Dentin basic structure and composition an overview

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1 bs_bs_banner Endodontic Topics 2012, 20, 3 29 All rights reserved 2012 John Wiley & Sons A/S ENDODONTIC TOPICS Dentin basic structure and composition an overview LEO TJÄDERHANE, MARCELA R. CARRILHO, LORENZO BRESCHI, FRANKLIN R. TAY & DAVID H. PASHLEY Dentin is the most voluminous structural component of human tooth. Dentin protects pulp tissue from microbial and other noxious stimuli. It also provides essential support to enamel and enables highly mineralized and thus fragile enamel to withstand occlusal and masticatory forces without fracturing. Furthermore, it is the first vital tissue to meet external irritation, and instead of being merely a passive mechanical barrier, dentin may in many ways participate in dentin pulp complex defensive reactions. Even though dentin is usually considered as an entity, different parts of dentin may have special qualitative properties that help dentin to meet all of its required demands. The aim of this review is to provide an overview of the basic structure and composition of dentin, including the collagenous components of the dentin organic matrix and minerals. We also describe the specific structural and functional features of the dentin enamel junction (DEJ), mantle dentin, inter- and peritubular dentin, and pulp stones. Received 21 August 2010; accepted 25 January Dentin, which comprises the bulk of teeth, is mineralized connective tissue that bears a strong resemblance to bone. Dentin has phylogenetically been thought to be derived from bone (1). In this view, the most primitive hard tissue was mesodentin, a cellular tissue with unpolarized cell processes reminiscent of cellular bone. That was followed by semidentin in which odontoblasts remained entrapped within the mineralized matrix, but the cell processes were strongly polarized in a single direction; and finally by orthodentin, the most advanced structure. It has also been suggested that the evolution of bone may stem from dentin-like tissue (2 4). Both of these theories have recently been questioned. There is no phylogenetic evidence to support Ørvig s model of dentin evolution since all grades of dentin are manifest among the earliest skeletonizing vertebrates (5). Hard tissues (bone, dentin, enamel/enameloid, and cartilage) in primitive vertebrate skeletons are fundamentally distinct from their first inceptions (5,6), although the reason for the high diversity early in vertebrate phylogeny remains to be answered (5). Dentin is formed by highly specialized, terminally differentiated cells odontoblasts that are believed to be almost exclusively responsible for the constitution of dentin. For decades, the sole function of the odontoblasts was believed to be the formation and maturation of dentin, but recent years have revealed that odontoblasts may have much more diverse functions. Odontoblasts may, for example, participate in the dentin pulp complex innate immune defense and transmittance, and in the regulation of pulpal pain. For the sake of clarity, the dentin pulp interface, including the various possible roles suggested for the odontoblasts, are discussed in detail in another article in this issue. Also the numerous and diverse dentin non-collagenous proteins, participating in various tasks from the regulation of dentin mineralization to non-specific defenses against microbes, deserve reviews of their own which are found in the next issue. The aim of this review is mainly to present the constitution and structural components of dentin. 3

2 Tjäderhane et al. a b Fig. 1. (a) Scanning electron microscope (SEM) image of the pulp chamber dentinal wall of mouse molar. Pulp tissue, odontoblasts, and predentin have been mechanically removed, exposing the tubule openings. Magnification = 1,000 ; bar = 10 mm. (b) Number and radius of tubules with respect to the coronal dentin depth in human teeth. Data adapted from Pashley, 1996 (8). Constitution of dentin The composition of dentin may be described in two ways: extracellular dentin consists of a mineralized organic extracellular matrix (mechanical approach), while functional dentin includes predentin and dentinforming cells (odontoblasts) with their cytoplasmic processes penetrating mineralized dentin, and dentinal fluid (the biological entity). The mineral phase comprises approximately 70% of the weight percentage and 45% of volume, and the organic matrix about 20% and 33%, respectively, the remaining fraction being water (7). However, since water is located primarily in dentinal tubules, and the tubule diameter increases significantly from the dentin enamel junction toward the pulp (for details, see below), these percentages are only average values. The water content or wetness of dentin is not uniform, but varies approximately 20-fold from superficial to deep dentin (8). Structurally, dentin can be described as a nanocrystalline reinforced composite, whereas enamel would be a dense ceramic with impurities, even though recent research indicates that enamel behaves more in a metal-like manner in terms of elastic and plastic properties (9 11). The composition and structure of dentin varies between the different parts of the tooth (12,13). Because of the tubular structure, with more or less patent tubules surrounded by intertubular dentin (Fig. 1a), dentin is a highly permeable structure in which not only outward flow of dentinal fluid but also inward movement of, for example, microbial components may occur. Dentinal tubules radiate from the dentin pulp border through the entire dentin, with the exception of the outermost layers in mantle dentin and in the dentin enamel junction (DEJ) and adjacent to the cementum. The tubular width is largest close to the pulp, and decreases toward the enamel (Fig. 1b). Consequently, the volume of the dentin 4

3 Overview of dentin structure occupied with open dentinal tubules decreases toward the DEJ. Dentin can be divided into several types according to the site, function, and origin of the dentin. Broad discrepancy in terminology exists; Cox et al. (14) reported 20 different terms of dentin used in 154 articles or book chapters. Most commonly, dentin is divided into five different types according to the formation phases: dentin enamel junction, mantle dentin, primary dentin, secondary dentin, and tertiary dentin. Tertiary dentin, representing defensive dentin formation to protect the pulp tissue, can be further divided into reactionary and reparative dentin depending on the cells forming the dentin (primary or replacement odontoblasts, respectively). Mineralized extracellular dentin is further divided into intertubular and peritubular dentin. Intertubular dentin is formed by odontoblast during dentinogenesis, and it forms through predentin mineralization. Intertubular dentin comprises most of the dentinal volume. Peritubular dentin is formed by mineralization inside the walls of dentin tubules within mineralized dentin, and may be totally absent near the pulp in human teeth. Dentin enamel junction The dentin enamel junction (DEJ) has traditionally been thought to be merely a simple anatomical interface between enamel and dentin, seen on sections as a scalloped line between two mineralized structures. However, recent studies have demonstrated that the DEJ may be much more than an inactive border between two different hard tissues. With laser-induced autofluorescence and emission spectroscopy, the DEJ appears as a 7 to 15 mm-wide structure distinct from both enamel and dentin, and composed of large amounts of organic and mineral matter (15). It has also been suggested that the DEJ forms a complex of two unique, thin adjacent layers: the inner aprismatic enamel, which differs to some extent from the prismatic enamel; and the mantle dentin, which also is similarly related but still distinct structurally compared to circumpulpal dentin (16). Even after dentin and enamel formation and mineralization are well underway, specific biological events may still occur at the DEJ, suggesting that the crosstalk between enamel and dentin continues throughout the formation of prismatic enamel and circumpulpal dentin. The presence of enzymes (16,17) and growth factors such as fibroblast growth factor-2 (FGF-2) (16) suggests that the DEJ region represents an area of biological activity. It may liberate and activate the stored growth factors and other potentially bioactive components that may exert their effects at a location distant from the DEJ (16). Based on phylogenetic, developmental, structural, and biological characteristics, it has been suggested that instead of the dentin enamel junction, this structure should be termed the dentin enamel junctional complex (16). The DEJ in human teeth is not smooth, but wavy or scalloped (18 22) (Fig. 2). This kind of an interface is believed to improve the mechanical interlocking between dentin and enamel. The size of the scallops ranges between 25 and 50 mm, and they are deeper and larger at the dentin cusps and incisal edges, leveling down toward the cervical region (18,21,23). This is in accordance with finite-element studies demonstrating that the mechanical interlocking between enamel and dentin is weaker in the cervical region (24). In addition, smaller (0.25 to 2 mm) secondary scallops within the primary scallops have been demonstrated (21,23), and upon close inspection the intermingling ridges of dentin and enamel, less than 1 mm wide, are clearly visible. It is generally thought that the scalloping structure of the DEJ can be explained as required for the tooth to withstand functional stress (7). This assumption has been questioned, though, as humans are among very few species in which the scalloped form of the DEJ has been demonstrated (23,25). In addition to the scalloped morphology of the DEJ, there are basically two possibilities to increase the mechanical interlocking between enamel and dentin: the continuity of mineral crystals from dentin to enamel, and organic interlocking material (25). The continuity of mineralization crystals between developing dentin and enamel at the DEJ has been a matter of debate for a long time. It has been suggested that enamel crystals grow epitaxially on the pre-existing dentin crystals because of an apparent high continuity between enamel and dentin crystals (26,27). Others have claimed that enamel crystals are formed at a given distance from the dentin surface (28) and could either grow into contact with dentin crystals (29) or remain distant (30,31). However, enamel and dentin have been demonstrated to be linked by nm diameter collagen fibrils inserted directly into the enamel 5

4 Tjäderhane et al. a b c d Fig. 2. Dentin enamel junction (DEJ) in human primary molar. (a) Enamel cap has been removed, revealing the scalloped structure of the DEJ. The size of individual scallops is approximately 25 to 50 mm. (b c) Lower primary canine of a human fetus: the box and arrow indicate the area examined at higher magnification in (c), where the ridge-like interface (dotted line) between enamel and dentin is clearly visible in the cross-section of the DEJ. (d) In primary scallops, smaller secondary scallops (0.25 to 2 mm) are located, further increasing the irregularity of the DEJ. Reproduced with permission from Radlanski et al., 2007 (23). and merging with the interwoven fibrillar network of the dentin matrix (32) (Fig. 3). Immunogold labeling indicated that the fibrils contain type I collagen, but it is not impossible that these coarse fibers would actually be so-called von Korff fibers, consisting of type III collagen and fibronectin (7), and frequently reported to occur during the initial phases of dentinogenesis. In any case, these findings indicate that the DEJ connection may be textural and structural, rather than biochemical, reinforced with fibrils traveling from dentin to enamel across the width of the DEJ. The authors suggest that the DEJ could be regarded as a fibrilreinforced bond mineralized to a moderate degree (32), which is believable due to the high biomechanical requirements of the junction. The collagen network could provide efficient stress transfer from enamel to dentin and resistance to the tensile and shear forces developed during masticatory function (Fig. 3). Mantle dentin The mantle dentin is a layer of 5 to 30 mm in thickness in humans (12) and differs from the rest of the dentin in that its organic matrix is more irregular. The von Korff fibers have been frequently reported in mantle dentin (25). These fibers consist of coarse, bundled collagen fibrils of type III, with a minor portion of type I (33), and run with their long axis parallel to that 6

5 Overview of dentin structure a b e c d Fig. 3. Dentin enamel junction (DEJ). (a) TEM image of rat incisor DEJ, where the inner aprismatic enamel (IAE) and the mantle dentin (MD) meet. Magnification = 21,600. From Goldberg et al., 2002 (16), reproduced with permission. (b) Field emission-sem (FE-SEM) image of human DEJ, following surface decalcification with EDTA. The image illustrates the scalloping outline of DEJ (asterisk). E, enamel; D, dentin. Original magnification = 1,000 ; bar = 10 mm. (c) Detailed image of DEJ, demonstrating the penetration of fibrillar structures (arrowheads) into the enamel (E). Collagen fibrils of the DEJ area exposed after partial decalcification with EDTA. Original magnification = 10,000 ; bar = 1 mm. (d) The collagen fibrils have a cross-banding appearance and are nm in diameter (small arrows). These coarse fibrils merge with finer fibrils from the dentin matrix (arrowhead) and split before entering into the enamel (large arrows). The fibrils were identified as type I collagen with immunogold labeling (not shown). E, enamel. Original magnification = 50,000 ; bar = 200 nm. Figures b to d are from Lin et al., 1993 (32), reproduced with permission. (e) The proposed collagen structure from dentin through the DEJ into mineralized enamel. Modified from Lin et al., 1993 (32). of the odontoblast processes (7). Mantle dentin is also different in biochemical composition (12): for example, it seems to be absent of phosphoproteins (34,35). The mineral content of mantle dentin has long been thought to be lower than that of circumpulpal dentin, but it has also been suggested that the differences in the degree of mineralization are not limited to mantle dentin but may be more gradual (36,37) (Fig. 4a). This seems to be contradicted by studies which demonstrate that the content of mineral elements does not vary markedly between mantle and circumpulpal dentin (38). This apparent discrepancy may be explained by the differences in dentin constitution. The volume percentage of peritubular dentin increases dramatically from the DEJ toward the pulp, while that of intertubular dentin decreases (8) (Fig. 4b). As peritubular dentin is much harder than intertubular dentin, and dentinal hardness is inversely related to tubule density (39), this change may contribute to the overall hardness differences seen in dentin. The totally different mode of formation of mantle dentin compared to the rest of dentin is clearly seen in patients with hypophosphatemic vitamin D-resistant rickets, in which dentin is mainly globular, but mantle dentin is not affected (16,40 43). Mantle dentin also differs from circumpulpal dentin as it does not contain dentinal tubules, only sometimes thin tubular branches (44). However, the atubular structure of mantle dentin does not result in a lack of permeability (45 47). The multiple branching of the odontoblast processes indicates that the mantle dentin matrix is heterogeneously secreted by differentiating or newly differentiated odontoblasts, which may initially lack odontoblast processes that create patent tubules. It must be noted, however, that the fate of the proteins of the degraded basement membrane between differentiating ameloblasts and odontoblasts is not known, and these degradation products may well be, at least partially, integrated into mantle dentin ground substance prior to mineralization. Also mineralized globular structures, about 2 mm in diameter, can be seen 7

6 Tjäderhane et al. a b Fig. 4. The gradual increase in mineralization, indicating the existence of a resilience zone beneath mantle dentin. (a) Back-scattered electron (BSE) image of human premolar slice. BSE image demonstrates the degree of mineralization in a manner similar to radiography: the higher the mineralization, the whiter the image. Enamel (top and right of the image) is all white; 150 to 200 mm of dentin under the DEJ appears darker, indicating a lower level of mineralization, with gradual increase in the mineralization level toward the pulp. Reproduced with permission from Wang et al., 1998 (36). (b) The relative proportion of intertubular dentin (ITD), peritubular dentin (PTD), and dentinal tubules filled with fluid in human teeth with respect to dentin depth. *: Peritubular dentin is not present at the immediate pulpal surface, but begins close to it. Data are from Pashley, 1996 (8) (see also Fig. 8). embedded in a network of interglobular dentin in crown mantle dentin (12). It has been proposed that mantle dentin is largely responsible for the elastic properties of teeth, allowing relatively high occlusal loads without fracture of enamel or dentin. However, recent findings indicate that this resilience zone is not clear-cut. Instead, it has been suggested to also include varying depths of sub-dej circumpulpal dentin (36,37,48,49). In this zone, both changes in collagen fibril direction in intertubular dentin (49) and a gradual increase in mineralization from the DEJ toward the pulp (36,37) have been detected, which may contribute to the mechanical properties in this area. The mechanical properties of dentin are discussed with detail elsewhere in this issue. Circumpulpal dentin Primary and secondary dentin The main portion of dentin is called primary dentin, and it is formed rapidly during tooth formation. There are several differences in primary and mantle dentin formation: the organic matrix is completely formed by odontoblasts and the collagen matrix is more compact. Primary dentin forms the bulk of the tooth and gives it the size and form determined genetically. After primary dentinogenesis, dentin formation continues as secondary dentin, which is formed at a much slower rate. To date, the absolute point of change in odontoblast activity reflecting the change from primary to secondary dentinogenesis is ill-defined and constitutes a terminological controversy. It has been postulated that primary dentinogenesis ends when the crown is complete. This assumption is supported by the finding that cell organelles undergo atrophy at that point in rat molars (50). Primary dentinogenesis has also been timed to end when teeth becomes functional (12) and when root formation is complete (7,51). The time span is large; for example, in human first upper molars it is longer than 6 years if counted from the completion of the crown at the age of years to the closure of the root apex at the age of years (52). The concept of a distinct change from primary to secondary dentinogenesis could also be challenged. Johannessen (53) calculated the dentin formation rate in molars of young albino rats and noticed that the increment of dentin in the mid-occlusal surface of the lower molars during weeks 0 3, 3 6, and 6 9 was 10.2, 7.3, and 5.1 mm, respectively. Thus, dentin formation seems to slow down gradually, even though all of the molars reach occlusion at 3 4 weeks of life. Also, the dentin formation rate in young rat molars (3 7 weeks) is about 10 times faster than that in older 8

7 Overview of dentin structure (15 30 weeks) rats (54,55). Overall, these studies indicate that the dentin formation rate decreases gradually, at least in rat molars. The reduction in dentinogenesis activity is also accompanied by a marked change in the odontoblast gene expression profile (56). Secondary dentin differs only slightly from primary dentin: the curvature of dentinal tubules may be slightly different and the tubular structure may be less regular. The deposition of dentin may also be uneven, as in human teeth the greatest dentin deposition is frequently seen in the floor and roof of the pulp chamber, especially in molar teeth (7). Composition of dentin ECM The organic extracellular matrix (ECM) of dentin is similar to soft tissue ECM and especially to bone ECM, but in some respects it is unique. The high level of collagen cross-linking in mineralized tissue (57,58) and virtual absence of type III collagen are the main differences with soft tissue collagens. Generalized conclusions of the significance and function of the different ECM components in dentin are caused by the findings that demonstrate differences in dentin ECM between species and even between the different parts of the tooth. About 90% of the dentinal organic matrix is collagen. The major component of dentin collagen is type I (59,60), of which the majority is a heteropolymer with two a1(i) chains and one a2(i) chain (60). Odontoblasts also synthesize and secrete a type I collagen homopolymer consisting of three a1(i) chains and commonly called type I trimer (60 64). The experiments with rat incisor odontoblast organ culture (61) and analysis of rat incisor predentin (63) indicate that approximately 25 to 30% of the synthesized type I collagen would be the type I trimer. However, in bovine extracts containing odontoblasts and predentin, only about 3% of the type I collagen was type I trimer (62). The presence of type I trimer in mineralized dentin has only been shown in lathyritic rat incisors in which the collagen cross-linking was inhibited by dietary lathyrogen (b-aminopropionitrile) (65,66), and it is not known if type I trimer would be present in normal dentin (60,65,66). Butler (60) concludes that type I trimer is important for dentin formation but that it may be involved in the maturation of predentin and degraded prior to cross-linking. Type III collagen, widely seen in soft connective tissues, is not normally seen in intertubular dentin matrix. In developing human teeth, Lukinmaa et al. (67) demonstrated the expression of type III pro-alpha collagen mrna, and type III collagen immunoreactivity was observed in early predentin and again in predentin toward the completion of dentinogenesis, when mrna was no longer detected via in situ methods. However, mature human odontoblasts express type III collagen mrna detected with PCR, and produce the protein in tissue culture (68). Several studies (69 71) have found that type III collagen localizes in dentinal tubules. Since it has frequently been related to von Korff fibers (70,72), it is possible that the staining seen in deeper areas of dentin (69,71) may represent intact or fragmented remnants of these fibers. With other collagens, the data are much more inconsistent. A small amount of type V collagen is synthesized by odontoblasts (63); it has been shown in rat and hamster predentin and dentin in developing teeth (73), and is weakly stained in human predentin (74), but it is absent in mineralized dentin (69,74). Similarly conflicting results have been presented for type VI collagen. Becker and co-workers (69) reported a type VI collagen staining distribution essentially similar to that of type III collagen, with relatively strong intensity in predentin and occasional fibrous staining in dentinal tubules. However, Lukinmaa et al. (74) could not detect type VI collagen in dentin. Type IV collagen has not been seen in normal dentin (71). These conflicting findings may reflect differences, for example, between species or in section pre-treatment, antibodies, or detection methods. Becker et al. (69) suggested that the role for collagens other than type I collagen in mineralized tissues may be related to ECM (bone and dentin) remodeling before hard tissue calcification. This is in accordance with the expression of various ECM protein-degrading enzymes, matrix metalloproteinases (MMPs) (75), and cathepsins (76) (for details, see the article on dentin non-collagenous proteins in the next issue). The role of enzymatic regulation of collagenous and noncollagenous components in the control of dentin ECM maturation prior to mineralization has also been suggested by Butler et al. (60,77). Type III collagen is also present in dentinogenesis imperfecta (78) and in reparative dentin under carious lesions (79,80), which may, at least partially, be related to the disturbances of 9

8 Tjäderhane et al. a b Fig. 5. (a) Intensive branching of dentinal tubules close to DEJ (arrows). (b) Intensive branching of dentinal tubules in the middle part of dentin. The tubules are visualized with Alizarin red. Bars = 20 mm. Reproduced with permission from Kagayama et al., 1999 (82). the enzymatic or other reactions controlling the matrix maturation rather than the expression of type III collagen. About 10% of the dentin organic matrix consists of proteoglycans and other non-collagenous proteins, and less than 2% is lipids. Non-collagenous proteins are also produced by odontoblasts; they are distributed between the collagen fibrils and accumulate along the dentinal tubule walls, and are supposed to serve important functions in the mineralization process of dentin (7). The presence and potential roles of noncollagenous proteins in dentin and dentinogenesis are discussed in detail in other articles in this double issue. Dentinal tubules Tubularity is a central characteristic of dentin, affecting, for example, its mechanical properties, its ability to withstand occlusal forces, and its behavior in dentin bonding. The understanding of dentin threedimensional structure may have a significant impact on optimal cavity design and restorative procedures. The common belief that dentinal tubules extend at right angles from the DEJ and run a fairly direct or slightly S-shaped course through the dentin was recently questioned in a study utilizing 3D phasecontrast microtomography (81). In dentin immediately beneath enamel (within 0.3 mm), a wide range of tubule angular tilts (up to 75% of the tubules tilting more than 10 degrees) was seen. Within this area, the tubules also seemed to twist or curl, occasionally up to 90 degrees. Slightly further (0.5 mm) into dentin, no more tilting or curling occurred, presumably because of odontoblast crowding (81). In addition, a difference in tubule orientation relative to the DEJ was observed between upper and lower teeth (81). While these findings require confirmation, it can be speculated that the difference in tubule orientation between upper and lower teeth can affect the response to teeth loading, which might, for example, cause differences in the deformation of the crown under mastication (81). The number of dentinal tubules in different locations in relation to the DEJ or cementum does not vary except under the cuspal area, where the number of dentinal tubules close to the DEJ is significantly higher (44). This may relate to the regulation of the pulp dentin defensive systems against wear (for details, please see the Tertiary dentin section below). In addition to the main tubule, dentinal tubules have numerous branches and ramifications (Fig. 5). The number of branches is higher in areas where the density of the main tubules is low (44,82), forming an abundant anastomosing system of canaliculi very much like osteocytes in bone (83) (Fig. 6). Mjör & Nordahl (44) identified three types of tubular branches: major, fine, and microbranches. Major branches (0.5 to 1.0 mm diameter) are abundant peripherally while fine branches (300 to 700 nm diameter) are abundant in areas where the density of the tubules is relatively low. Microbranches (25 to 200 nm diameter) extend at right angles from the tubules in all parts of the dentin (44). 10

9 Overview of dentin structure Fig. 6. SEM images of resin-embedded, acid-etched dentin (a) and mandibular bone (b), demonstrating marked similarity between the odontoblast process and osteocyte lacuno-canalicular networks. Reproduced with permission from Lu et al., 2007 (83). An interesting finding of bamboo-like dentinal tubules with many nodules in the longitudinal sections, which appear as circular tubules surrounding the main tubule, was seen with Alizarin red staining (82). The circular tubules of the nodules adhere to one side of the dentinal tubules and resemble that of the peritubular dentin (Fig. 7). The nodules correspond to the tubular branching penetrating through thick peritubular dentin observed with SEM (44). Peritubular dentin, even though highly mineralized, is porous and the nodules observed with Alizarin red indicate a suggested function for peritubular dentin in regulating the degree of communication between the intertubular dentin and odontoblasts via dentinal fluid (84). Peritubular dentin The commonly used name peritubular is actually misleading, since the prefix peri ( around, surrounding, enclosing ) refers to material formed around the tubules. Since peritubular dentin in most species (including humans) is deposited on the inner surface of the tubular lumen by the odontoblasts only after the formation of intertubular dentin, a more 11

10 Tjäderhane et al. a c b d Fig. 7. (a) Longitudinal section of dentinal tubules in the middle part of dentin observed with Alizarin red demonstrates numerous circular nodular structures around main tubules. (b) In cross-section the staining is located at the interface between peritubular and intertubular dentin, with occasional branches penetrating into intertubular dentin. The nodules were absent in dentin close to DEJ, at the pulpal part of dentin (c and d), and in teeth extracted from young patients. Bar = 5 mm, applies to all figures. Reproduced with permission from Kagayama et al., 1999 (82). correct phrase would be intratubular dentin (12,13). However, peritubular is still most commonly used, and therefore will also be used in this review. Peritubular dentin is sharply demarcated from intertubular dentin. It is considered to be more mineralized and practically free of collagenous matrix, although both symmetry and degree of mineralization may vary significantly. The deposition of peritubular dentin causes a progressive reduction in the tubule lumen (dentin sclerosis). During environmental stimulation and irritation, the formation of peritubular dentin may be accelerated (12,13). Within mantle dentin, where dentinal tubules terminate in small branches, very little peritubular dentin is present and the tubules appear as empty channels penetrating the intertubular dentin. A thin lining of peritubular dentin may already be present at 20 mm from the DEJ (49) (Fig. 8). A gradual thickening of peritubular dentin occurs with increasing distance from the DEJ until it reaches the normal thickness of approximately 1 mm (Fig. 8). Concomitant with this thickening, the tubular density per unit volume increases (8,49). The mineral content of human peritubular dentin is approximately 40% higher compared to intertubular dentin (7). The differences in mineral structures between inter- and peritubular dentin indicated in earlier studies (85,86) have been, at least to some extent, questioned by more recent studies indicating little difference in the nature, size, and organization of the mineral phase between inter- and peritubular dentin (84,87 89). Peritubular dentin is spatially more homogenous than intertubular dentin, with different hardness (39), elastic properties (90), optical anisotropy (91), and fracture properties (92). All of these observations indicate completely different mechanical and structural properties of these intimately associated forms of mineralized dentin structures. The formation of peritubular dentin begins in dentinal tubules close to (but not at) the mineralization 12

11 Overview of dentin structure a b Fig. 8. (a) Thin peritubular lining inside the tubules approximately 20 mm from the DEJ in fractured dentin seen with SEM. (b) Coronal dentin 800 mm below the DEJ. Highly mineralized peritubular dentin (P) is clearly distinguishable from intertubular dentin. A crack in the right-hand side of the tubule is frequently seen in SEM preparations of tooth crown dentin and most likely represents an artifact due to sample handling. Modified with permission from Zaslansky et al., 2006 (49). front (93), presumably with the accumulation of noncollagenous proteins along the tubule walls (7) (Fig. 9). Recent studies with combined SEM and time-of-flight secondary ion mass spectroscopy (TOF- SIMS) analyses show that peritubular dentin is a separate phase from intertubular dentin, forming a distinct annulus within each tubule (84,94), thus contradicting previous studies which have indicated that crystallization within the peritubular dentin would be mediated by a collagenous matrix interaction as in intertubular dentin (88,89,95). The contradictory results may, at least partially, be caused by the heterogenous intratubular mineralization occasionally shown to contain collagenous and other proteins (13). However, those types of mineralization do not represent true peritubular dentin, and may represent atypical intratubular calcification, which has been suggested to be a non-vital process (96). Indeed, one of the main differences between inter- and peritubular dentins is that peritubular dentin is essentially collagen-free (13,84,88,97). Overall, peritubular dentin is very low in organic components, which are a mixture of acidic proteins, phospholipids, or possibly proteolipid complexes, with small amounts of glycoproteins and proteoglycans. Even though the sulfate content of peritubular dentin proteins seems to be low, the presence of chondroitin 4-sulfate (CS-A), chondroitin 6-sulfate (CS-C), and dermatan sulfate (CS-B) types have been indicated (84). Peritubular dentin is perforated in addition to tubular branches (44) by many small pores and fenestrations (84,94), allowing the passage of tubular fluid and intertubular dentin components across the peritubular dentin (Fig. 9). Based on the analysis of the organic components of peritubular dentin (84), there may be a potential for the calcium-phospholipidproteolipid components of peritubular dentin to be involved in the signaling and ion transport processes. Thus, peritubular dentin may also have direct role(s) in active transport and other regulatory activities between vital intertubular dentin matrix and odontoblasts, participating in retaining the vitality of dentin. The active processes in mineralized intertubular dentin would offer an explanation of some previously poorly understood findings regarding the age-related changes in dentin, e.g. the disappearance of matrix metalloproteinase-2 (MMP-2) from intertubular dentin with age (76,98) (discussed in more detail in the next issue). This proposal offers a completely new view not only on the role of peritubular dentin (usually thought to act as a passive blockage in dentinal tubules), but also on the vitality of mineralized dentin as a whole. If signaling and active transport between intratubular structures and components (odontoblast processes, tubular fluid) and intertubular dentin actually occur, the nature and function of dentin as a tissue should be revisited. Dentin sclerosis The main dentin response under carious lesions and restorations is reactive dentin sclerosis, seen as a translucent or transparent zone. The first phase of its formation in the initial stage of dentinal caries seems to be 13

12 Tjäderhane et al. a b Fig. 9. (a) Field emission scanning electron microscope (FE-SEM) image of a cross-section of a mouse second lower molar. Pulp tissue with odontoblasts, odontoblast processes, and predentin have been mechanically removed. Dense collagen network covers the tubular walls immediately below the orifice. In most intertubular dentin ridges, the individual collagen fibrils cannot be seen, presumably due to mineralization (arrows). The ridges thus represent the mineralization front. Collagenous mesh is apparent just below the tubule orifice. Slightly deeper, the collagen fibrils are masked with more homogenous structure, representing either non-collagenous proteins or the initial formation of peritubular dentin. Magnification = 10,000 ; bar = 1 mm. (b) Higher magnification of the area marked with a square in (a). Relatively sharp borderline between visible fibrils and homogenous surface of non-collagenous proteins or peritubular dentin indicates highly regulated process in this area. The fibril diameter varies between approximately 20 and 100 nm. Globular structures in connection with fibrils and in the homogenous surface either uniform layer of non-collagenous proteins or the outermost layer of mineralized peritubular dentin are readily seen (arrowheads). Few pores with approximately 70 nm diameter or less (arrows) are visible, potentially representing the peritubular dentin porosity suggested by Gotliv & Veis (84,94). Magnification = 35,000 ; bar = 1 mm. dependent on odontoblastic processes (99). However, acid etching in vivo and in extracted teeth produces a transparent layer, suggesting that it is not a vital defensive reaction (100). Instead, calcium phosphate dissolved from apatite crystals diffuses more deeply into the dentin tubules and precipitates ( ), at least at the side of the lesion where the irritation of associated odontoblasts is not so intense (100), into what has been termed caries crystals. Massler (104) proposed that superficial sclerosis is due to the re-precipitation of minerals from carious lesions and saliva, while deeper sclerosis requires calcium from the pulp. The degree of sclerosis is directly related to the success in arresting the lesion progression (103), but after the successful arrest of the lesion, mineral uptake from the saliva is very limited (105). Reactive dentin sclerosis occurs at all ages but increases both in prevalence and intensity with age, and is reported to be seen more often in males than in females (106). Similar to tertiary dentin formation, sclerosis is a non-specific reaction and physiological dentin sclerosis especially occurs in older teeth ( ). It is typically observed to decrease in the direction of the pulp (107), while most tubules in the translucent zone may be totally occluded (108). It has been proposed that physiological sclerosis should be regarded as being different from the tubular sclerosis seen in relation to caries (100,109). Physiological dentin sclerosis may even reduce the formation of tertiary dentin, possibly by reducing the permeability of dentin before the irritation occurs, whereas reactive dentin sclerosis does not prevent tertiary dentin formation. Physiological dentin sclerosis also develops in areas without carious lesions or irritation, for example on the floor of the pulp chamber and root canals. In roots, dentin sclerosis progresses with age from the apex toward the cervical portion (Fig. 10) (106,110,111). Tertiary dentin Tertiary dentin forms as a response to external irritation attrition, abrasion, erosion, trauma, caries, or cavity preparation in order to increase the thickness of the mineralized tissue barrier between the oral microbes and the pulp tissue. It has also been called irritation dentin, irregular dentin, irregular secondary dentin, etc. (14). Defensive reactions can be observed 14

13 Overview of dentin structure very early, with enzymatic changes being seen during the earliest stage of enamel caries both in the dentin pulp border (104, ) and in dentin itself (104, ), but not in pulpal tissues (120,121). The dentinogenic potential of the dental pulp, including different ex vivo and in vivo experimental models and the chemical and biomolecular agents used in these experiments, has very recently been extensively reviewed by Tziafas (122). Therefore, tertiary dentin is only briefly reviewed here. The aim of tertiary dentin is to protect pulpal tissue by increasing the thickness of dentin between the pulp and external wear or irritation (Fig. 11). Attrition is suggested to be more prone to induce tertiary dentin formation than caries (106). From a physiological point of view, this is acceptable, since caries is more or less related to the modern diet containing excess refined sugars, which favors caries formation. More natural, coarse diets are prone to cause abrasion and attrition, and it is tempting to speculate that tertiary a b Fig. 10. The classical image demonstrating, for the first time, the age-related root dentinal sclerosis advancing from the apical part toward the crown. Longitudinal central 0.5 mm section of an adult tooth photographed by transmitted light shows the transition between transparent apical root dentin (dark) and opaque dentin (white). Reproduced with permission from Nalbandian et al., 1960 (110). c Fig. 11. (a) Pulp chamber obliteration in a lower first molar by tertiary dentin formation, as seen in the apical radiograph. The radio-opaque area around the calcification in the second molar (black arrows), as well as irregular calcification in the distal wall of the second premolar pulp chamber (white arrow), indicate the presence of pulp stones rather than tertiary dentin formation in these teeth. (b) Removal of the filling from the first molar exposed the site of the original pulp chamber, with a clear demarcation line between primary/ secondary dentin and tertiary dentin (arrows). (c) Removal of the tertiary dentin, following the outline of the demarcation line, exposed the pulp chamber floor and allowed instrumentation of the root canals. 15

14 Tjäderhane et al. dentin formation as a defensive reaction to increase the dentinal width between the pulp and the oral cavity is designed to protect the pulp from extensive wear. According to Stanley et al. (106), the prevalence and intensity of tertiary dentin formation under caries is proposed to be independent of age in humans. However, this conclusion can be criticized because the group of young teeth in that particular study contained only three specimens and the age scale was wide (11 to 19 years). In young (3 to 8 weeks) rat molars with ongoing primary dentinogenesis, a non-linear relationship (123) or even a negative correlation (124) between dentin formation and extension of dentinal caries has been observed, indicating that the tertiary dentin-like response does not occur in teeth with primary dentinogenesis. The form and regularity of tertiary dentin depends on the intensity and duration of the stimulus. Generally, two forms of tertiary dentin are recognized: reactionary dentin (produced by original primary odontoblasts) and reparative dentin (produced by newly differentiated replacement odontoblasts) (109, ). In addition, it has been suggested that a third type of mineralization be distinguished as a merely defensive non-specific production of mineralized matrix. This would be produced by so-called fibrodentinoblasts, and the product would be called fibro- or osteodentin (122). In fact, Taintor et al. (129) already challenged the term reparative dentin in They argued that since repair, by definition, consists of the replacement of damaged tissue, the irregular dentin formed in response to external irreversible damage would be comparable to scar tissue formation (129,130), and should rather be called irritation or irritational dentin (129). While this would be, strictly speaking, a more exact term, the present terminology (tertiary dentin and reactionary or reparative dentin) is commonly accepted and is therefore also used in this review. In clinical situations, tertiary dentin usually contains atypical fibrodentin, reparative dentin, and reactionary dentin. Since the criteria for the assessment of differentiated odontoblast-like cells (or the level of differentiation) are not well defined (131), the presence of different types of tertiary dentin at the same site may reflect the process of odontoblast-like cell differentiation from non-specific, hard-tissue forming cells into fully differentiated odontoblast-like cells. In general, reactionary dentin has a more or less tubular continuity with secondary dentin, while the structure, organization, and mineralization of reparative dentin vary significantly. Since reparative dentin is generally atubular, it may form a relatively impermeable barrier between tubular dentin and pulp tissue. The regularity of reparative dentin is supposed to be inversely related to the degree of irritation (122). The goal of pulpal treatment procedures aimed at preserving pulp vitality could be the reduction of dentin permeability beneath the injury, thus isolating the pulp from further irritation. The junction between primary and reparative dentin is considered to act as a protective barrier against carious stimuli (109,132). It can, however, also be argued that the complete isolation of the pulp by non-tubular reparative dentin is not necessarily desirable. Pulpal sensory nerve fibers undergo extensive sprouting in response to injury, and it is commonly accepted that pulpal nerves are protective in nature and are involved in the recruitment of inflammatory and immunocompetent cells to the injured pulp (133). The degree and state of the response seem to be highly dependent on the changes in dentin permeability. Interestingly, the number of dentinal tubules close to the DEJ is significantly higher in the cuspal area than in other parts of dentin (44). Under the cusps, the dentinal tubules are also straighter and the odontoblast processes penetrate deeper to the dentin pulp border (134,135) or even to the DEJ (136). Since the cusps are the first area to be worn due to abrasion or attrition, it is tempting to speculate that the reason may be related to the regulation of defensive mechanisms in the dentin pulp complex. The dentinal tubules may be more direct and odontoblast processes may penetrate more deeply in order to deliver the message of dental wear and induce tertiary dentin formation so as to maintain the hard tissue barrier between dental pulp and the oral cavity. The coronal dentin pulp border has other distinctive histological features: a dense innervation of inner dentin and the odontoblast layer (137), pulp cells producing nerve growth factor and its receptor (137), and an extremely dense capillary network (138,139). The co-localization of these tissue components together with the straight tubules and long odontoblast processes may indicate a role in sensing the external irritation and controlling defensive reactions. The connection of pulpal histological features with odontoblasts and their processes is at least indirectly supported by the finding that the dense 16

15 Overview of dentin structure innervations normally seen under the odontoblast layer (the subodontoblastic plexus of Raschkow) with some axons passing into dentinal tubules (7) is not seen in the case of reparative dentin (140). Since the nociceptive fibers contribute to normal homeostatic regulation and vasoregulation and to healing after injury (133,137), this significant reduction in innervations may affect the inflammatory and immune responses under reparative dentin. As well, the initial immunodefensive reaction (measured as the accumulation of antigen-presenting cells) occurs beneath the dentinal tubules communicating with superficial caries lesions (141,142). However, after substantial formation of sound reparative dentin, the inflammatory response to the microbial burden subsides (142,143). The re-accumulation of antigen-presenting cells occurs only after bacterial invasion of reparative dentin, close to the pulp (142). We conclude that this supports the concept that the junction between the primary and reparative dentin may act as a barrier to prevent carious stimuli (109,132). However, even if reparative dentin prevents microbial component penetration into the pulp, it also allows caries progression close to the pulp without inducing normal defensive reactions occurring in the dentin pulp complex (142). Therefore, the formation of reactionary dentin with tubular continuity is preferable (122). In summary, it must be emphasized that the understanding of the mechanisms regulating reparative dentin formation is still limited. Excessive dentin repair may also be detrimental when the response is not limited to the site immediately below the dentinal injury but results in generalized root canal system calcification (129) (Fig. 11). The decrease of vital pulp tissue may reduce the defensive potential of the pulp (for details, see next paragraph), and obliteration of the root canal system certainly makes endodontic procedures more challenging. The dentin matrix contains biologically active molecules such as growth factors (for details, see the article on dentin non-collagenous proteins in the next issue), which can induce dentinogenic events both in vivo and in vitro. These dentin matrix bioactive factors are supposed to possess a similar inductive potential for tertiary dentin formation as seen by the enamel epithelium and basement membrane during physiological dentinogenesis in embryonic conditions (122,128). An appropriate pulpal environment (such as the absence of severe inflammation and adequate vascularity) and mechanical support with a favorable surface for cell attachment (such as dentin, or sufficiently solid calcium hydroxide, or perhaps even better MTA in the case of pulp capping) are considered absolute requirements for appropriate tertiary dentin formation (122). The dentin pulp complex may possess a remarkable capacity to survive even intensive dentinal damage, with reactionary and reparative dentin formation occurring under a remaining dentin thickness of as thin as 0.5 mm (144,145). In the absence of microbial infection, injuries to the pulp coincident with dentinal injuries are presumed to be reversible. After experimental exposure of dentinal tubules, repair and healing of the pulp occurs in spite of continuous exposure of cut dentinal cavities to the salivary microflora (143), suggesting that dentin is able to oppose bacterial threats even when a small rim ( 1.5 mm) remains. Clinically, the dentin pulp complex is a target for repeated microbial, mechanical, and chemical insults such as primary and secondary caries, replacement of restorations, and attrition. Empirical clinical experience has indicated that the pulpal healing potential is reduced by a repeated series of stimuli. For example, the tendency for teeth restored with full crowns ( ) or traumatized teeth with pulp canal obliterations ( ) to develop pulpal necrosis has been observed in numerous studies. It is likely that repeated dentinal irritation affects the mechanisms for reactionary dentin synthesis and replacement of odontoblasts as well as causing pulpal scarring and loss of perivascular stem cells. While there is ample evidence demonstrating increased pulpal cell proliferation, collagenous protein deposition, and reparative dentin formation after a single dentinal injury (cavity preparation) ( ), after a double injury, the increase in cell proliferation and collagenous protein deposition is significantly less than after the single injury (163). Although the response may have been decreased by pulp dentin defensive reactions to the first injury (such as occlusion of dentinal tubules or impermeable reactionary dentin formation), the study still supports the decreased ability of the dentin pulp complex to respond to repeated insults. The author suggests that the timing of sequential episodes of dentinal irritation could be used to minimize pulpal damage after extensive restorative dental treatment (163). However, more research is needed to validate this suggestion. 17

16 Tjäderhane et al. Root dentin The epithelial cells of Hertwig s epithelial root sheath (HERS) initiate odontoblast differentiation very similar to that in the coronal area (7,12). In principle, root dentin forms in a manner similar to coronal dentin, even though some differences may exist. For example, the coarse fibrils of mantle dentin at the cementum dentin junction (CDJ) are usually parallel to the basal lamina, not perpendicular as often seen in the crown (25). The terminology and even the structure of root mantle dentin are also controversial: it has been called the hyaline layer and reported to be absent in some animals and to vary in thickness in others (25). Structurally, the human CDJ represents a region of interspaced 15 to 30 mm-wide collagen fibril bridges (25,164) formed after the breakdown of HERS (165). In the cementum, the collagen fibril bridges from the dentin intermingle with collagen fibers parallel to the root surface, ensuring a tight attachment of cementum to the dentin (Fig. 12). The CDJ also contains pores, possibly representing HERS remnants (164). The granular layer of Tomes is located in the outermost part of the root dentin. It is believed to represent the mantle dentin in the root surface or be located immediately below the root mantle dentin (25). Similar to coronal mantle dentin, it also displays thin canaliculi and poorly fused globules (12,25). However, the granular structures can only be seen in ground sections, not in histological staining or electron microradiographs (7). Most likely they represent the mineralization pattern in the initial phases of root dentin and cementum formation. The density of tubules in root dentin is still a somewhat controversial issue. Some studies have indicated a rather moderate decrease in tubular density from the cemento enamel junction (CEJ) toward the apex (44,166) while others have indicated a more pronounced decrease (167). The reason for the differences may be the different methods or different teeth used for the analyses, as Schellenberg et al. (167) found markedly fewer tubules in mesio-distal than buccolingual surfaces of premolars, but not in third molars. The main part of the root dentin is rich with both fine tubular branches and microbranches, with occasional major branching (Fig. 13) (44). Apical dentin It has been suggested that the dentin in the most apical part of the roots differs from the rest of the a b c Fig. 12. FE-SEM images of human cement dentinal junction (CDJ). (a) 10 to 15 mm cementum layer in intimate contact with dentin. Magnification = 500 ; bar = 10 mm. (b,c) Higher magnification demonstrates the mineralized collagen fiber continuity from cementum to underlying dentin. Magnifications = 2,500 (b) and 5,000 (c); Bars = 10 mm (b) and 1 mm (c). 18

17 Overview of dentin structure a b c d Fig. 13. Dentinal tubule branching in root dentin. (a) Typical major branching, with numerous fine branches (Fb). (b) The dentinal tubule in the center has numerous fine branches, giving it the appearance of an interdental toothbrush. H-E staining of a premolar from a 12-yearold. (c) Dentinal tubules in cervical area showing variations of fine branches from the same tooth as in (b). (d) Variation in branching of dentinal tubules in root dentin. Hematoxylin-eosin (H-E) staining (a c); Masson staining (d). Reproduced with permission from Mjör & Nordahl, 1996 (44). root dentin in many respects. The number and regularity of tubules markedly decreases in the apical root (44,166,168,169). Apical primary dentinal tubules may have an irregular direction and density, and in some areas they are missing (168,169). Apical dentin also differs in tertiary dentin formation (168). Since there should not be external irritation causing the formation of reparative dentin, the dentin formed after primary dentinogenesis should, by definition, be secondary dentin. However, a distinct border between primary and secondary dentin, with discontinuity of dentinal tubules, has been demonstrated. The apical portion of human teeth also shows other marked variations in structure, such as accessory root canals, areas of resorption and repaired resorption, and cementum-like tissue lining the apical root canal wall. The causative factors for these kinds of dentin formation could be, for example, pulp inflammation or a response to occlusal loading. In addition, age-related root tubular sclerosis that starts in the third decade of life from the apical region and advances coronally (111) has recently been suggested to be the main factor influencing the permeability of root dentin (170,171) (Fig. 14). Root dentin seems to have regional differences in its permeability: buccal and lingual curvatures of root canals show patent tubules that take up dyes, while the mesial and distal pulpal borders seem to be occluded with minerals (170,171) (Fig. 15). One wonders how this pattern of tubule patency corresponds to local stress distributions in the functioning of these roots, and whether these stress distributions translate into regional differences in dentinal fluid shifts (172). Pulp stones Pulp stones are discrete or diffuse pulp calcifications that can be classified structurally as well as based on location (173). Structurally, there are true and false pulp stones, the distinction being morphological. A third type, diffuse or amorphous pulp stones, are more irregular in shape than false pulp stones and occur in close association with blood vessels (174). The cells forming the pulp stones may also vary, as true pulp stones contain dentinal tubules and are lined with odontoblasts (or rather odontoblast-like cells), while false pulp stones with atubular calcification have been considered to be formed from degenerating cells of the pulp that mineralize. The distinction between the true and false pulp stones may be somewhat artificial, as both tubular and atubular dentin can be found in a single pulp stone (Fig. 16). 19

18 Tjäderhane et al. Fig. 14. Relative mean dye penetration (in percentage of complete dentin area) after extensive (60 day) incubation of Methylene blue in instrumented root canals. Data are from Thaler et al., 2008 (171). Fig. 15. (a) A light microscope view of a cross-section of a human tooth, showing the typical bucco-lingual barbell shape and dye penetration pattern of Patent blue. Magnification = 16. (b d) Back-scattered electron micrograph of areas with (b) and without (c,d) dye penetration, demonstrating patent dentinal tubules in (b) and tubular sclerosis in (c,d). Magnifications = 1,000 (b,c); 3,000 (d). Reproduced with permission from Paqué et al., 2006 (170). 20

19 Overview of dentin structure a b c d e f Fig. 16. Heterogenous structure of pulp stone. (a) Pulp stone in human dental pulp, stained with hematoxylin-eosin staining. The pulp stone appears as a solid calcified mass. The width of the stone is approximately 3 mm. Original magnification = 50. (b) Adjacent section stained with Toluidine blue. The pulp stone appears much more heterogenous. Original magnification = 50. (c) Higher magnification of the lower left corner of (a). Odontoblast-like cells line the lower border of the pulp stone, while the right side is devoid of odontoblast-like cells. Original magnification = 100. (d) Same area stained with Toluidine blue demonstrates tubular structure in the pulp stone at the site of the odontoblast-like cells, while the area without cells is essentially free of tubules. Original magnification = 100. (e) Higher magnification of (d). Well-formed longitudinally cut tubules are on the left side, while the tubules on the right side are cut across the tubule direction and appear less organized. Original magnification = 200. (f) In another part of the pulp stone, tubules appear more sparse and with numerous fine branches and microbranches. Original magnification =

20 Tjäderhane et al. a b c d Fig. 17. Pulp stones. (a) Pulp stone obliterating most of the coronal pulp chamber in a lower molar. (b) Higher magnification of (a). The pulp chamber contains both loose pulp stone a originally surrounded by pulpal tissue, and pulp stone attached to the pulp chamber wall b. (c) Pulp stone filling a premolar pulp chamber. The uneven form of the pulp stone indicates that several pulp stones have developed independently and grown in size until being united. (d) The size of the pulp stone presented in (a) and (b), with some necrotic pulp tissue still attached to the stone. It must be noted that some material was lost as the pulp stone had to be drilled out of the pulp chamber. Therefore, the original size of the stone may have been even larger. A single tooth may contain one or several pulp stones of varying size, most often found in coronal pulp but sometimes also present in radicular pulp. Despite a number of studies, the exact cause of such pulp calcifications remains largely unknown. External irritation (caries, attrition) has been suggested as a cause, but pulp stones also appear in teeth with no apparent cause (e.g. impacted third molars). Pulp stones have also been noted in relation to systemic or genetic diseases, including dentin dysplasia [especially dentin dysplasia type II (175,176)], and certain syndromes (176). Large pulp stones in the pulp chamber may obstruct the canal orifices (Fig. 17), and in the root canal they may complicate access to the apical canal. For a comprehensive description of pulp stones, the reader is referred to a recent review article by Goga et al. (176). Words of caution: dentin differences between species It is important to realize that differences in the structure and chemistry of dentin between different species have long been recognized (12). For example, the cellular junctions between odontoblasts, the amount of peritubular matrix, the structure and mechanism of formation of peritubular dentin, and the presence and thickness of root mantle dentin vary between species (25). These differences may affect the usability of certain animal models. Because many (maybe most) biochemical studies have been performed on bovine and rat dentin, available data often directly concern only these species. In general, in terms of protein composition of the organic matrix, dentin species can be divided into two main 22