to the dentinoenamel junction, the predentin grew wider than control predentin as deposition of collagen increased. Cell proliferation in the subodontoblastic layer accompanied changes in activity of the odontoblasts.

It was suggested that in early enamel caries, the odontoblasts respond to stimuli transmitted along partially demineralized enamel rods and the dentinal tubules.²⁰⁴ When the caries process involves dentin, fibronectin and a 165-kDa fibronectin-binding protein are deposited on the surface of the odontoblastic process and along the walls of the dentinal tubules.²⁰⁵ It was suggested that fibronectin and 165-kDa proteins regulate reactive dentinogenesis, perhaps by playing a signaling role similar to that which occurs during initial odontoblast differentiation.

If the injury is severe, as in rapidly advancing dental caries or in a dental operative procedure producing excessive heat, the odontoblasts undergo necrosis. In this case, repair (the formation of reparative dentin) must await the differentiation of new odontoblast-like cells from precursors, either from the cell-rich zone or from deeper regions of the pulp.²⁰⁶ The presence of a fibronectin-rich surface, permitting adhesion of pulp cells, appears essential to the differentiation of odontoblast-like cells.²⁰⁷

Replacement odontoblasts are generally produced in fewer numbers than the original complement. The reparative dentin that they deposit is characterized by irregular tubules. Under less favorable conditions, new odontoblasts may fail to differentiate, and repair is carried out by fibroblastic cells that deposit a fibrodentin type of matrix. In either case, the process requires several weeks.

Recent experiments have shown that certain growth factors, namely bone morphogenetic protein (BMP-2, BMP-4, and BMP-7), TGF-β1, and components of dentin matrix, stimulate the development of the odontoblast phenotype and the expression of type I collagen and osteocalcin in pulp cells.²⁰⁸–210 Cultured human pulp fibroblasts express BMPs and BMP receptor.²⁰⁹^,²¹¹^,²¹² In the presence of fibronectin, TGF-β1 and BMP-2 trigger odontoblast development from embryonic dental papilla cells.⁸⁹^,²¹³ It has been proposed that these growth factors have a regulatory role in the initiation of reparative dentin by activating the differentiation of new odontoblasts.²¹⁴^–²¹⁶

The potential value of BMPs as pulp-capping agents is under investigation by several research groups. Recombinant human BMP-2, BMP-4, and BMP-7 have been incorporated in pulp-capping preparations applied to the pulps of dogs and monkeys. Significant increases in reparative dentin were noted after several months in teeth capped with BMP preparations. ²¹⁰^,²¹⁷^–²¹⁹ High–molecular weight hyaluronic acid also promotes formation of reparative dentin in amputated dental pulp.²²⁰

Sclerotic dentin

Empty dentinal tubules result from either the physiologic retraction of the odontoblastic process or from the death of the odontoblasts. These tubules appear as dark bands (dead tracts) in ground mineralized sections when viewed under transmitted light. Open dentinal tubules, especially at the cervical region, often lead to dental hypersensitivity. Occlusion of the tubules by precipitation of calcium salts or with composite resin reduces the flow of fluid and decreases the sensation of pain.²²¹^–²²³

During mild irritation, dentinal tubules may become obliterated by mineral deposition, a process known as dentinal sclerosis (Fig 2-16). Some investigators have suggested that continued or excessive deposition of peritubular dentin is the basis of dentinal sclerosis. Sclerotic dentin is usually present under chronic carious lesions, dental restorations, and areas of attrition. It has been suggested that sclerosis of dentinal tubules is a defense mechanism for protection of the pulp. In ground sections, sclerotic dentin appears translucent, blending in closely with adjacent mineralized intertubular dentin. The distal portion of the odontoblastic process may become mineralized in carious dentin.¹⁰³

In the caries process, dentin demineralization begins when the enamel lesion reaches the dentinoenamel junction. The caries process does not spread preferentially along the dentinoenamel junction but rather progresses into the dentin along the dentinal tubules.²²⁴ In sclerotic tubules, bacteria advance more slowly because they must remove hydroxyapatite crystals by acid dissolution. Nevertheless, because of the small crystallites (large surface area for exchange) in sclerotic and peritubular dentin, and the absence of a collagen fibril matrix, bacteria are able to advance preferentially along the tubules (see Fig 2-16). Destruction of the collagenous matrix of intertubular dentin proceeds more slowly, because proteolytic enzymes must gain access to and degrade the collagen matrix after it has been demineralized. Bacterial invasion of the dentinal tubules is a complex process involving bacterial adhesions to extracellular matrix molecules, proteolytic enzymes, and the ability of bacteria to survive in an environment of limited oxygen and nutrients.²²⁵

Fig 2-16 In a caries lesion, cariogenic bacteria invade the dentinal tubules, demineralizing sclerotic and peritubular dentin in the process. Intertubular dentin is slower to degrade because of its dense collagenous matrix and larger hydroxyapatite crystals.

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