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Odontoblasts: the cells forming and maintaining dentine
The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
Cells in focus
Odontoblasts: the cells forming and maintaining dentine Victor E. Arana-Chavez∗ , Luciana F. Massa Laboratory of Mineralized Tissue Biology, Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524 Cidade Universitaria, 05508-900 São Paulo, SP, Brazil Received 13 September 2003; received in revised form 19 December 2003; accepted 13 January 2004
Abstract Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth. © 2004 Elsevier Ltd. All rights reserved. Keywords: Odontoblasts; Dentine; Cell differentiation; Dentinogenesis; Mineralization
Cell facts • Odontoblasts form the dentine. • Odontoblasts may deposit new layers of reactionary/reparative dentine. • Odontoblasts may play a role in dentine sensitivity.
1. Introduction Odontoblasts are the cells responsible for the formation of dentine, the collagen-based mineralized tissue that forms the bulk of teeth. Odontoblasts derive from ectomesenchymal cells, exhibit a tall columnar shape, and establish a continuous single layer with a clear epithelioid appearance. After dentinogenesis, they are aligned along the periphery of the dental pulp thus playing a role in the maintenance of the tooth integrity owing to their capacity of depositing new layers ∗ Corresponding author. Tel.: +55-11-3091-7308; fax: +55-11-3091-7402. E-mail address: [email protected] (V.E. Arana-Chavez).
of dentine throughout life. In addition, newly differentiated odontoblast-like cells may also form a layer of reparative dentine after some tissue injuries. Since dentine is a tissue analogous to bone, its extracellular matrix shares many similarities with the bone matrix. Thus, whereas collagen type I is the major organic component, the extracellular dentine matrix also contains a variety of noncollagenous proteins. Odontoblasts synthesize and secrete all the matrix constituents and therefore they exhibit well develop synthesis organelles. The odontoblast layer is separated from the mineralized dentine by a 10–40 m-thick layer of unmineralized matrix, the predentine, which is similar to the osteoid that separates the osteoblasts/bone lining cells from the bone mineralized matrix. However, the
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.01.006
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V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
odontoblast differs from the osteoblast by possessing a single cell process that arises from its tall columnar cell body and penetrates into the dentinal tubule. Thus, the formed dentine is crossed by thousands of dentinal tubules and canaliculae, which contain the odontoblast processes and their branches for playing a role in communication with the dentine matrix; the cell bodies form the odontoblast layer at the periphery of dental pulp. Although the exact mechanisms on dentine sensitivity and dental pain are still unknown, it is believed that odontoblasts play a key role either by establishing a dentinal/predentinal compartment in which the dentinal fluid is confined, thus permitting a hydrodynamic phenomenon; other evidences, however, consider the possibility of odontoblast might be a direct sensory receptor or even they may conduct the nervous impulses to neural pathways.
they start their differentiation into ameloblasts, perhaps modifying the inner basal lamina (bl) in order to induce the post-mitotic outer cells of the dental papilla to differentiate into odontoblasts (Ruch, Lesot, & Begue-Kirn, 1995). Some growth factors, as TGF-1, BMP 2 and 4, as well as IGF, have been shown to be expressed by the epithelial cells at this early moment of development in which the carefully orchestrated sequence of epithelial-mesenchymal interactions continues (Thesleff, 2003). It is also possible that some small amounts of amelogenin molecules that are secreted by the differentiated ameloblasts or amelogenin isoforms or even peptides may participate in that inductive phenomenon (Veis, 2003). The differentiating odontoblasts start the secretion of dentine before the initiation of enamel matrix secretion by ameloblasts.
2. Cell origin
3. Functions: the formation of normal and reactionary/reparative dentine
Odontoblasts are derived from embryonic connective cells that are called ectomesenchymal cells because of their well-established origin from the neural crest. Cells from the cranial neural crest early migrate to populate the first branchial (pharyngeal) arch regions forming the ectomesenchyme that gives arise to all the soft and hard connective craniofacial tissues. First, the primitive oral epithelium thickens and proliferates into the underlying ectomesenchyme that undergoes a conspicuous cellular condensation, which results in the establishment of dental papilla. A variety of regulated and reciprocal epithelial-ectomesenchymal interactions are responsible for the initiation of tooth formation (Cobourne & Sharpe, 2003). When odontogenesis starts, although signaling continues, proliferation of epithelial cells is a noticeable event occurring at the bud, cap, and bell stages of odontogenesis. The differentiation of odontoblasts begins at the tip of the future cusps only after the tooth germ has reached the bell stage and the shape of the crown has been established. The ectomesenchymal cells of dental papilla exhibit a high nucleus to cytoplasm ratio. They possess several short processes and contain relatively abundant polyribosomes but few cisternae of rough endoplasmic reticulum (RER), Golgi saccules and mitochondria. Signaling molecules arise from the epithelial cells as
Dentine is the mineralized tissue that constitutes the bulk of the tooth. It is intimately related to the dental pulp with which it shares the same embryological origin from the dental papilla. As mentioned above, deposition of the extracellular dentine matrix begins when the outer cells from dental papilla are induced to differentiate into odontoblasts. Then, they start their polarization while elongating and developing synthesis organelles. Differentiating odontoblasts establish a cylindrical shape and the nucleus locates at the proximal pole of the cell body (Fig. 1). The Golgi apparatus becomes located in the supranuclear region while numerous reticulum endoplasmic cisternae occupy the majority of the distal cytoplasm. As polarization and differentiation of odontoblasts start, few small and short processes arise at the odontoblast surface facing the enamel organ. Differentiating odontoblasts then secrete the organic matrix for establishing the first dentinal layer—the mantle dentine. The earlier unmineralized matrix is mainly composed by collagen fibrils, which are deposited into the preexisting ground substance of the dental papilla. The mantle dentine collagen fibrils that are conspicuously large and long, align to form right angles to the basal lamina. As the differentiating odontoblasts deposit collagen and other mantle dentine components, numerous small, spherical, membrane-limited bodies, 50–150 nm in diameter
V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
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Fig. 1. Light micrograph from a tooth germ at the early stage of dentinogenesis. The differentiation of odontoblasts (O) and the onset of dentine deposition are gradually more advanced from the left to the right side of the micrograph. Odontoblasts exhibit a tall columnar shape, with their nucleus located at their basal region. Whereas at early stages the forming matrix is unmineralized (asterisk), when it becomes mineralized (arrows), a layer of newly formed matrix (the predentine, Pd) is always interposed between the odontoblasts and the mineralizing front. A, differentiating ameloblasts. Hematoxylin-eosin staining (900×).
that are known as matrix vesicles (MV), bud off from their distal end. It is owing to their small size, the matrix vesicles are only visible under the transmission electron microscope where they show a variable amorphous content (Fig. 2). They establish a close relation to proteoglycans/glycosaminoglycans that are thought to bind calcium ions. The first mineral crystals appear within matrix vesicles, as occurs in immature bone and in calcifying cartilage (Bonucci, 2002). In the meantime, the collagen fibrils, as well as the rest of the mantle dentine extracellular matrix remain devoid of mineral. As differentiating odontoblasts secrete the first layers of mantle dentine, they reach around 30–40 m in length, become more closely packed and develop numerous gap and adherents junctions, thus establishing a cellular layer with a well defined epithelioid appearance. The differentiating odontoblasts begin to move toward the center of the dental papilla whereas one
of the cell processes gradually becomes accentuated. This cell process that is left behind into the forming dentine matrix constitutes the odontoblast process around which the dentine matrix mineralizes, thus forming a dentinal tubule. As each odontoblast leaves behind one single cell process, thousands of dentinal tubules cross the dentine from the dentinoenamel junction to the pulp. The odontoblast process lacks cell organelles but contains numerous longitudinally arranged actin filaments and microtubules (Fig. 3). At stages in which most matrix vesicles are fully mineralized, odontoblasts appear taller (50–60 m in length) than at initial stages and focal tight junctions assemble between them. Different from those in epithelia, the tight junctions between differentiating odontoblasts play a role that is more related to the crescent cell polarization and differentiation than to permeability functions (Arana-Chavez & Katchburian, 1997). Recent studies have shown that the tight
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Fig. 2. Electron micrograph of a region of early dentine mineralization showing numerous matrix vesicles (MV), some of them containing fine mineral crystals (arrowheads), among the organic mantle dentine matrix rich in collagen fibrils (C). At these stages, the basal lamina (bl) is partially degraded, while the differentiating ameloblasts (A) present some short projections towards the developing mantle dentine. The differentiating odontoblasts also possess cell processes (P), which appear sectioned in different directions (26,000×). The inset shows a high-resolution post-embedding colloidal gold immunocytochemical preparation for dentin matrix protein 1 (DMP 1). The gold particles are present around a mineralization foci of the mantle dentin in a more advanced stage than that of the large micrograph, in which the mineral is spreading from matrix vesicles to the surrounding matrix (28,000×).
junctions between differentiating odontoblasts contain more ZO-1, a cytoplasmic junctional protein that is close related to actin filaments, than occludin and claudins, two classes of transmembrane proteins that are related to sealing properties of tight junctions (João & Arana-Chavez, 2003,2004). Nevertheless, with the assembly of tight junctions between odontoblasts an apical domain is established on their plasma membrane and they become fully differentiated. Matrix vesicles are no longer released and the secretion of several noncollagenous matrix proteins begins (Arana-Chavez & Katchburian, 1998). The differentiated odontoblasts that exhibit a exuberant secreting apparatus (Fig. 4) continue to deposit a somewhat different dentine—that is called circumpulpal, which forms the bulk of the whole dentine. In circumpulpal dentine, the collagen fibrils are thinner and they are more densely packed than those found in mantle dentine, many of them running around the dentinal tubules. The noncollagenous dentine matrix proteins
synthesized and secreted by the fully differentiated odontoblasts include dentine sialophosphoprotein (DSPP)—a large parental protein that subsequently suffers cleavage to originate two products, dentine sialoprotein (DSP) and dentine phosphoprotein (DPP), dentine matrix proteins 1, 2, and 3 (DMP-1, DMP-2, and DMP-3, respectively), and small amounts of four noncollagenous proteins (osteopontin, bone sialoprotein, osteonectin, and osteocalcin) that are abundant in bone (Butler & Ritchie, 1995; He, Dahl, Veis, & George, 2003; Papagerakis et al., 2002;). Because mineralization of circumpulpal dentine occurs without the presence of matrix vesicles, it is likely that noncollagenous matrix proteins play a key role to establish favorable conditions for the mineral deposition spreading through the fibrilar and interfibrilar regions of matrix (Fig. 2, inset). The dentine formed by secretion of collagen and noncollagenous proteins is called intertubular dentine. After formation of mantle dentine, each odontoblast
V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
Fig. 3. Electron micrograph showing the mineralized dentine/predentine/odontoblast layer interface. Whereas the mineralized layer of dentine (MD) exhibits higher electron opacity, the underlying unmineralized predentine (Pd) exhibits numerous collagen fibrils. The density of collagen in predentine gradually decreases from the mineralization front, where there is no matrix vesicles, to the lower regions, in which there are abundant proteoglycans and other noncollagenous matrix components. An odontoblasts processes (P) that arises from the odontoblast cell body (O) crosses the entire predentine and penetrates into a tubule of the mineralized dentine. Some branches (double arrowheads) of the odontoblast processes are clearly observed in other odontoblast process (5400×).
secrete additional noncollagenous components from the end of its cell process. This matrix mineralizes rapidly between the previously formed intertubular dentine and the odontoblast process and constitutes the peritubular dentin. Peritubular dentine is hypermineralized in relation to intertubular dentine and forms the wall of the dentinal tubule. It is owing to the peritubular dentine is secreted throughout life of the odontoblast, it occurs a gradual obliteration of the dentinal tubules with aging. The dentine formed up to the completion of root development is defined as primary dentine. Because
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Fig. 4. Electron micrograph showing the Golgi region of a secretory odontoblast in which well-developed synthesis organelles such as cisternae of rough endoplasmic reticulum (RER), Golgi stacks (G), and secretory granules (g) are abundant. M, mitochondria (32,500×).
the odontoblasts deposit dentine throughout life, a secondary dentine is laid down at a much slower rate than the primary dentine. Both primary and secondary dentine posses a similar structure. Formation of secondary dentine mainly occurs at the roof and floor of the pulp chamber, thus leading to an increasing reduction of the pulp volume, as well as of the height of pulp horns. At these stages the odontoblasts exhibit less developed synthesis organelles than at stages of primary dentine formation. It is important to say that the odontoblasts are post-mitotic cells that remain for tooth life. Odontoblasts might, however, form a third type of dentine—the tertiary dentine. There are two subtypes of tertiary dentine because sometimes the original odontoblasts die and then a new generation of odontoblast-like cells forms a new dentinal layer. Thus, the tertiary dentine formed by the original odontoblasts in response to attrition, caries or other stimuli such as some restorative procedures is called
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reactionary dentine. In general, the tubules of reactionary dentine continue with those of secondary dentine, while the thickness of the newly formed layer is related to the intensity and duration of the stimulus. The reactionary dentine possesses an organic matrix as well as a mineral content similar to those found in primary and secondary dentine. In other cases, however, some external factors such as dental caries may irreversibly affect the odontoblast layer in a given region. Cavity preparation during restorative procedures in dentistry may also affect the odontoblast layer. Whereas shallow cavities produce disruption of the junctions between odontoblasts, deep cavity preparation has been shown to cause aspiration of the odontoblast body up into the dentinal tubule thus disrupting its cytoplasmic content and resulting in cell death. Although these situations ultimately promote several pulp pathologies, when they have a reversible behavior, newly differentiated odontoblast-like cells may arise to forming another subtype of tertiary dentine that is called reparative dentine (Smith et al., 1995). It is assumed that undifferentiated ectomesenchymal cells present at the pulp, especially at the subodontoblastic layer, become the cells that produce the reparative dentine. Although the exact mechanisms that induce the differentiation of pulpal cells into odontoblast-like cells are still unknown, the new cells arise after a growth factor signaling that triggers a cascade of events involving cell division, chemotaxis, cell migration, cell adhesion and cytodifferentiation. Reparative dentine is in the majority of cases quite different morphologically from reactionary dentine. It may contain cellular inclusions, which resemble the osteocytes from bone. In addition, their extracellular matrix contains some noncollagenous proteins (osteopontin, for example) that are more typical for bone than of dentine. For these reasons, sometimes the reparative dentine shows an osteodentine appearance. It is important to say that differentiation of pulpal cells into odontoblast-like cells takes place in absence of enamel organ epithelium and basement membrane. It is believed that cells from the subodontoblastic layer—and perhaps other undifferentiated pulpal cells—may acquire a latent capability for this specific differentiation during the morphogenetic epithelial-ectomesenchymal interactions that took place at the bell stage of odontogenesis, despite some
components of dentine matrix have also been involved in these mechanisms. Nevertheless, it appears that absence of inflammation and sufficient oxygen supply within the pulp may improve the promotion of odontoblast-like cells cytodifferentiation (Tziafas, 1995).
4. Associated pathologies Several genetic disorders exhibiting an autosomal dominant pattern of inheritance may affect the dentine formation by odontoblasts. They are classified into two main groups, dentinogenesis imperfecta and dentine dysplasia, both with several subtypes. Dentinogenesis imperfecta type I (DGI-I) is associated with osteogenesis imperfecta that is caused by mutations of genes coding for pro-␣ 1(1) chains and pro-␣2(1) chains of type I collagen, which occur on the long arm of chromosomes 7 and 17. These mutations result in helix-destabilizing glycine substitutions, single exon-skipping mutations, and premature stop codons, which produce shortened and elongated collagen fibrils. In DGI-I, the dentine is hypomineralized and the overlying enamel is frequently brittle and easily fractures from it. The dentinogenesis imperfecta type II (DGI-II) is considered the classical dentinal type; dentinogenesis imperfecta type III (DGI-III) is also restricted to the dentine and is the most severe but it appears to affect restricted populations from the south of Maryland, USA. Both DGI-II and DGI-III are not collagen defects but two disorders of dentine mineralization; the basic defects appear to be originated within the human chromosome 4, on the gene cluster 4q21, from which the main noncollagenous dentine matrix proteins DSPP and DMP 1 and 2, as well as the novel protein—matrix extracellular phosphoglycoprotein/osteoblast-osteocyte factor 45 kDa (MEPE/OF45)—are encoded. Dentine dysplasia, on the other hand, is a rarer disease characterized by the obliteration of the pulp chamber and by resulting in teeth with short and cone-shaped root(s); the mineralization in the coronal dentine is normal or sometimes higher. Some disorders related to the gene expression of the noncollagenous matrix proteins, especially DSPP, are likely to play a role in the etiology of dentine dysplasia (MacDougall, Simmons, Gu, & Dong, 2002).
V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
Acknowledgements The authors would like to thank Sabrina S. Tessi and Gaspar F. de Lima by their technical assistance as well as Fapesp and CNPq (Brazil) by their financial support during the studies on odontoblast biology. References Arana-Chavez, V. E., & Katchburian, E. (1997). Development of tight junctions between odontoblasts in early dentinogenesis as revealed by freeze-fracture. The Anatomical Record, 248, 332– 338. Arana-Chavez, V. E., & Katchburian, E. (1998). Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarization. European Journal of Oral Sciences, 106, 132–136. Bonucci, E. (2002). Crystal ghosts and biological mineralization: Fancy spectres in an old castle, or neglected structures worthy of belief? Journal of Bone Mineral and Metabolism, 20, 249–265. Butler, W. T., & Ritchie, H. H. (1995). The nature and functional significance of dentin extracellular matrix proteins. International Journal of Developmental Biology, 39, 169–179. Cobourne, M. T., & Sharpe, P. T. (2003). Tooth and jaw: Molecular mechanisms of patterning in the first branchial arch. Archives of Oral Biology, 48, 1–14. He, G., Dahl, T., Veis, A., & George, A. (2003). Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1. Nature Materials, 2, 552–558.
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João, S. M., & Arana-Chavez, V. E. (2003). Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs. Histochemistry and Cell Biology, 119, 21–26. João, S. M., & Arana-Chavez, V. E. (2004). Tight junctions in differentiating ameloblasts and odontoblasts differentially express ZO-1, occludin and claudin-1 in early odontogenesis of rat molars. The Anatomical Record, 274, in press. MacDougall, M., Simmons, D., Gu, T. T., & Dong, J. (2002). MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21. Connective Tissue Research, 43, 320–330. Papagerakis, P., Berdal, A., Mesbah, M., Peuchmaur, M., Malaval, L., & Nydegger, J. et al., (2002). Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth. Bone, 30, 377–385. Ruch, J. V., Lesot, H., & Begue-Kirn, C. (1995). Odontoblast differentiation. International Journal of Developmental Biology, 39, 51–68. Smith, A. J., Cassidy, N., Perry, H., Begue-Kirn, C., Ruch, J. V., & Lesot, H. (1995). Reactionary dentinogenesis. International Journal of Developmental Biology, 39, 273–280. Thesleff, I. (2003). Epithelial-mesenchymal signaling regulating tooth morphogenesis. Journal of Cell Science, 116, 1647– 1648. Tziafas, D. (1995). Basic mechanisms of cytodifferentiation and dentinogenesis during dental pulp repair. International Journal of Developmental Biology, 39, 281–290. Veis, A. (2003). Amelogenin splice gene products: Potential signaling molecules. Cellular and Molecular Life Sciences, 60, 30–55.
Cells in focus
Odontoblasts: the cells forming and maintaining dentine Victor E. Arana-Chavez∗ , Luciana F. Massa Laboratory of Mineralized Tissue Biology, Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524 Cidade Universitaria, 05508-900 São Paulo, SP, Brazil Received 13 September 2003; received in revised form 19 December 2003; accepted 13 January 2004
Abstract Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth. © 2004 Elsevier Ltd. All rights reserved. Keywords: Odontoblasts; Dentine; Cell differentiation; Dentinogenesis; Mineralization
Cell facts • Odontoblasts form the dentine. • Odontoblasts may deposit new layers of reactionary/reparative dentine. • Odontoblasts may play a role in dentine sensitivity.
1. Introduction Odontoblasts are the cells responsible for the formation of dentine, the collagen-based mineralized tissue that forms the bulk of teeth. Odontoblasts derive from ectomesenchymal cells, exhibit a tall columnar shape, and establish a continuous single layer with a clear epithelioid appearance. After dentinogenesis, they are aligned along the periphery of the dental pulp thus playing a role in the maintenance of the tooth integrity owing to their capacity of depositing new layers ∗ Corresponding author. Tel.: +55-11-3091-7308; fax: +55-11-3091-7402. E-mail address: [email protected] (V.E. Arana-Chavez).
of dentine throughout life. In addition, newly differentiated odontoblast-like cells may also form a layer of reparative dentine after some tissue injuries. Since dentine is a tissue analogous to bone, its extracellular matrix shares many similarities with the bone matrix. Thus, whereas collagen type I is the major organic component, the extracellular dentine matrix also contains a variety of noncollagenous proteins. Odontoblasts synthesize and secrete all the matrix constituents and therefore they exhibit well develop synthesis organelles. The odontoblast layer is separated from the mineralized dentine by a 10–40 m-thick layer of unmineralized matrix, the predentine, which is similar to the osteoid that separates the osteoblasts/bone lining cells from the bone mineralized matrix. However, the
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.01.006
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V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
odontoblast differs from the osteoblast by possessing a single cell process that arises from its tall columnar cell body and penetrates into the dentinal tubule. Thus, the formed dentine is crossed by thousands of dentinal tubules and canaliculae, which contain the odontoblast processes and their branches for playing a role in communication with the dentine matrix; the cell bodies form the odontoblast layer at the periphery of dental pulp. Although the exact mechanisms on dentine sensitivity and dental pain are still unknown, it is believed that odontoblasts play a key role either by establishing a dentinal/predentinal compartment in which the dentinal fluid is confined, thus permitting a hydrodynamic phenomenon; other evidences, however, consider the possibility of odontoblast might be a direct sensory receptor or even they may conduct the nervous impulses to neural pathways.
they start their differentiation into ameloblasts, perhaps modifying the inner basal lamina (bl) in order to induce the post-mitotic outer cells of the dental papilla to differentiate into odontoblasts (Ruch, Lesot, & Begue-Kirn, 1995). Some growth factors, as TGF-1, BMP 2 and 4, as well as IGF, have been shown to be expressed by the epithelial cells at this early moment of development in which the carefully orchestrated sequence of epithelial-mesenchymal interactions continues (Thesleff, 2003). It is also possible that some small amounts of amelogenin molecules that are secreted by the differentiated ameloblasts or amelogenin isoforms or even peptides may participate in that inductive phenomenon (Veis, 2003). The differentiating odontoblasts start the secretion of dentine before the initiation of enamel matrix secretion by ameloblasts.
2. Cell origin
3. Functions: the formation of normal and reactionary/reparative dentine
Odontoblasts are derived from embryonic connective cells that are called ectomesenchymal cells because of their well-established origin from the neural crest. Cells from the cranial neural crest early migrate to populate the first branchial (pharyngeal) arch regions forming the ectomesenchyme that gives arise to all the soft and hard connective craniofacial tissues. First, the primitive oral epithelium thickens and proliferates into the underlying ectomesenchyme that undergoes a conspicuous cellular condensation, which results in the establishment of dental papilla. A variety of regulated and reciprocal epithelial-ectomesenchymal interactions are responsible for the initiation of tooth formation (Cobourne & Sharpe, 2003). When odontogenesis starts, although signaling continues, proliferation of epithelial cells is a noticeable event occurring at the bud, cap, and bell stages of odontogenesis. The differentiation of odontoblasts begins at the tip of the future cusps only after the tooth germ has reached the bell stage and the shape of the crown has been established. The ectomesenchymal cells of dental papilla exhibit a high nucleus to cytoplasm ratio. They possess several short processes and contain relatively abundant polyribosomes but few cisternae of rough endoplasmic reticulum (RER), Golgi saccules and mitochondria. Signaling molecules arise from the epithelial cells as
Dentine is the mineralized tissue that constitutes the bulk of the tooth. It is intimately related to the dental pulp with which it shares the same embryological origin from the dental papilla. As mentioned above, deposition of the extracellular dentine matrix begins when the outer cells from dental papilla are induced to differentiate into odontoblasts. Then, they start their polarization while elongating and developing synthesis organelles. Differentiating odontoblasts establish a cylindrical shape and the nucleus locates at the proximal pole of the cell body (Fig. 1). The Golgi apparatus becomes located in the supranuclear region while numerous reticulum endoplasmic cisternae occupy the majority of the distal cytoplasm. As polarization and differentiation of odontoblasts start, few small and short processes arise at the odontoblast surface facing the enamel organ. Differentiating odontoblasts then secrete the organic matrix for establishing the first dentinal layer—the mantle dentine. The earlier unmineralized matrix is mainly composed by collagen fibrils, which are deposited into the preexisting ground substance of the dental papilla. The mantle dentine collagen fibrils that are conspicuously large and long, align to form right angles to the basal lamina. As the differentiating odontoblasts deposit collagen and other mantle dentine components, numerous small, spherical, membrane-limited bodies, 50–150 nm in diameter
V.E. Arana-Chavez, L.F. Massa / The International Journal of Biochemistry & Cell Biology 36 (2004) 1367–1373
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Fig. 1. Light micrograph from a tooth germ at the early stage of dentinogenesis. The differentiation of odontoblasts (O) and the onset of dentine deposition are gradually more advanced from the left to the right side of the micrograph. Odontoblasts exhibit a tall columnar shape, with their nucleus located at their basal region. Whereas at early stages the forming matrix is unmineralized (asterisk), when it becomes mineralized (arrows), a layer of newly formed matrix (the predentine, Pd) is always interposed between the odontoblasts and the mineralizing front. A, differentiating ameloblasts. Hematoxylin-eosin staining (900×).
that are known as matrix vesicles (MV), bud off from their distal end. It is owing to their small size, the matrix vesicles are only visible under the transmission electron microscope where they show a variable amorphous content (Fig. 2). They establish a close relation to proteoglycans/glycosaminoglycans that are thought to bind calcium ions. The first mineral crystals appear within matrix vesicles, as occurs in immature bone and in calcifying cartilage (Bonucci, 2002). In the meantime, the collagen fibrils, as well as the rest of the mantle dentine extracellular matrix remain devoid of mineral. As differentiating odontoblasts secrete the first layers of mantle dentine, they reach around 30–40 m in length, become more closely packed and develop numerous gap and adherents junctions, thus establishing a cellular layer with a well defined epithelioid appearance. The differentiating odontoblasts begin to move toward the center of the dental papilla whereas one
of the cell processes gradually becomes accentuated. This cell process that is left behind into the forming dentine matrix constitutes the odontoblast process around which the dentine matrix mineralizes, thus forming a dentinal tubule. As each odontoblast leaves behind one single cell process, thousands of dentinal tubules cross the dentine from the dentinoenamel junction to the pulp. The odontoblast process lacks cell organelles but contains numerous longitudinally arranged actin filaments and microtubules (Fig. 3). At stages in which most matrix vesicles are fully mineralized, odontoblasts appear taller (50–60 m in length) than at initial stages and focal tight junctions assemble between them. Different from those in epithelia, the tight junctions between differentiating odontoblasts play a role that is more related to the crescent cell polarization and differentiation than to permeability functions (Arana-Chavez & Katchburian, 1997). Recent studies have shown that the tight
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Fig. 2. Electron micrograph of a region of early dentine mineralization showing numerous matrix vesicles (MV), some of them containing fine mineral crystals (arrowheads), among the organic mantle dentine matrix rich in collagen fibrils (C). At these stages, the basal lamina (bl) is partially degraded, while the differentiating ameloblasts (A) present some short projections towards the developing mantle dentine. The differentiating odontoblasts also possess cell processes (P), which appear sectioned in different directions (26,000×). The inset shows a high-resolution post-embedding colloidal gold immunocytochemical preparation for dentin matrix protein 1 (DMP 1). The gold particles are present around a mineralization foci of the mantle dentin in a more advanced stage than that of the large micrograph, in which the mineral is spreading from matrix vesicles to the surrounding matrix (28,000×).
junctions between differentiating odontoblasts contain more ZO-1, a cytoplasmic junctional protein that is close related to actin filaments, than occludin and claudins, two classes of transmembrane proteins that are related to sealing properties of tight junctions (João & Arana-Chavez, 2003,2004). Nevertheless, with the assembly of tight junctions between odontoblasts an apical domain is established on their plasma membrane and they become fully differentiated. Matrix vesicles are no longer released and the secretion of several noncollagenous matrix proteins begins (Arana-Chavez & Katchburian, 1998). The differentiated odontoblasts that exhibit a exuberant secreting apparatus (Fig. 4) continue to deposit a somewhat different dentine—that is called circumpulpal, which forms the bulk of the whole dentine. In circumpulpal dentine, the collagen fibrils are thinner and they are more densely packed than those found in mantle dentine, many of them running around the dentinal tubules. The noncollagenous dentine matrix proteins
synthesized and secreted by the fully differentiated odontoblasts include dentine sialophosphoprotein (DSPP)—a large parental protein that subsequently suffers cleavage to originate two products, dentine sialoprotein (DSP) and dentine phosphoprotein (DPP), dentine matrix proteins 1, 2, and 3 (DMP-1, DMP-2, and DMP-3, respectively), and small amounts of four noncollagenous proteins (osteopontin, bone sialoprotein, osteonectin, and osteocalcin) that are abundant in bone (Butler & Ritchie, 1995; He, Dahl, Veis, & George, 2003; Papagerakis et al., 2002;). Because mineralization of circumpulpal dentine occurs without the presence of matrix vesicles, it is likely that noncollagenous matrix proteins play a key role to establish favorable conditions for the mineral deposition spreading through the fibrilar and interfibrilar regions of matrix (Fig. 2, inset). The dentine formed by secretion of collagen and noncollagenous proteins is called intertubular dentine. After formation of mantle dentine, each odontoblast
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Fig. 3. Electron micrograph showing the mineralized dentine/predentine/odontoblast layer interface. Whereas the mineralized layer of dentine (MD) exhibits higher electron opacity, the underlying unmineralized predentine (Pd) exhibits numerous collagen fibrils. The density of collagen in predentine gradually decreases from the mineralization front, where there is no matrix vesicles, to the lower regions, in which there are abundant proteoglycans and other noncollagenous matrix components. An odontoblasts processes (P) that arises from the odontoblast cell body (O) crosses the entire predentine and penetrates into a tubule of the mineralized dentine. Some branches (double arrowheads) of the odontoblast processes are clearly observed in other odontoblast process (5400×).
secrete additional noncollagenous components from the end of its cell process. This matrix mineralizes rapidly between the previously formed intertubular dentine and the odontoblast process and constitutes the peritubular dentin. Peritubular dentine is hypermineralized in relation to intertubular dentine and forms the wall of the dentinal tubule. It is owing to the peritubular dentine is secreted throughout life of the odontoblast, it occurs a gradual obliteration of the dentinal tubules with aging. The dentine formed up to the completion of root development is defined as primary dentine. Because
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Fig. 4. Electron micrograph showing the Golgi region of a secretory odontoblast in which well-developed synthesis organelles such as cisternae of rough endoplasmic reticulum (RER), Golgi stacks (G), and secretory granules (g) are abundant. M, mitochondria (32,500×).
the odontoblasts deposit dentine throughout life, a secondary dentine is laid down at a much slower rate than the primary dentine. Both primary and secondary dentine posses a similar structure. Formation of secondary dentine mainly occurs at the roof and floor of the pulp chamber, thus leading to an increasing reduction of the pulp volume, as well as of the height of pulp horns. At these stages the odontoblasts exhibit less developed synthesis organelles than at stages of primary dentine formation. It is important to say that the odontoblasts are post-mitotic cells that remain for tooth life. Odontoblasts might, however, form a third type of dentine—the tertiary dentine. There are two subtypes of tertiary dentine because sometimes the original odontoblasts die and then a new generation of odontoblast-like cells forms a new dentinal layer. Thus, the tertiary dentine formed by the original odontoblasts in response to attrition, caries or other stimuli such as some restorative procedures is called
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reactionary dentine. In general, the tubules of reactionary dentine continue with those of secondary dentine, while the thickness of the newly formed layer is related to the intensity and duration of the stimulus. The reactionary dentine possesses an organic matrix as well as a mineral content similar to those found in primary and secondary dentine. In other cases, however, some external factors such as dental caries may irreversibly affect the odontoblast layer in a given region. Cavity preparation during restorative procedures in dentistry may also affect the odontoblast layer. Whereas shallow cavities produce disruption of the junctions between odontoblasts, deep cavity preparation has been shown to cause aspiration of the odontoblast body up into the dentinal tubule thus disrupting its cytoplasmic content and resulting in cell death. Although these situations ultimately promote several pulp pathologies, when they have a reversible behavior, newly differentiated odontoblast-like cells may arise to forming another subtype of tertiary dentine that is called reparative dentine (Smith et al., 1995). It is assumed that undifferentiated ectomesenchymal cells present at the pulp, especially at the subodontoblastic layer, become the cells that produce the reparative dentine. Although the exact mechanisms that induce the differentiation of pulpal cells into odontoblast-like cells are still unknown, the new cells arise after a growth factor signaling that triggers a cascade of events involving cell division, chemotaxis, cell migration, cell adhesion and cytodifferentiation. Reparative dentine is in the majority of cases quite different morphologically from reactionary dentine. It may contain cellular inclusions, which resemble the osteocytes from bone. In addition, their extracellular matrix contains some noncollagenous proteins (osteopontin, for example) that are more typical for bone than of dentine. For these reasons, sometimes the reparative dentine shows an osteodentine appearance. It is important to say that differentiation of pulpal cells into odontoblast-like cells takes place in absence of enamel organ epithelium and basement membrane. It is believed that cells from the subodontoblastic layer—and perhaps other undifferentiated pulpal cells—may acquire a latent capability for this specific differentiation during the morphogenetic epithelial-ectomesenchymal interactions that took place at the bell stage of odontogenesis, despite some
components of dentine matrix have also been involved in these mechanisms. Nevertheless, it appears that absence of inflammation and sufficient oxygen supply within the pulp may improve the promotion of odontoblast-like cells cytodifferentiation (Tziafas, 1995).
4. Associated pathologies Several genetic disorders exhibiting an autosomal dominant pattern of inheritance may affect the dentine formation by odontoblasts. They are classified into two main groups, dentinogenesis imperfecta and dentine dysplasia, both with several subtypes. Dentinogenesis imperfecta type I (DGI-I) is associated with osteogenesis imperfecta that is caused by mutations of genes coding for pro-␣ 1(1) chains and pro-␣2(1) chains of type I collagen, which occur on the long arm of chromosomes 7 and 17. These mutations result in helix-destabilizing glycine substitutions, single exon-skipping mutations, and premature stop codons, which produce shortened and elongated collagen fibrils. In DGI-I, the dentine is hypomineralized and the overlying enamel is frequently brittle and easily fractures from it. The dentinogenesis imperfecta type II (DGI-II) is considered the classical dentinal type; dentinogenesis imperfecta type III (DGI-III) is also restricted to the dentine and is the most severe but it appears to affect restricted populations from the south of Maryland, USA. Both DGI-II and DGI-III are not collagen defects but two disorders of dentine mineralization; the basic defects appear to be originated within the human chromosome 4, on the gene cluster 4q21, from which the main noncollagenous dentine matrix proteins DSPP and DMP 1 and 2, as well as the novel protein—matrix extracellular phosphoglycoprotein/osteoblast-osteocyte factor 45 kDa (MEPE/OF45)—are encoded. Dentine dysplasia, on the other hand, is a rarer disease characterized by the obliteration of the pulp chamber and by resulting in teeth with short and cone-shaped root(s); the mineralization in the coronal dentine is normal or sometimes higher. Some disorders related to the gene expression of the noncollagenous matrix proteins, especially DSPP, are likely to play a role in the etiology of dentine dysplasia (MacDougall, Simmons, Gu, & Dong, 2002).
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