Odontoblasts and Dentin Formation

Chapter 30

Odontoblasts and Dentin Formation Françoise Bleicher, Béatrice Richard, Béatrice Thivichon-Prince, Jean-Christophe Farges, and Florence Carrouel Team Evo-Devo of vertebrate dentition, Institute of functional genomics of Lyon, UMRCNRS 5242, University Lyon 1 Faculty of Odontology, Lyon, France

Chapter Contents 30.1 Key Concepts 30.2 Odontoblast Life Cycle 30.2.1 From Neural Mesenchymal Cells to Aged Odontoblasts 30.2.2 Regulation of Odontoblast Terminal Differentiation 30.3 Primary and Secondary Dentinogenesis 30.3.1 Primary Dentinogenesis 30.3.1.1 Matrix Molecules Synthesis and Secretion 30.3.1.2 Mineralization Process 30.3.2 Secondary Dentinogenesis

379 380 380 383 384 384 384 386 387

30.4 Tertiary Dentinogenesis 30.4.1 Reactionary Dentinogenesis 30.4.2 Reparative Dentinogenesis 30.5 Non-Dentinogenic Function of Odontoblasts 30.5.1 Odontoblasts in the Dental Pulp Immune and Inflammatory Response 30.5.2 Odontoblasts as Sensor Cells 30.6 Conclusion Acknowledgment Abbreviations References

30.1  KEY CONCEPTS l

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Odontoblasts are highly differentiated, post-mitotic cells originating from the neural crest, which are organized at the periphery of the pulp as a cellular palisade. Odontoblasts synthesize the components of the predentin (type I collagen, glycoproteins, and other non-­collagenous proteins), and are responsible for its mineralization. They synthesize the primary dentin during organogenesis, but also secondary dentin during the life of the tooth and tertiary dentin when tooth undergoes pathological stimulations. Primary dentin is made up of a network of type I collagen fibers that mineralize due to non-collagenous proteins (SIBLINGs and glycoproteins). The external part of the primary dentin (the mantle dentin) is less mineralized than the circumpulpal dentin. This primary dentin is synthesized until the complete formation of the root. Secondary dentin is synthesized at a lower rate than primary dentin all over the life of the tooth. The chemical composition and the structural organization of this secondary dentin are identical to the primary dentin. Tertiary dentin is synthesized by odontoblasts when teeth are injured. It includes two types of tissues, reactionary and reparative dentins, which differ by the ­nature of the pulp response, the type of cells involved in

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387 387 388 390 390 391 392 392 392 393

the dentinogenic process, and the structure of the deposited mineralized tissue. Reactionary dentin is produced by primary odontoblasts, i.e., odontoblasts that have originally differentiated during the development of the tooth germ. It is deposited in response to a low-grade environmental stimulus. Local demineralization of the primary dentin releases entrapped growth factors and ECM peptides, which diffuse toward the odontoblasts and stimulate them in order to synthesize a new layer of dentin that protects the pulp from the external stimulus (bacteria, irritants, etc.). Reparative dentin is produced by odontoblast-like cells that have differentiated from a pool of mesenchymal pulp cells after the necrosis of the primary odontoblasts. This situation occurs when caries progression accelerates and gains access to deep dentin, for example. Odontoblasts are sensor cells. They represent, in the tooth, the first line of defense for the host. Due to the presence of Toll-like receptors at their cell surface, odontoblasts can recognize a bacterial invasion and consequently can elicit inflammatory and immune reactions in the pulp by secreting chemokines. Odontoblasts can also sense noxious stimuli such as cold, heat, mechanical stress, etc. Several lines of evidence have demonstrated that odontoblasts express mechano- and/or thermosensitive transient receptor potential ion c­ hannels that are

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likely to sense heat and/or cold or movements of dentinal fluid within tubules. As odontoblasts are in close association with nerve endings, odontoblasts could transduce the noxious signal to the surrounding nerve fibers by the release in the gap space of mediators such as eATP or galanin, or by depolarization of the neuron membrane. Odontoblasts are a good target for new therapeutics applied to the treatment of caries and dental pain.

30.2  ODONTOBLAST LIFE CYCLE Odontoblasts are post-mitotic cells aligned at the edge of the dental pulp. Their life cycle follows several successive steps. Initially, neural crest cells from the midbrain and ­rostral hindbrain migrate toward the first visceral arch and become preodontoblasts along with mesenchymal pulp cells. Preodontoblasts will generate functional odontoblasts, then secretory odontoblasts, and finally aged odontoblasts. Differentiation is initiated at each cusp tip and is carried out according to a specific temporo-spatial pattern. In the mouse, the first differentiated odontoblast appears on the 18th day of embryonic development, on the top of the main cusp of the first lower molar. The differentiation process gradually extends to the apical area, and leads to the formation of a gradient of differentiation [1].

30.2.1  From Neural Mesenchymal Cells to Aged Odontoblasts Neural crest cells from the midbrain and rostral hindbrain migrate toward the first visceral arch and provide dental papilla cells. At the late bell stage of tooth development, these undifferentiated ectomesenchymal cells are small and exhibit a central nucleus and few organelles. Their ­differentiation into preodontoblasts occurs after a specific

number of cell divisions. All the cells of the dental papilla have the potential to differentiate; however, only those in contact with the basement membrane at the interface with the inner dental epithelium undergo terminal differentiation. In mice, the preodontoblasts, located near the basement membrane, undergo 14 to 15 mitoses before leaving the cell division cycle [1]. The number of cell divisions necessary for this process in humans, however, has not yet been demonstrated. During the last division, the mitotic spindle, originally parallel to the basement membrane, realigns to become perpendicular to this membrane [2]. After the last mitosis, only the cell in contact with the basement membrane that contains some fine collagen fibrils is able to enter into the differentiation process, while the other daughter cell (not in contact with the basement membrane), becomes part of the Höhl’s cell layer. Preodontoblasts (Figure 30.1), which are cells in contact with the basement membrane, stop dividing, increase in size, and hang on anchoring fibrils on the surface of the mesenchymal basement membrane by their plasma membrane. Their organelles and cytoskeletal components are uniformly distributed in the cytoplasm. To differentiate into functional odontoblasts (Figure 30.2), preodontoblasts begin to polarize. Their nucleus rolls away from the basement membrane, while granular endoplasmic reticulum and Golgi complex are aggregated into a supranuclear position. Granular reticulum cisternae, whose number increases, flatten and orientate parallel to the long axis of the cell. The Golgi complex, more central compared to the reticulum, turns to the pole in contact with the basement membrane [3,4]. The part of the cell which contains the nucleus is called the basal pole, whereas the opposite region, close to the basement membrane, is named the apical pole (secretory). Mitochondria are ­dispersed throughout the cell. Elements of the c­ ytoskeleton

FIGURE 30.1  Schematic representation of preodontoblast differentiation. Dental papilla cells in contact with the basement membrane stop dividing, increase in size, and hang on anchoring fibrils on the surface of the mesenchymal basement membrane by their plasma membrane. These preodontoblasts have their organelles and cytoskeletal components uniformly distributed in the cytoplasm.

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FIGURE 30.2  Schematic representation of functional odontoblasts. Functional odontoblasts are composed of two parts: the basal pole that contains the nucleus; and the apical pole (secretory) which is closed to the basement membrane. Granular endoplasmic reticulum (parallel to the long axis of the cell) and Golgi complex are aggregated into a supranuclear position. The Golgi complex, more central compared to the reticulum, turns to the pole in contact with the basement membrane. Mitochondria are dispersed throughout the cell. Elements of the cytoskeleton (microtubules, microfilaments, and intermediate filaments) accumulate at the apical pole of the odontoblasts.

FIGURE 30.3  Schematic representation of interodontoblast junctions. (a) An area of intercellular junctions appears at the boundary between the odontoblast cell body and process. This area is crossed by large diameter fibers which are mainly composed of collagen type I and III, and fibronectin (Von Korff fibers). Tight and gap junctions appear between odontoblasts, and between odontoblasts and subodontoblast cells. (b) The terminal web is composed of cytoplasmic filaments connected to the inner side of the junctional complex.

(microtubules, microfilaments, and intermediate filaments) accumulate at the apical pole of the odontoblasts. The cell body grows to a height of about 50 microns, due to a high increase in the level of synthesis organelles [4,5]. A process develops in contact with the anchoring fibrils. Sometimes this trunk divides into several branches. The extension and its main branches ramify to give many secondary branches. This process is limited by a plasma membrane and contains predominantly cytoskeletal ­components such as microfilaments, intermediate filaments,

and ­microtubules, with no synthesis organelles except a few small mitochondria at the base of the main extensions in the neighboring region of the cell body. It contains secretory vesicles enclosing the components of the predentin, and endocytic vesicles with molecular fragments from the maturation and partial degradation of the predentin. At the boundary between the odontoblast cell body and process, an area of intercellular junctions appears (Figure 30.3). It is circular and separates the ­extracellular subodontoblast compartment and the extracellular

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p­ redentinary compartment. This area is crossed by a large diameter fiber matrix synthesized by the subodontoblast pulpal cells. These fibers are mainly composed of collagen type I and III, and fibronectin. They are called Von Korff fibers. Many cytoplasmic filaments are connected to the inner face of the junction region to form a structure called the terminal web. They separate the cytoplasm of the extension and that of the cell body. The terminal web acts as a selective filter. It maintains the synthesis organelles in the cell body, but the secretory vesicles and endocytes which are smaller are able to pass through. The transit occurs especially in the central area, because the network is looser at this level. The edges of the terminal web, tight junctions, and gap junctions appear between odontoblast cells, and between odontoblast cells and subodontoblast cells [6–10]. Tight junctions appear just before the start of the predentin mineralization. They control the passage of ions toward the mineralization front, and allow adequate ionic concentration at this level. The occurrence of various types of junctions leads to the formation of the odontoblast layer that isolates the central pulp (Figure 30.4) from where the predentin will be secreted and mineralized (organic matrix of dentin). Once polarized, odontoblasts differ in functional terms and are named secretory odontoblasts. These cells synthesize the predentin at an average speed of 4 microns/ day (Figure 30.5). Differentiation is characterized by the amplification of the synthesis of type I collagen, the suppression of synthesis of collagen type III, the synthesis of non-collagenous protein (which occurs mostly during the phenomena of mineralization), and the release of growth factors (which are stored in the dentin). Synthesis of components of the predentin is facilitated by the arrival in the odontoblast layer of fenestrated capillaries that bring nutrients to the odontoblasts in large q­ uantities. Fenestrations disappear when the primary dentin is ­completely formed.

D

PD

Dentin tubule with odontoblast process

Terminal web

Odontoblasts

P FIGURE 30.4  Section of human dental pulp stained by Masson’s trichrome. D: dentin; PD: predentin; P: pulp.

Once secreted, the predentin undergoes a maturation phenomenon which results in the supramolecular organization of the collagen network and the degradation of various components. Then it becomes mineralized in the distal part of the predentin far from the cell body, where the maturation is complete. Mineralization occurs when the predentin reaches a thickness of 20-30 microns. The continuous deposit of predentin pushes the body of the odontoblast cell to the center of the dental papilla. This centripetal movement gradually increases the size of the extension around which the predentin is mineralized to form dentin. Aged odontoblasts are secretory odontoblasts whose synthetic and secretory capacities have decreased. Nevertheless, the secretory activity of these cells can be reactivated after injury. These cells are shorter than secretory ones and crowded, giving the odontoblasts layer a pseudo-stratified appearance. The organelles of

FIGURE 30.5  Predentin secretion by odontoblasts. The secretion begins at the odontoblast apical pole between the anchoring fibrils of the basement membrane. It continues near the mineralization front localized at the interface between predentin and dentin along the odontoblast process.

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the cells which are reduced in number are mainly located in the infranuclear region. The supranuclear region is devoid of organelles, except for large lipid-filled vacuoles (autophagic vacuoles) which are characteristic of aged odontoblasts. The decrease in size of the rough endoplasmic reticulum and Golgi complex, with the absence of ­secretory granules, is associated with the decrease of the activity of these cells.

Functional odontoblasts display a primary cilium in the vicinity of the Golgi apparatus [11]. Primary cilia have been described in almost every eukaryotic cell type as a non-motile antenna extending into the extracellular space [12]. It forms a single organelle consisting of a membrane-bound cylinder surrounding the axoneme made of nine microtubule doublets, extended from the nine triplet microtubules of the basal body, a centriole-derived microtubule-organizing center. Underneath the ciliary membrane, the assembly and disassembly of the axoneme is possible because of the shuttle of ciliary proteins by intraflagellar transport particles (IFT) attached to two molecular motors: the heterotrimeric kinesin-2 for anterograde transport (from the base to the tip of the cilium); and cytoplasmic dynein-2 for the retrograde transport (from the tip to the base) [13]. A striated cytoskeleton structure (the ciliary rootlet) extends from the basal body toward the cell nucleus. In many tissues, primary cilia are essential for sensing various mechanical or biochemical signals [14,15]. Primary cilia may also control fundamental aspects of cellular physiology and development, via its implication in different signaling pathways such as Wnt, PDGFRα, and Hedgehog [16]. In mice tooth development, primary cilium control molar tooth number via a negative regulatory effect on the Hedgehog pathway in the mesenchyme [17]. Concerning mice tooth morphogenesis, mutation of certain primary cilium components could be associated with molars morphologically disturbed showing undifferentiated odontoblasts and ameloblasts in upper molars [18]. In humans some ciliopathies (diseases associated with primary cilium defects) are associated with dental defects. For example, hypodontia has been reported in BBS patients (Bardet-Biedl syndrome), and teeth abnormalities including missing/supernumerary teeth, malposition of teeth, and enamel hypoplasia are observed in 42% of patients displaying mutation in the OFD1 (oro-facio-digital syndrome type 1) transcript [19].

30.2.2  Regulation of Odontoblast Terminal Differentiation During tooth development, only mesenchymal cells in contact with the basement membrane are able to differentiate into odontoblasts. Odontoblast differentiation is regulated by reciprocal molecular interactions between the inner dental epithelium and the mesenchyme. The extracellular matrix

and the basement membrane play a critical role in this regulation, serving as a reservoir of paracrine and autocrine factors. Only one specific dental basement membrane is able to induce the differentiation of odontoblasts. The ectomesenchymal cells can only respond to epigenetic signals after having carried out a determined number of cell divisions. Moreover, to play its role, the basement membrane is subject to modifications, regulated by the inner dental epithelium. These changes are essential for the regulation of dental development. The basement membrane is mainly composed of collagen IV, fibronectin, laminin [20,21], nidogen [22], tenascin [23,24], hyaluronic acid, and proteoglycans including heparan sulfate [21]. These components are present before and during odontoblast differentiation. As odontoblasts polarize, collagen III in the epithelial-­mesenchymal junction gradually disappears [20]. Fibronectin surrounding preodontoblasts will accumulate in the apical pole of polarized odontoblasts [20,21,25]; collagen type I, decorin, and biglycan present in the extracellular matrix also aggregate to the secretory pole of elongated cells [26]. During differentiation, chondroitin-4-sulfate has the same profile of evolution as fibronectin [27]. However, chondroitin sulfates do not seem to play a role in the control of odontoblast differentiation [28]. Studies have also shown changes in the pattern of distribution of glycosaminoglycans [29,30] and polysaccharides [31]. Tenascin associated with the basement membrane increases during terminal differentiation of odontoblasts [32]. These ­observations illustrate some aspects of the evolution of the composition of the basement membrane during the differentiation of odontoblasts. The extracellular matrix molecules are also involved in odontoblast differentiation process via receptors. Fibronectin interacts with plasma membrane proteins including one receptor of 165 kDa. The 165 kDa receptor and the fibronectin accumulate at the apical pole of polarizing odontoblasts [33,34]. A monoclonal antibody specifically recognizing an extracellular epitope of the 165 kDa protein interferes with the organization of microfilaments and blocks the elongation of the odontoblasts and polarization without affecting microtubules [35,36]. Various growth factors and their receptors have been shown to be present at the enamel organ-dental papilla interface during tooth development and have been implicated in odontoblast differentiation: l

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GH (growth hormone) and IGF-1 (of the family of IGF: insulin-like growth factors) could play a paracrine and/ or autocrine role in dental development [37–39] TGFβ1, -2, -3 (transforming growth factor) [40–43], and BMP-2, -4, -6 (bone morphogenetic protein) [44–47] play a role in the polarization and differentiation of odontoblasts [48].

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30.3  PRIMARY AND SECONDARY DENTINOGENESIS Following mesenchymal cell differentiation into secretory odontoblasts, dentinogenesis occurs with the synthesis and secretion by secretory odontoblasts of an extracellular matrix named predentin that subsequently becomes mineralized into dentin, including the cell processes in tubules. The dentin formed up to the completion of root development is defined as primary dentin. It constitutes the major part of the dentin bulk, and is produced at a relatively high rate.

30.3.1  Primary Dentinogenesis The mantle dentin is the first layer of the primary dentin; it is deposited peripherally in the dental papilla beneath the enamel organ at the onset of dentin formation (Figure 30.6). The thickness of the mantle dentin varies in humans between 5 and 30 microns. In the root, a similar superficial layer is observed. The rest of the primary dentin is named circumpulpal dentin. The circumpulpal dentin is divided into intertubular dentin, which constitutes the largest volume of primary dentin, and peritubular dentin that forms the wall of the dentinal tubules. A gradual obliteration of the dentinal tubules occurs with aging. The layer of predentin is a constant feature in pulped teeth. Located between the dental pulp and the mineralized tissue, limited at its proximal side by the cell bodies of the odontoblasts, firmly connected by distal junctional complexes and at its distal side by the mineralization front, it is a sealed compartment for the formation and maturation of

the collagen network of the dentinal matrix. It has a rather constant thickness, about 20 to 30 microns at the coronal part of the tooth, and only a few microns at the root part.

30.3.1.1  Matrix Molecules Synthesis and Secretion The organic matrix of dentin is, with the exception of minor serum protein content, entirely produced by the odontoblasts [49]. As polarization and differentiation of odontoblasts start, a small process arises at the odontoblast surface facing the enamel organ which will extend and participate in the secretion. The synthesis of extracellular matrix molecule (ECM) components occurs in the cell body. The secretion begins in the apical pole adjacent to the basement membrane and continues near the mineralization front at the interface between dentin and predentin, and along the odontoblast process. Almost 90% of the dentin organic matrix consists of collagen, whereas the remainder consists of non-­collagenous proteins, proteoglycans, and some lipidcontaining components [50]. Large molecule (collagen and proteoglycans) secretion takes place in the predentin at the base of the odontoblasts process, whereas other smaller molecules (glycoproteins) are transported along the process to the front and secreted more distally near the mineralization front, or even further within the lumen of tubules, they are not present in predentin. The collagen fibrils function as a scaffold for the deposition of hydroxyapatite crystals, whereas dentin matrix mineralization is highly controlled by the expression of the non-collagenous proteins.

FIGURE 30.6  Primary dentinogenesis. The primary dentin constitutes the major part of the dentin bulk. Beneath the enamel organ, the odontoblasts first synthetize the mantle dentin, followed by the circumpulpal dentin when the odontoblast process appear. A constant layer of predentin is located between the dental pulp and the mineralized tissue.

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30.3.1.1.1 Collagens Collagen is the major protein found in dentin and constitutes about 90% of the dentin organic matrix. The majority is type I, although trace amounts of type III, IV V, VI, XI, and XII collagens have been reported. Type I collagen results from the self-assembly of two alpha1 chains and one alpha2 chain, or three alpha1 chains. The molecules are synthesized by the odontoblasts and released in predentin near the cell bodies at the base of the odontoblast process. They progressively aggregate to form collagen fibers and become a dense fibrillar network near the mineralization front. In the mantle dentin, near the basement membrane, the collagen fibers align between the anchoring fibers, forming right angles to the membrane. This disposition strengthens the cohesion of the enamel-dentin junction. In the circumpulpal dentin fibers lie parallel to the basement membrane. The secretion of phosphoproteins near the fibers creates a microenvironment that triggers the deposition of hydroxyapatite. 30.3.1.1.2  Non-Collagenous Proteins The non-collagenous ECM proteins are believed to play important roles in the biomineralization process that forms

hard tissues. Even if they make up less than 10% of the ­dentin matrix, non-collagenous proteins (NCPs) have important functions in processes such as induction and regulation of mineral formation, cell attachment, and collagen fibrillogenesis. They are secreted at the mineralization front and are implicated in the nucleation and growth of the mineral phase, or in its inhibition. 30.3.1.1.3 SIBLINGs The Small Integrin-Binding Ligand, N-linked Glycoprotein family is the major group of non-collagenous proteins in dentin. Their genes are clustered along chromosome 4q2123 in the human. SIBLING structure shows RGD motifs able to bind integrin receptors and to mediate cell attachment and signaling. SIBLING proteins are extremely acidic and highly phosphorylated extracellular proteins. The family includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) (Table 30.1). DSPP is immediately cleaved after secretion into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP);

TABLE 30.1  Matrix Molecules Involved in Dentinogenesis Matrix Molecules

Origin

Location

Role

Collagens

Odontoblasts

Predentine

Scaffold

Odontoblasts

Mineralization front

Cell attachment and signaling mediation

Non-Collagenous Proteins SIBLINGs DPP

Intrafibrillar mineralization induction

DMP-1

Calcium binding

BSP

Apatite crystal nucleation and crystal growth guidance

OPN

Crystal nucleation inhibition

MEPE

Phosphate metabolism and mineralization regulation

Gla-proteins

Odontoblasts

Mineralization front

Osteocalcin

Crystal nucleation inhibition

Matrix gla protein

Mineralization inhibition

Proteoglycans

Odontoblasts

Predentine

Collagen matrix structuring; hydroxyapatite binding and crystal growth guidance

Biglycan

Mineralization nucleation

Decorin, Fibromodulin, Asporin, Mimecan, Lumican

Collagen fibrillogenesis regulation

Glycosaminoglycans

Odontoblasts

Predentine

Matrix metalloproteinases

Odontoblasts

Serum-derived proteins

Serum

Predentine and dentine

Enamel proteins

Ameloblasts

Mantle dentine

Mineralization inhibition by binding calcium Degradation of some matrix constituents Mineral transport

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a third protein named dentin glycoprotein (DGP) has been identified in the pig. DPP is the major NCP component (more than 50%); it is a highly acidic phosphoserine-rich protein with a high content of aspartic acid. DPP binds to hydroxyapatite and calcium ions with a high affinity, and binds to collagen to induce intrafibrillar mineralization. DSP is a less-phosphorylated molecule, rich in aspartic acid, glutamic acid, glycine and serine, related to sialoproteins. DMP-1 is a highly phosphorylated acidic NCP that is potentially glycosylated. A post-translational proteolytic cleavage leads to two fragments: a NH2-terminal found in non-mineralized predentin, and a COOH-terminal found in the mineralization front and mineralized dentin which has a direct effect on mineral formation, and crystal growth. DMP-1 regulates DSPP gene transcription and interacts with other molecules. In particular, DMP1 contains domains rich in serine, glutamate, and aspartate, and is highly acidic, which gives the molecule a strong calcium-binding potential and provides an appropriate microenvironment for mineral precipitation. BSP is a highly sulfated, phosphorylated, and glycosylated protein characterized by its ability to bind to hydroxyapatite through polyglutamic acid sequences. It promotes apatite crystal nucleation and directs the growth of the crystals. OPN, a highly acidic protein, binds collagen and HA and inhibits crystal nucleation. MEPE, an acidic phosphorylated protein, regulates phosphate metabolism and mineralization via its C-terminal proteolytic cleavage product (acidic-serine-aspartate-rich-MEPE-associated). 30.3.1.1.4  Other Non-Collagenous Proteins Gla (gamma-carboxyglutamic acid) proteins are directly secreted at the mineralization front. Osteocalcin regulates the growth of apatite crystals in forming dentin. It reduces dentin mineralization in inhibiting crystal nucleation. Matrix gla protein (MGP) binds the hydroxyapatite crystal and inhibits mineralization. Thrombospondin, osteonectin, and tenascin are also found in predentin. 30.3.1.1.5 Proteoglycans Proteoglycans (PG) are known to be involved in the organization of the ECM prior to mineral deposition. In particular, small leucine-rich proteoglycans (SLRPs) facilitate the structuring of the ECM collagen matrix and direct hydroxyapatite binding and crystal growth. SLRPs consist of two different structural components, a protein core involved in protein/ protein interactions and varying numbers and types of GAG chains. SLRPs appear to interact with type I collagen fibrils by the leucine rich region of the protein core. Some of them have been shown to be involved in dentinogenesis [51,52]. Biglycan acts as a nucleator of mineralization. Decorin, fibromodulin, asporin, mimecan and lumican regulate collagen fibrillogenesis. Osteoadherin has a high affinity for hydroxyapatite and accumulates at the mineralization front.

30.3.1.1.6 Glycosaminoglycans Glycosaminoglycans (GAGs) are most abundant in predentin, and are barely detectable in dentin. They inhibit mineralization by binding calcium, and are removed and degrade where mineralization is initiated. 30.3.1.1.7  Matrix Metalloproteinases and Other Enzymes Matrix metalloproteinases (MMP) are implicated in degradation of some matrix constituents. In particular, MMP2 and MMP20 have been shown play a role in predentin maturation to allow mineralization. Odontoblast processes are implicated in the re-internalization of fragments after the degradation of ECM molecules. SOD3, an antioxidant enzyme, may mediate scavenging in the dentinal tubules to protect dentin itself or after its secretion into the dentinal tubule space. Dentin also contains collagenases. Enamel proteins are found in mantle dentin. Serum-derived proteins including growth factors, albumin, and immunoglobulins are also present in predentin and dentin, as well as lipids, comprising phospholipids, cholesterol, cholesterol esters, and triacylglycerols. They migrate directly from the serum to the dentin compartment, albumin and phospholipids being implicated in the transport of mineral toward and therefore in the mineralization process of intertubular dentin.

30.3.1.2  Mineralization Process In mantle dentin, the odontoblasts secrete ECM, and matrix vesicles with an amorphous content bud off from their distal end and establish a close relation to proteoglycans and glycosaminoglycans that bind calcium ions. The first mineral crystals appear within these matrix vesicles [50]. In circumpulpal dentin, mineralization occurs without the presence of matrix vesicles, and non-collagenous matrix proteins then play a key role to regulate the mineral deposition into the collagen scaffold. Calcium is transferred from the vascular network in the subodontoblastic area to the proximal end of the odontoblasts, across the odontoblast cell layer, to be incorporated in the mineral phase at the interface between the non-­mineralized predentin and the mineralized dentin, the mineralization front. The intercellular junctions between the odontoblasts do not allow the diffusion of calcium ions into the predentin layer. Calcium ions are actively transported by intracellular pathways in the functional odontoblast cytoplasm. Odontoblast calcium transport mechanisms probably imply a Ca2+-activated ATPase, Na+/Ca2+ exchanger, and calcium channels of L7, N7, and T types. Ca2+ binding proteins like calmodulin, the 28 kDa calbindin, parvalbumin, and annexins III to VI have been shown to be present in functional odontoblasts. Ca2+ and PO 4 2− combine to initiate the nucleation process and hydroxyapatite is deposited in the e­ xtracellular

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matrix. Mineral formation in the collagenous network occurs at the advancing mineralization front, which moves forward at a rate varying between 4 and 20 μm per day.

may allow specific molecules to be exploited in the future to develop biological approaches to dental pulp repair and healing.

30.3.2  Secondary Dentinogenesis

30.4.1  Reactionary Dentinogenesis

After root formation, the odontoblasts form secondary dentin which is responsible for the progressive reduction of the canal space during the life of the tooth. The chemical composition and the structural organization of this secondary dentin are identical to the primary dentin. The only difference is that the secondary dentin is deposited at a much reduced rate of 0.4 microns/day

Reactionary dentin (RcD) is produced by primary odontoblasts, i.e., odontoblasts that have originally differentiated during the development of the tooth germ and that produce primary and secondary dentins [54]. It is deposited in response to a low-grade environmental stimulus. It is commonly found beneath initial, shallow dentin caries lesions which progress slowly along the dentin-enamel junction [61]. RcD is supposed to protect the pulp from external injuries by isolating it from invading irritants. It includes two entities distinct at the topographical level, which are called sclerotic and circumpulpal reactionary dentins. Sclerotic reactionary dentin (SRcD) is deposited by injured odontoblasts in the tubule lumen (Figure 30.7) [62]. It contains few extracellular matrix, almost no collagen, and is hypermineralized and translucent. Its formation is believed to result from the acceleration of the slow physiological process of peritubular dentinogenesis. It often leads to complete sealing of the most external part of the dentin tubule. By reducing dentin permeability beneath the caries lesion, SRcD prevents intratubular dissemination of bacteria, toxins, and salivary fluids and molecules. Circumpulpal reactionary dentin (CRcD) is formed by primary odontoblasts at the pulp-dentin interface adjacent to the lesion (Figure 30.8). It results from the acceleration of primary or secondary den-

30.4  TERTIARY DENTINOGENESIS When teeth are injured by caries, trauma, excessive wear, cavity preparation, or filling materials, the dental pulp seeks to preserve its vitality and health by synthesizing, at the pulp-dentin interface, a new layer of dentin whose function is to isolate the living tissue from the external irritant agent [53]. This dentin, formed in pathological conditions, is called tertiary dentin. It includes two types of tissues, reactionary and reparative dentins, which differ by the nature of the pulp response, the type of cells involved in the dentinogenic process, and the structure of the deposited mineralized tissue [26]. Formation of either of these dentins depends on the extent and severity of the injury and its progression speed. For instance, slowly progressing, moderatesized caries promote the formation of reactionary dentin by adjacent surviving odontoblasts. However, acceleration in the progression and/or increase in the severity of the lesion results in odontoblast death and elaboration of reparative dentin by odontoblast-like cells differentiating from pulp cells locally recruited [54,55]. Tertiary dentin formation is enhanced by low-grade pulp inflammation, but is inhibited by intense inflammatory events that prevent dentin matrix synthesis by odontoblasts [56,57]. In this case, odontoblast metabolism becomes primarily oriented toward the production of proinflammatory and innate immune effectors intended to increase pulp defense capacity [58,59]. If dentin infection and associated pulp inflammation continue to increase, odontoblasts end up undergoing necrosis or apoptosis [60]. Studies have shown that tertiary dentin formation is promoted by low amounts of proinflammatory cytokines and/or biologically active molecules responsible for the induction of embryonic odontoblast differentiation. These active molecules include growth factors and extracellular matrix components sequestered in dentin during tooth germ formation that may be solubilized or exposed following dentin demineralization by cariogenic bacteria. Improved understanding of the nature of biological processes involved in the pulp immune/inflammatory and dentinogenic responses

FIGURE 30.7  Schematic representation of reactionary dentin in a human carious tooth. Sclerotic reactionary dentin (SRcD) is deposited in the lumen of the tubule (T) to prevent the diffusion of microbial components from the caries lesion. Circumpulpal reactionary dentin (CRcD) is deposited at the interface between pulp and primary dentin (PD), which takes the living primary odontoblasts (PO) and the underlying pulp tissue far from the lesion (black arrows).

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factors exert their effects in association with extracellular matrix proteins. Among these, fibronectin, an extracellular matrix adhesive glycoprotein known to be involved in embryonic odontoblast differentiation, has been localized in dentin tubules connecting the caries lesion to the odontoblast layer, whereas it is absent from tubules in healthy dentin [70]. As it is able to bind TGF-β1, fibronectin may modulate the stimulation of odontoblast-dependent RcD formation by this growth factor.

30.4.2  Reparative Dentinogenesis

FIGURE 30.8  Circumpulpal reactionary dentin in a human carious tooth. Masson trichrome staining. Circumpulpal reactionary dentin (CRcD) is deposited at the interface between pulp (P) and primary dentin (PD) by primary odontoblasts (PO). White arrows show the processes of PO extending in PD tubules after having crossed CRcD.

tinogenesis, depending on whether the caries lesion gains access to the dentin before or after the completion of root formation. CRcD deposition tends to progressively reduce the volume of the pulp tissue. By increasing the dentin thickness separating the pulp from the lesion, CRcD takes pulp far from the lesion, which protects it from harmful effects of microorganisms or other kinds of oral irritants. Experiments in animal models have shown that reactionary dentinogenesis may be promoted by biologically active (bioactive) molecules released or exposed from the dentin matrix by acid demineralization [63,64]. Lyophilized preparations of demineralized dentin matrix implanted into dentin cavities promote circumpulpal dentinogenesis in areas where tubules directly connect the cavity to the odontoblast layer. This suggests that bioactive molecules diffuse from the demineralized matrix through dentin tubules in the pulp direction. The diffusion distance between the cavity and the odontoblast layer influences the level of odontoblast stimulation, since CRcD is deposited in greater amounts in areas where the distance is the shortest, and reciprocally. Among endogenous molecules potentially able to diffuse transdentinally upon dentin demineralization are members of the Transforming Growth Factor-beta (TGF-β) family, including TGF-β1 and 3, and Bone Morphogenetic Proteins (BMP) 2, 4, and 7. These growth factors are all capable, at varying degrees, of promoting dentin matrix production when added to odontoblast or odontoblast-like cell cultures, or when implanted in dentin cavities in experimental animal models [65–69]. In vivo data have shown that the amount of CRcD produced by odontoblasts is proportional to the amount of growth factor introduced into the cavity, and is inversely related to the residual dentin thickness separating the cavity from the odontoblast layer. In general, growth

When caries progression accelerates and gains access to deep dentin, odontoblasts may be destroyed, which generates an area of necrosis in the peripheral pulp adjacent to the lesion [61]. If caries evolution is not too fast to irreversibly degrade the pulp tissue, the latter has the possibility to implement a dentin barrier, called reparative dentin (RpD), to protect itself from invading microorganisms. The main criteria allowing for the implementation of this barrier are the absence of severe inflammatory events, a sufficient cell density, and adequate pulp vascularization. Following the disappearance of the odontoblast layer, undifferentiated cells in the adjacent subodontoblast region migrate to the necrosis area, where they adhere to the primary dentin wall and progressively differentiate into odontoblast-like cells often called replacement odontoblasts. Two main hypotheses have been put forward regarding the origin of these cells [26]: l

l

First, they could be daughter cells of primary odontoblasts that have differentiated during the embryonic development of the tooth germ. These daughter cells are generated during the last mitosis that precedes odontoblast differentiation of the cells in contact with the basement membrane separating the dental mesenchymal papilla from the enamel organ. They have the same developmental history as odontoblasts but, as they are located to some distance from the basement membrane, they lack the final step in the induction process to become odontoblasts. In the mature tooth, these post-mitotic cells are present in the subodontoblast cell-rich zone, the so-called Höhl’s layer. Second, they could originate from subodontoblast and/ or pulp core cells surrounding blood vessels, possibly pericytes or dental pulp progenitor/stem cells. Pulp stem cells generally proliferate before migrating toward the necrosis area in order to maintain the pool of perivascular undifferentiated pulp cells.

Migration to the dentin wall is made easier by a modification in the composition of the local pulp extracellular matrix. Indeed, the route followed by migrating cells becomes enriched in type III collagen and fibronectin [61]. After having crossed the odontoblast necrosis area, cells adhere to the dentin wall and progressively differentiate, first by acquiring a nonpolarized spindle or cuboidal shape, then a spinous one. They rapidly secrete an extracellular matrix essentially composed

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of type I and type III collagens and fibronectin [71,72]. Once mineralized, this matrix is called fibrodentin. It is less mineralized than primary and secondary dentins, and is atubular. Cells responsible for its formation are called fibrodentinoblasts. As fibrodentin formation is generally fast, some cells may be trapped within the tissue, mainly at the dentin-fibrodentin junction. In this case, fibrodentin displays a structure more or less resembling bone and is called osteodentin. Cells totally surrounded by osteodentin are named ­ osteodentinocytes, whereas those lining osteodentin at the osteodentin-pulp interface are called osteodentinoblasts. Fibrodentin or osteodentin formation is considered as providing a seal of the pulp with regard to intradentinal irritants to allow repair conditions to prevail. It is generally characteristic of pulps that have undergone severe but temporary inflammation. Although not always present, fibrodentin is considered by most authors a prerequisite for the differentiation of replacement odontoblasts and the formation of underlying tubular reparative dentin (see below and Figure 30.9). It would be necessary to immobilize and/or adequately present bioactive molecules such as growth factors involved in this differentiation. By restraining the diffusion of growth factors to the most peripheral pulp, fibrodentin would limit their ­action to the nearest cells and thereby would prevent unwanted pulp core mineralization [26]. Fibronectin in the fibrodentin matrix constitutes an adhesive surface for pulp cells, and thus is believed to play a role similar to the anchoring fibrils of the basement membrane during embryonic differentiation of primary odontoblasts.

FIGURE 30.9  Schematic representation of osteodentin-like fibrodentin in a human carious tooth. Fibrodentin (FD) is formed by fibrodentinoblasts (FDB) that originate from undifferentiated subodontoblast/pulp cells after the death of primary odontoblasts (PO). It is deposited at the interface between primary dentin and the odontoblast necrosis area to seal tubules that communicate with the caries lesion. When cells are entrapped in the FD matrix, they become fibrodentinocytes (FDC). Continuous FD deposition takes FDB and the underlying pulp tissue far from the lesion (black arrows) and protects them from invading pathogens (intratubular brown dots).

After a variable amount of fibrodentin is deposited, some fibrodentinoblasts and/or migrating pulp cells ­arriving on-site adhere to the dentin matrix, elongate and adopt an odontoblast-like morphology with a long cell process and a cell body in which organelles are polarized. These cells produce an extracellular matrix which is close to fibrodentin in composition, with a relatively large amount of type III collagen and fibronectin in addition to type I collagen. Once mineralized, this tissue is called orthodentin, because of its tubular structure that results from the progressive entrapment of the cell processes. Replacement odontoblasts have a morphological aspect more or less similar to primary odontoblasts, but their cell body is less elongated and polarized. They secrete, in a polarized fashion, most of the dentin components produced by primary odontoblasts, but their functional properties appear to be different as primary odontoblasts do not synthesize type III collagen and fibronectin. Therefore they must be regarded more as odontoblast-like cells than true odontoblasts. Orthodentin resembles primary and secondary dentins, but its structure is coarser, tubules are less numerous, and are more irregular and tortuous. Tubules generally have a non-parallel orientation. It is considered by most authors that orthodentin is secreted after fibrodentin, but the situation is often more complex. Indeed, fibrodentin and orthodentin can be deposited simultaneously by adjacent fibrodentinoblasts and replacement odontoblasts, and are thus impossible to distinguish from each other. This reflects the great variability of the pulp response to injury and the diversity of dentin-like

FIGURE 30.10  Schematic representation of orthodentin in a human carious tooth. Orthodentin (OD) is formed by odontoblast-like cells (ODL) that originate from undifferentiated subodontoblast/pulp cells after the death of primary odontoblasts (PO). It is most often secreted after the deposition of a layer of fibrodentin (FD). Reparative orthodentin takes the pulp tissue far from the lesion (black arrows) to protect it from invading pathogens (intratubular brown dots). FDB: Fibrodentinoblasts; FDC: Fibrodentinocytes.

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FIGURE 30.11  Complex reparative dentin structure in a human carious tooth. Masson trichrome staining. (a) When the pulp tissue (P) is injured by a dentin caries lesion, primary odontoblasts (PO) in direct contact with the lesion through dentin tubules (white arrow) may die (DO) and be replaced by fibrodentinoblasts (FDB), odontoblast-like cells (OLC), or both, that deposit reparative dentin (RD). In all cases, the RD structure differs from that of primary dentin (PD). It is much less organized and may contain entrapped cells (white arrowheads). Dilated blood vessels (DBV) are present beneath dying odontoblasts and reflect the inflammatory status of the pulp and the activation of the local immune defense system. (b) At higher magnification, the few tubules present (white arrows) have an irregular shape and are oriented in a non-parallel manner.

tissues secreted by dental pulp cells to protect the living tissue from oral invaders (Figures 30.10, 30.11). Studies have demonstrated that bioactive molecules in the fibrodentin matrix are able to promote the differentiation of replacement odontoblasts, a phenomenon that generally takes place as part of the pulp healing process in an inflammation-controlled environment. These molecules include fibronectin and TGF-β1 that provide the molecular interacting network controlling the initiation of orthodentin formation [65]. Molecules potentially released from inflammatory cells, including TNF-alpha and interleukin1β, might also influence RpD formation, since they are able to increase the dentinogenic process in dental pulp cells in vitro when used at low concentration [73]. A variety of molecular signals may thus contribute to the dentinogenic response after complex dentin-pulp injury. All these molecules and their related signaling pathways are considered potential targets for future therapeutic strategies designed to stimulate reparative dentinogenesis necessary to pulp vitality preservation.

30.5  NON-DENTINOGENIC FUNCTION OF ODONTOBLASTS As seen above, the main function of odontoblasts is to synthesize dentin. However, new functions for these cells are now emerging. The in vitro differentiation of dental pulp cells into odontoblasts has permitted the study of the phenotype of odontoblasts at the transcriptome level using molecular biology techniques such as cDNA libraries and microarrays [74,75]. These techniques have revealed new

genes expressed by odontoblasts. Some of them were known to be involved in the immune and inflammatory responses (coding for chemokines, major histocompatibility complex, interleukins, etc.), and others were associated with a ­neuronal phenotype (encoding reelin, neuronal ion channels, neuropeptides, etc.). These discoveries allowed investigation of new fields concerning the function of mature odontoblasts.

30.5.1  Odontoblasts in the Dental Pulp Immune and Inflammatory Response Due to their situation at the dentin-pulp interface and entrapment of their long cell processes in dentin tubules, odontoblasts are the first cells encountered by dentin invading pathogens and/or their released components. They thus represent, in the tooth, the first line of defense for the host [76]. Experiments have shown that odontoblasts express membrane and cytoplasmic receptors to bacterial byproducts, and that they are able to elicit inflammatory and immune reactions in the pulp when these receptors are activated [58,77–79]. In particular, odontoblasts express several pathogen recognition molecules (PRMs) of the Toll-like receptor (TLR) family including TLR2, TLR4, and TLR9. TLR2 and TLR4, present in the odontoblast cell membrane, are able to bind bacterial cell wall components. TLR2 binds lipoteichoic acid and lipoproteins from Gram-positive bacteria, whereas TLR4 binds lipopolysaccharide from Gramnegative ones. TLR9, present in the endosomal membrane, binds bacterial deoxyribonucleic acid. Odontoblasts also express cytoplasmic PRMs including the NOD-like receptor NOD2 that recognizes dipeptide units of the bacterial cell

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wall component peptidoglycan [80]. Sensing of these various components by odontoblast PRMs was shown to induce activation of Nuclear Factor (NF)-κB and p38 intracellular signaling pathways, as well as production of inflammatory cytokines, including interleukin 6 and chemokines such as CC-chemokine ligand (CCL2) and CXC-chemokine ligand (CXCL1, CXCL2, CXCL8, and CXCL10) [78,81,82]. By early attraction of immune cells at the pulp-dentin interface, odontoblast chemokines would participate to the triggering of the immune and inflammatory pulp responses intended to combat dentin-invading pathogens. In particular, they would be involved in the recruitment and accumulation of antigenpresenting dendritic cells in the odontoblast layer, which is a strategic location to sample foreign antigens diffusing through affected dentin tubules [58]. Some of these chemokines (CXCL2, CXCL8) are strongly angiogenic, and may be involved in the increase of vascularization observed in bacteria-challenged inflamed pulps (Figure 30.12). In vitro stimulation of odontoblasts by high concentrations of purified bacterial cell wall components, which aims at mimicking the action of bacteria in rapidly progressing dentin caries, leads to down-regulation of collagen type I and dentin sialophosphoprotein, two major dentin matrix components, and TGF-β1, a known inducer of dentin formation. Collectively, these data suggest that odontoblasts, upon severe bacterial stimulation, undergo a functional switch to decrease their specialized functions of dentin matrix synthesis and mineralization while orienting their metabolic activity toward the production of molecules responsible for the triggering and development of pulp immune and inflammatory responses.

Therapeutic strategies intended to rapidly reverse this switch after caries tissue removal by the dental practitioner might be useful to dampen the odontoblast immune response and promote the formation of a new dentin layer protective for the underlying healing pulp.

30.5.2  Odontoblasts as Sensor Cells Emerging evidence supports a role for odontoblasts as sensory cells that directly mediate the tooth pain sensation [83]. The idea that odontoblasts may sense external stimuli and transduce the signal to nearby nerves cells is supported by the close apposition of the odontoblasts to the dentinal nerve terminals [84]. In addition, odontoblasts are known to express several classes of ion channels which have been implicated in nociception and signal propagation, including L-type Ca2+ channels, mechanosensitive K+ channels, and voltage-gated Na+ channels [85–90]. More recently, several members of the transient receptor potential (TRP) superfamily of ion channels have been identified in odontoblasts. These receptors play a critical role in sensory physiology, where they act as transducers for thermal, mechanical, and chemical stimuli [91]. Subfamilies of heat-sensing TRP channels, such as TRPV1 (> 43 °C), TRPV2 (> 52 °C), and TRPV3 (33-39 °C), are functionally expressed in odontoblasts. In addition, human odontoblasts express functional TRPM8 (< 25 °C) and TRPA1 (< 17 °C), which are respectively cool and cold sensors [92–94]. Moreover, TRPP1 and TRPP2, which have been shown to act together as a mechanical receptor,

FIGURE 30.12  Putative role of odontoblasts in the initiation of dental pulp immunity to cariogenic bacteria. The growth of bacteria present in dentin caries lesions leads to the release of various components from their cell wall. It has been postulated that these components (brown dots) can diffuse through the tubule lumen (T) to the odontoblast layer (PO), where they are sensed by specific pathogen recognition receptors (yellow cup) at the cell surface of the odontoblast process and/or cellular body. Activation of specific odontoblast intracellular pathways (black dotted line) leads to the production of proinflammatory mediators, including chemokines (green squares) secreted at the basal pole of the cell. In parallel, odontoblasts strongly decrease their production of predentin (Pd). Odontoblast chemokines, upon binding to cell membrane-specific receptors (orange boxes), attract responsive immune cells which reside in the subodontoblast area, mainly antigen-presenting immature dendritic cells (iDC) that ensure pulp immunosurveillance. IDC then migrate to the odontoblast layer (open black arrows) for uptaking bacterial components that arrive at the pulpal tubule end, thus amplifying the local immune response.

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are present at the surface of odontoblasts, and appear to be located at the base of the primary cilium of the cell [18]. Altogether, these observations make odontoblasts suitable candidates to sense external stimuli (thermal and mechanical stimuli) in a manner similar to that reported in skin keratinocytes [95,96]. Therefore, an important question arises concerning this putative role of odontoblasts as sensory receptor cells involved in the nociceptive signal transduction mechanism: how can odontoblasts transduce the noxious signal to the surrounding nerve fiber, and then participate in the generation of tooth pain? Even if this point is not elucidated today, several tracks are emerging. One can keep in mind that even if no synapses have been identified between these two cell types, a close association of odontoblast and nerve fiber has been observed by electron microscopy, with a restricted cleft delimited by the neuronal and odontoblast membranes of about 20 nm [84]. 1. It has been shown that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na+ currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of sodium channels which clustered at the site of odontoblast-nerve fiber contact. Therefore, a firing odontoblast may induce a supraliminal depolarization in the unmyelinated axon to impulse a spike and then propagate the signal in the nerve fiber [87]. 2. The release in the gap space of mediators from stimulated odontoblasts can be another way to communicate with the nerve ending. A promising mediator is extracellular ATP (eATP). eATP has been shown to function as a signaling molecule in taste bud cells by activating P2X receptors expressed on sensory afferent nerves. In teeth, P2X3 are expressed on afferent nerve terminals, particularly in the odontoblastic layer, and odontoblasts can release ATP through pannexin-3, an ATP-permeable hemi-channel recently identified. Thus, ATP released from stimulated odontoblasts could activate P2X receptors on nearby nerves and generate the pain sensation [83]. More recently, microarray analysis focused on gene expression profiles of human native and cultured odontoblasts revealed a set of neuronal genes including galanin (GAL) [75] known to be involved in sensory signal transduction. Obviously, identification of galanin receptor 1 confined to nerve fibers of the dental pulp near the odontoblasts also suggests a possible involvement of this peptide as a neuromediator.

30.6 CONCLUSION Odontoblasts are key cells of the tooth. Their primary role is to synthesize the dentin of the crown and the root. However, contrary to ameloblasts, these cells remain living during the whole life of the tooth, and they can be ­reactivated during

pathological stimulations such as caries or ­cavity preparation to synthesize a new layer of dentin. This property has been taken into account in order to develop new filling biomaterials designed to stimulate the reparative d­ entinogenesis necessary to pulp vitality preservation. The next step for these biomaterials will be to functionalize them with ­biomolecules (growth factors or dentin ECM peptides) in order to strengthen their reparative activity. Moreover, new functions for these cells have been shown that will open new fields for the treatment of caries with attempts to limit the inflammatory process by reducing the synthesis of chemokines by odontoblasts, and the treatment of dental pain.

ACKNOWLEDGMENT The authors acknowledge the iCAP service of the University Lyon 1 for its help with the realization of Figures 30.1-30.3, 30.5 and 30.6.

ABBREVIATIONS BMP bone morphogenetic protein BSP bone sialoprotein CRcD circumpulpal reactionary dentin DMP1 dentin matrix protein 1 DSPP dentin sialophosphoprotein DSP dentin sialoprotein DPP dentin phosphoprotein DGP dentin glycoprotein ECM extracellular matrix eATP extracellular ATP GAG glycosaminoglycan GAL galanin GH growth hormone IGF-1 insulin-like growth factor-1 Kda kilo dalton MEPE matrix extracellular phosphoglycoprotein MMP matrix metalloproteinase NCPs non-collagenous proteins OPN osteopontin PG proteoglycans PRMs pathogen recognition molecules RcD reactionary dentin RGD motifs arginine-glycine-aspartic acid motifs RpD reparative dentin SIBLINGs  small integrin-binding ligand, N-linked glycoproteins family SLRPs small leucine-rich proteoglycans SRcD sclerotic reactionary dentin TGFβ transforming growth factor TLR toll-like receptor TNF tumor necrosis factor TRP transient receptor potential

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