Dentin basic structure andcomposition—an overviewLEO 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 microbialand other noxious stimuli. It also provides essential support to enamel and enables highly mineralized and thusfragile enamel to withstand occlusal and masticatory forces without fracturing. Furthermore, it is the first vitaltissue to meet external irritation, and instead of being merely a passive mechanical barrier, dentin may in manyways 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 requireddemands. 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 specificstructural and functional features of the dentin–enamel junction (DEJ), mantle dentin, inter- and peritubulardentin, and pulp stones.

Received 21 August 2010; accepted 25 January 2011.

Dentin, which comprises the bulk of teeth, is mineral-ized connective tissue that bears a strong resemblanceto bone. Dentin has phylogenetically been thought tobe derived from bone (1). In this view, the most primi-tive hard tissue was mesodentin, a cellular tissue withunpolarized cell processes reminiscent of cellular bone.That was followed by semidentin in which odontoblastsremained entrapped within the mineralized matrix, butthe cell processes were strongly polarized in a single di-rection; and finally by orthodentin, the most advancedstructure. It has also been suggested that the evolutionof bone may stem from dentin-like tissue (2–4). Both ofthese theories have recently been questioned. There isno phylogenetic evidence to support Ørvig’s model ofdentin evolution since all grades of dentin are manifestamong the earliest skeletonizing vertebrates (5). Hardtissues (bone, dentin, enamel/enameloid, and carti-lage) in primitive vertebrate skeletons are fundamen-tally distinct from their first inceptions (5,6), althoughthe reason for the high diversity early in vertebratephylogeny remains to be answered (5).

Dentin is formed by highly specialized, terminallydifferentiated cells—odontoblasts—that are believedto be almost exclusively responsible for the constitu-tion of dentin. For decades, the sole function of theodontoblasts was believed to be the formation andmaturation of dentin, but recent years have revealedthat odontoblasts may have much more diversefunctions. Odontoblasts may, for example, participatein the dentin–pulp complex innate immune defenseand transmittance, and in the regulation of pulpalpain. For the sake of clarity, the dentin–pulp inter-face, including the various possible roles suggestedfor the odontoblasts, are discussed in detail inanother article in this issue. Also the numerous anddiverse dentin non-collagenous proteins, partici-pating in various tasks from the regulation of den-tin mineralization to non-specific defenses againstmicrobes, deserve reviews of their own which arefound in the next issue. The aim of this review ismainly to present the constitution and structuralcomponents of dentin.

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ENDODONTIC TOPICS 20121601-1538

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Constitution of dentin

The composition of dentin may be described in twoways: extracellular dentin consists of a mineralizedorganic extracellular matrix (mechanical approach),while functional dentin includes predentin and dentin-forming cells (odontoblasts) with their cytoplasmicprocesses penetrating mineralized dentin, and dentinalfluid (the biological entity). The mineral phase com-prises approximately 70% of the weight percentage and45% of volume, and the organic matrix about 20% and33%, respectively, the remaining fraction being water(7). However, since water is located primarily in den-tinal tubules, and the tubule diameter increases signifi-cantly from the dentin–enamel junction toward thepulp (for details, see below), these percentages areonly average values. The water content—or wetness—of dentin is not uniform, but varies approximately20-fold from superficial to deep dentin (8).

Structurally, dentin can be described as a nanocrys-talline reinforced composite, whereas enamel would bea dense ceramic with impurities, even though recentresearch indicates that enamel behaves more in ametal-like manner in terms of elastic and plastic prop-erties (9–11). The composition and structure ofdentin varies between the different parts of the tooth(12,13). Because of the tubular structure, with moreor less patent tubules surrounded by intertubulardentin (Fig. 1a), dentin is a highly permeable structurein which not only outward flow of dentinal fluid butalso inward movement of, for example, microbialcomponents may occur. Dentinal tubules radiate fromthe dentin–pulp border through the entire dentin,with the exception of the outermost layers in mantledentin and in the dentin–enamel junction (DEJ) andadjacent to the cementum. The tubular width is largestclose to the pulp, and decreases toward the enamel(Fig. 1b). Consequently, the volume of the dentin

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 adaptedfrom Pashley, 1996 (8).

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occupied with open dentinal tubules decreases towardthe DEJ.

Dentin can be divided into several types according tothe site, function, and origin of the dentin. Broaddiscrepancy in terminology exists; Cox et al. (14)reported 20 different terms of dentin used in 154articles or book chapters. Most commonly, dentin isdivided into five different types according to the for-mation phases: dentin–enamel junction, mantledentin, primary dentin, secondary dentin, and tertiarydentin. Tertiary dentin, representing defensive dentinformation to protect the pulp tissue, can be furtherdivided into reactionary and reparative dentin depend-ing on the cells forming the dentin (primary orreplacement odontoblasts, respectively).

Mineralized extracellular dentin is further dividedinto intertubular and peritubular dentin. Intertubulardentin is formed by odontoblast during dentinogen-esis, and it forms through predentin mineralization.Intertubular dentin comprises most of the dentinalvolume. Peritubular dentin is formed by mineraliza-tion inside the walls of dentin tubules within mineral-ized dentin, and may be totally absent near the pulp inhuman teeth.

Dentin–enamel junctionThe dentin–enamel junction (DEJ) has traditionallybeen thought to be merely a simple anatomical inter-face between enamel and dentin, seen on sections as ascalloped line between two mineralized structures.However, recent studies have demonstrated that theDEJ may be much more than an inactive borderbetween two different hard tissues. With laser-inducedautofluorescence and emission spectroscopy, the DEJappears as a 7 to 15 mm-wide structure distinct fromboth enamel and dentin, and composed of largeamounts of organic and mineral matter (15). It hasalso been suggested that the DEJ forms a complex oftwo unique, thin adjacent layers: the inner aprismaticenamel, which differs to some extent from the pris-matic enamel; and the mantle dentin, which also issimilarly related but still distinct structurally comparedto circumpulpal dentin (16).

Even after dentin and enamel formation and miner-alization are well underway, specific biological eventsmay still occur at the DEJ, suggesting that the cross-talk between enamel and dentin continues throughoutthe formation of prismatic enamel and circumpulpal

dentin. The presence of enzymes (16,17) and growthfactors such as fibroblast growth factor-2 (FGF-2)(16) suggests that the DEJ region represents an area ofbiological activity. It may liberate and activate thestored growth factors and other potentially bioactivecomponents that may exert their effects at a locationdistant from the DEJ (16). Based on phylogenetic,developmental, structural, and biological characteris-tics, it has been suggested that instead of the dentin–enamel junction, this structure should be termed thedentin–enamel junctional complex (16).

The DEJ in human teeth is not smooth, but wavy orscalloped (18–22) (Fig. 2). This kind of an interface isbelieved to improve the mechanical interlockingbetween dentin and enamel. The size of the scallopsranges between 25 and 50 mm, and they are deeperand larger at the dentin cusps and incisal edges, lev-eling down toward the cervical region (18,21,23).This is in accordance with finite-element studies dem-onstrating that the mechanical interlocking betweenenamel and dentin is weaker in the cervical region(24). In addition, smaller (0.25 to 2 mm) “secondaryscallops” within the “primary” scallops have beendemonstrated (21,23), and upon close inspection theintermingling ridges of dentin and enamel, less than1 mm wide, are clearly visible. It is generally thoughtthat the scalloping structure of the DEJ can beexplained as required for the tooth to withstand func-tional stress (7). This assumption has been questioned,though, as humans are among very few species inwhich the scalloped form of the DEJ has been dem-onstrated (23,25).

In addition to the scalloped morphology of the DEJ,there are basically two possibilities to increase themechanical interlocking between enamel and dentin:the continuity of mineral crystals from dentin toenamel, and organic interlocking material (25). Thecontinuity of mineralization crystals between develop-ing dentin and enamel at the DEJ has been a matter ofdebate for a long time. It has been suggested thatenamel crystals grow epitaxially on the pre-existingdentin crystals because of an apparent high continuitybetween enamel and dentin crystals (26,27). Othershave claimed that enamel crystals are formed at a givendistance from the dentin surface (28) and could eithergrow into contact with dentin crystals (29) or remaindistant (30,31). However, enamel and dentin havebeen demonstrated to be linked by 80–120 nm diam-eter collagen fibrils inserted directly into the enamel

Overview of dentin structure

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and merging with the interwoven fibrillar network ofthe dentin matrix (32) (Fig. 3). Immunogold labelingindicated that the fibrils contain type I collagen, but itis not impossible that these coarse fibers would actuallybe so-called von Korff fibers, consisting of type IIIcollagen and fibronectin (7), and frequently reportedto occur during the initial phases of dentinogenesis. Inany case, these findings indicate that the DEJ connec-tion may be textural and structural, rather than bio-chemical, reinforced with fibrils traveling from dentinto enamel across the width of the DEJ. The authorssuggest that the DEJ could be regarded as a fibril-reinforced bond mineralized to a moderate degree(32), which is believable due to the high biomechani-

cal requirements of the junction. The collagennetwork could provide efficient stress transfer fromenamel to dentin and resistance to the tensile and shearforces developed during masticatory function (Fig. 3).

Mantle dentinThe mantle dentin is a layer of 5 to 30 mm in thicknessin humans (12) and differs from the rest of the dentinin that its organic matrix is more irregular. The vonKorff fibers have been frequently reported in mantledentin (25). These fibers consist of coarse, bundledcollagen fibrils of type III, with a minor portion oftype I (33), and run with their long axis parallel to that

a

c d

b

Fig. 2. Dentin–enamel junction (DEJ) in human primary molar. (a) Enamel cap has been removed, revealing thescalloped structure of the DEJ. The size of individual scallops is approximately 25 to 50 mm. (b–c) Lower primarycanine of a human fetus: the box and arrow indicate the area examined at higher magnification in (c), where theridge-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 theDEJ. Reproduced with permission from Radlanski et al., 2007 (23).

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of the odontoblast processes (7). Mantle dentin is alsodifferent in biochemical composition (12): forexample, it seems to be absent of phosphoproteins(34,35). The mineral content of mantle dentin haslong been thought to be lower than that of circumpul-pal dentin, but it has also been suggested that thedifferences in the degree of mineralization are notlimited to mantle dentin but may be more gradual(36,37) (Fig. 4a). This seems to be contradicted bystudies which demonstrate that the content of mineralelements does not vary markedly between mantle andcircumpulpal dentin (38). This apparent discrepancymay be explained by the differences in dentin consti-tution. The volume percentage of peritubular dentinincreases dramatically from the DEJ toward the pulp,while that of intertubular dentin decreases (8)(Fig. 4b). As peritubular dentin is much harder thanintertubular dentin, and dentinal hardness is inverselyrelated to tubule density (39), this change may con-tribute to the overall hardness differences seen indentin.

The totally different mode of formation of mantledentin compared to the rest of dentin is clearly seen inpatients with hypophosphatemic vitamin D-resistantrickets, in which dentin is mainly globular, but mantledentin is not affected (16,40–43). Mantle dentin alsodiffers from circumpulpal dentin as it does not containdentinal tubules, only sometimes thin tubularbranches (44). However, the atubular structure ofmantle dentin does not result in a lack of permeability(45–47). The multiple branching of the odontoblastprocesses indicates that the mantle dentin matrix isheterogeneously secreted by differentiating or newlydifferentiated odontoblasts, which may initially lackodontoblast processes that create patent tubules. Itmust be noted, however, that the fate of the proteinsof the degraded basement membrane between differ-entiating ameloblasts and odontoblasts is not known,and these degradation products may well be, at leastpartially, integrated into mantle dentin ground sub-stance prior to mineralization. Also mineralized globu-lar structures, about 2 mm in diameter, can be seen

a b

c d

e

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 withpermission. (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 theenamel (E). Collagen fibrils of the DEJ area exposed after partial decalcification with EDTA. Original magnifica-tion = 10,000 ¥; bar = 1 mm. (d) The collagen fibrils have a cross-banding appearance and are 80–120 nm in diameter(small arrows). These coarse fibrils merge with finer fibrils from the dentin matrix (arrowhead) and split beforeentering into the enamel (large arrows). The fibrils were identified as type I collagen with immunogold labeling (notshown). 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 mineralizedenamel. Modified from Lin et al., 1993 (32).

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embedded in a network of interglobular dentin incrown mantle dentin (12).

It has been proposed that mantle dentin is largelyresponsible for the elastic properties of teeth, allowingrelatively high occlusal loads without fracture ofenamel or dentin. However, recent findings indicatethat this “resilience zone” is not clear-cut. Instead, ithas been suggested to also include varying depths ofsub-DEJ circumpulpal dentin (36,37,48,49). In thiszone, both changes in collagen fibril direction in inter-tubular dentin (49) and a gradual increase in miner-alization from the DEJ toward the pulp (36,37) havebeen detected, which may contribute to the mechani-cal properties in this area. The mechanical propertiesof dentin are discussed with detail elsewhere in thisissue.

Circumpulpal dentin

Primary and secondary dentin

The main portion of dentin is called primary dentin,and it is formed rapidly during tooth formation. Thereare several differences in primary and mantle dentinformation: the organic matrix is completely formed byodontoblasts and the collagen matrix is more compact.

Primary dentin forms the bulk of the tooth and givesit the size and form determined genetically. After

primary dentinogenesis, dentin formation continues assecondary dentin, which is formed at a much slowerrate. To date, the absolute point of change in odon-toblast activity reflecting the change from primary tosecondary dentinogenesis is ill-defined and constitutesa terminological controversy. It has been postulatedthat primary dentinogenesis ends when the crown iscomplete. This assumption is supported by the findingthat cell organelles undergo atrophy at that point in ratmolars (50). Primary dentinogenesis has also beentimed to end when teeth becomes functional (12) andwhen root formation is complete (7,51). The timespan is large; for example, in human first upper molarsit is longer than 6 years if counted from the comple-tion of the crown at the age of 2.6–2.7 years tothe closure of the root apex at the age of 9.2–10.1years (52).

The concept of a distinct change from primary tosecondary dentinogenesis could also be challenged.Johannessen (53) calculated the dentin formation ratein molars of young albino rats and noticed that theincrement of dentin in the mid-occlusal surface of thelower molars during weeks 0–3, 3–6, and 6–9 was10.2, 7.3, and 5.1 mm, respectively. Thus, dentin for-mation seems to slow down gradually, even though allof 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

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 minerali-zation in a manner similar to radiography: the higher the mineralization, the whiter the image. Enamel (top and rightof the image) is all white; 150 to 200 mm of dentin under the DEJ appears darker, indicating a lower level ofmineralization, with gradual increase in the mineralization level toward the pulp. Reproduced with permission fromWang et al., 1998 (36). (b) The relative proportion of intertubular dentin (ITD), peritubular dentin (PTD), anddentinal tubules filled with fluid in human teeth with respect to dentin depth. *: Peritubular dentin is not present atthe immediate pulpal surface, but begins close to it. Data are from Pashley, 1996 (8) (see also Fig. 8).

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(15–30 weeks) rats (54,55). Overall, these studiesindicate that the dentin formation rate decreas-es gradually, at least in rat molars. The reductionin dentinogenesis activity is also accompanied by amarked change in the odontoblast gene expressionprofile (56).

Secondary dentin differs only slightly from primarydentin: the curvature of dentinal tubules may beslightly different and the tubular structure may be lessregular. The deposition of dentin may also be uneven,as in human teeth the greatest dentin deposition isfrequently seen in the floor and roof of the pulpchamber, especially in molar teeth (7).

Composition of dentin ECM

The organic extracellular matrix (ECM) of dentin issimilar to soft tissue ECM and especially to boneECM, but in some respects it is unique. The high levelof collagen cross-linking in mineralized tissue (57,58)and virtual absence of type III collagen are the maindifferences with soft tissue collagens. Generalized con-clusions of the significance and function of the differ-ent ECM components in dentin are caused by thefindings that demonstrate differences in dentin ECMbetween species and even between the different partsof the tooth.

About 90% of the dentinal organic matrix is colla-gen. The major component of dentin collagen is typeI (59,60), of which the majority is a heteropolymerwith two a1(I) chains and one a2(I) chain (60).Odontoblasts also synthesize and secrete a type I col-lagen homopolymer consisting of three a1(I) chainsand commonly called type I trimer (60–64). Theexperiments with rat incisor odontoblast organ culture(61) and analysis of rat incisor predentin (63) indicatethat approximately 25 to 30% of the synthesized typeI collagen would be the type I trimer. However, inbovine extracts containing odontoblasts and preden-tin, only about 3% of the type I collagen was type Itrimer (62). The presence of type I trimer in mineral-ized dentin has only been shown in lathyritic rat inci-sors in which the collagen cross-linking was inhibitedby dietary lathyrogen (b-aminopropionitrile) (65,66),and it is not known if type I trimer would be present innormal dentin (60,65,66). Butler (60) concludes thattype I trimer is important for dentin formation butthat it may be involved in the maturation of predentinand degraded prior to cross-linking.

Type III collagen, widely seen in soft connectivetissues, is not normally seen in intertubular dentinmatrix. In developing human teeth, Lukinmaa et al.(67) demonstrated the expression of type III pro-alphacollagen mRNA, and type III collagen immunoreac-tivity was observed in early predentin and again inpredentin toward the completion of dentinogenesis,when mRNA was no longer detected via in situmethods. However, mature human odontoblastsexpress type III collagen mRNA detected with PCR,and produce the protein in tissue culture (68). Severalstudies (69–71) have found that type III collagenlocalizes in dentinal tubules. Since it has frequentlybeen related to von Korff fibers (70,72), it is possiblethat the staining seen in deeper areas of dentin (69,71)may represent intact or fragmented remnants of thesefibers.

With other collagens, the data are much more incon-sistent. A small amount of type V collagen is synthe-sized by odontoblasts (63); it has been shown in ratand 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). Similarlyconflicting results have been presented for type VIcollagen. Becker and co-workers (69) reported a typeVI collagen staining distribution essentially similar tothat of type III collagen, with relatively strong inten-sity in predentin and occasional fibrous staining indentinal tubules. However, Lukinmaa et al. (74) couldnot detect type VI collagen in dentin. Type IV colla-gen has not been seen in normal dentin (71). Theseconflicting findings may reflect differences, forexample, between species or in section pre-treatment,antibodies, or detection methods.

Becker et al. (69) suggested that the role for colla-gens other than type I collagen in mineralized tissuesmay be related to ECM (bone and dentin) remodelingbefore hard tissue calcification. This is in accordancewith the expression of various ECM protein-degradingenzymes, matrix metalloproteinases (MMPs) (75), andcathepsins (76) (for details, see the article on dentinnon-collagenous proteins in the next issue). The roleof enzymatic regulation of collagenous and non-collagenous components in the control of dentinECM maturation prior to mineralization has also beensuggested by Butler et al. (60,77). Type III collagen isalso present in dentinogenesis imperfecta (78) and inreparative dentin under carious lesions (79,80), whichmay, at least partially, be related to the disturbances of

Overview of dentin structure

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the enzymatic or other reactions controlling thematrix maturation rather than the expression of typeIII collagen.

About 10% of the dentin organic matrix consists ofproteoglycans and other non-collagenous proteins,and less than 2% is lipids. Non-collagenous proteinsare also produced by odontoblasts; they are distributedbetween the collagen fibrils and accumulate along thedentinal tubule walls, and are supposed to serveimportant functions in the mineralization process ofdentin (7). The presence and potential roles of non-collagenous proteins in dentin and dentinogenesis arediscussed in detail in other articles in this double issue.

Dentinal tubules

Tubularity is a central characteristic of dentin, affect-ing, for example, its mechanical properties, its abilityto withstand occlusal forces, and its behavior in den-tin bonding. The understanding of dentin three-dimensional structure may have a significant impact onoptimal cavity design and restorative procedures.

The common belief that dentinal tubules extend atright angles from the DEJ and run a fairly direct—orslightly S-shaped—course through the dentin wasrecently questioned in a study utilizing 3D phase-contrast microtomography (81). In dentin immedi-ately beneath enamel (within 0.3 mm), a wide range oftubule angular tilts (up to 75% of the tubules tiltingmore than 10 degrees) was seen. Within this area, thetubules also seemed to twist or curl, occasionally up to

90 degrees. Slightly further (0.5 mm) into dentin, nomore tilting or curling occurred, presumably becauseof odontoblast crowding (81). In addition, a differ-ence in tubule orientation relative to the DEJ wasobserved between upper and lower teeth (81). Whilethese findings require confirmation, it can be specu-lated that the difference in tubule orientation betweenupper and lower teeth can affect the response to teethloading, which might, for example, cause differences inthe deformation of the crown under mastication (81).The number of dentinal tubules in different locationsin relation to the DEJ or cementum does not varyexcept under the cuspal area, where the number ofdentinal 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 havenumerous branches and ramifications (Fig. 5). Thenumber of branches is higher in areas where thedensity of the main tubules is low (44,82), forming anabundant anastomosing system of canaliculi very muchlike osteocytes in bone (83) (Fig. 6). Mjör & Nordahl(44) identified three types of tubular branches: major,fine, and microbranches. Major branches (0.5 to1.0 mm diameter) are abundant peripherally while finebranches (300 to 700 nm diameter) are abundant inareas where the density of the tubules is relatively low.Microbranches (25 to 200 nm diameter) extend atright angles from the tubules in all parts of thedentin (44).

a b

Fig. 5. (a) Intensive branching of dentinal tubules close to DEJ (arrows). (b) Intensive branching of dentinal tubulesin the middle part of dentin. The tubules are visualized with Alizarin red. Bars = 20 mm. Reproduced with permissionfrom Kagayama et al., 1999 (82).

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An interesting finding of bamboo-like dentinaltubules with many nodules in the longitudinal sec-tions, which appear as circular tubules surrounding themain tubule, was seen with Alizarin red staining (82).The circular tubules of the nodules adhere to one sideof the dentinal tubules and resemble that of the peri-tubular dentin (Fig. 7). The nodules correspond tothe tubular branching penetrating through thick peri-tubular dentin observed with SEM (44). Peritubulardentin, even though highly mineralized, is porous andthe nodules observed with Alizarin red indicate a sug-gested function for peritubular dentin in regulating

the degree of communication between the intertubu-lar dentin and odontoblasts via dentinal fluid (84).

Peritubular dentin

The commonly used name “peritubular” is actuallymisleading, since the prefix “peri” (“around”, “sur-rounding”, “enclosing”) refers to material formedaround the tubules. Since peritubular dentin in mostspecies (including humans) is deposited on the innersurface of the tubular lumen by the odontoblasts onlyafter the formation of intertubular dentin, a more

Fig. 6. SEM images of resin-embedded, acid-etched dentin (a) and mandibular bone (b), demonstrating markedsimilarity between the odontoblast process and osteocyte lacuno-canalicular networks. Reproduced with permissionfrom Lu et al., 2007 (83).

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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 inter-tubular dentin. It is considered to be more mineralizedand practically free of collagenous matrix, althoughboth symmetry and degree of mineralization may varysignificantly. The deposition of peritubular dentincauses a progressive reduction in the tubule lumen(dentin sclerosis). During environmental stimulationand irritation, the formation of peritubular dentin maybe accelerated (12,13).

Within mantle dentin, where dentinal tubules termi-nate in small branches, very little peritubular dentin ispresent and the tubules appear as empty channels pen-etrating the intertubular dentin. A thin lining of peri-tubular dentin may already be present at 20 mm fromthe DEJ (49) (Fig. 8). A gradual thickening of peritu-bular dentin occurs with increasing distance from theDEJ until it reaches the normal thickness of approxi-

mately 1 mm (Fig. 8). Concomitant with this thicken-ing, the tubular density per unit volume increases(8,49).

The mineral content of human peritubular dentin isapproximately 40% higher compared to intertubulardentin (7). The differences in mineral structuresbetween inter- and peritubular dentin indicated inearlier studies (85,86) have been, at least to someextent, questioned by more recent studies indicatinglittle difference in the nature, size, and organization ofthe mineral phase between inter- and peritubulardentin (84,87–89). Peritubular dentin is spatially morehomogenous than intertubular dentin, with differenthardness (39), elastic properties (90), optical aniso-tropy (91), and fracture properties (92). All of theseobservations indicate completely different mechanicaland structural properties of these intimately associatedforms of mineralized dentin structures.

The formation of peritubular dentin begins in den-tinal tubules close to (but not at) the mineralization

a c

b d

Fig. 7. (a) Longitudinal section of dentinal tubules in the middle part of dentin observed with Alizarin reddemonstrates numerous circular nodular structures around main tubules. (b) In cross-section the staining is locatedat the interface between peritubular and intertubular dentin, with occasional branches penetrating into intertubulardentin. The nodules were absent in dentin close to DEJ, at the pulpal part of dentin (c and d), and in teeth extractedfrom young patients. Bar = 5 mm, applies to all figures. Reproduced with permission from Kagayama et al., 1999 (82).

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front (93), presumably with the accumulation of non-collagenous proteins along the tubule walls (7)(Fig. 9). Recent studies with combined SEM andtime-of-flight secondary ion mass spectroscopy (TOF-SIMS) analyses show that peritubular dentin is a sepa-rate phase from intertubular dentin, forming a distinctannulus within each tubule (84,94), thus contradict-ing previous studies which have indicated that crystal-lization within the peritubular dentin would bemediated by a collagenous matrix interaction as inintertubular dentin (88,89,95). The contradictoryresults may, at least partially, be caused by the heter-ogenous intratubular mineralization occasionallyshown to contain collagenous and other proteins (13).However, those types of mineralization do not repre-

sent true peritubular dentin, and may represent atypi-cal intratubular calcification, which has been suggestedto be a non-vital process (96). Indeed, one of the maindifferences between inter- and peritubular dentins isthat peritubular dentin is essentially collagen-free(13,84,88,97). Overall, peritubular dentin is very lowin organic components, which are a mixture of acidicproteins, phospholipids, or possibly proteolipid com-plexes, with small amounts of glycoproteins and pro-teoglycans. Even though the sulfate content ofperitubular dentin proteins seems to be low, the pres-ence of chondroitin 4-sulfate (CS-A), chondroitin6-sulfate (CS-C), and dermatan sulfate (CS-B) typeshave been indicated (84).

Peritubular dentin is perforated—in addition totubular branches (44)—by many small pores and fen-estrations (84,94), allowing the passage of tubularfluid and intertubular dentin components across theperitubular dentin (Fig. 9). Based on the analysis ofthe organic components of peritubular dentin (84),there may be a potential for the calcium-phospholipid-proteolipid components of peritubular dentin to beinvolved in the signaling and ion transport process-es. Thus, peritubular dentin may also have directrole(s) in active transport and other regulatory activi-ties between vital intertubular dentin matrix andodontoblasts, participating in retaining the vitality ofdentin. The active processes in mineralized intertu-bular dentin would offer an explanation of some pre-viously poorly understood findings regarding theage-related changes in dentin, e.g. the disappearanceof matrix metalloproteinase-2 (MMP-2) from intertu-bular dentin with age (76,98) (discussed in more detailin the next issue). This proposal offers a completelynew view not only on the role of peritubular dentin(usually thought to act as a passive blockage in den-tinal tubules), but also on the vitality of mineralizeddentin as a whole. If signaling and active transportbetween intratubular structures and components(odontoblast processes, tubular fluid) and intertubulardentin actually occur, the nature and function ofdentin as a tissue should be revisited.

Dentin sclerosis

The main dentin response under carious lesions andrestorations is reactive dentin sclerosis, seen as a trans-lucent or transparent zone. The first phase of its for-mation in the initial stage of dentinal caries seems to be

a

b

Fig. 8. (a) Thin peritubular lining inside the tubulesapproximately 20 mm from the DEJ in fractured dentinseen with SEM. (b) Coronal dentin 800 mm below theDEJ. Highly mineralized peritubular dentin (P) isclearly distinguishable from intertubular dentin. A crackin the right-hand side of the tubule is frequently seen inSEM preparations of tooth crown dentin and most likelyrepresents an artifact due to sample handling. Modifiedwith permission from Zaslansky et al., 2006 (49).

Overview of dentin structure

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dependent on odontoblastic processes (99). However,acid etching in vivo and in extracted teeth produces atransparent layer, suggesting that it is not a vital defen-sive reaction (100). Instead, calcium phosphate dis-solved from apatite crystals diffuses more deeply intothe dentin tubules and precipitates (100–103), at leastat the side of the lesion where the irritation of associ-ated odontoblasts is not so intense (100), into whathas been termed “caries crystals.”

Massler (104) proposed that superficial sclerosis isdue to the re-precipitation of minerals from cariouslesions and saliva, while deeper sclerosis requirescalcium from the pulp. The degree of sclerosis isdirectly related to the success in arresting the lesionprogression (103), but after the successful arrest of thelesion, mineral uptake from the saliva is very limited(105). Reactive dentin sclerosis occurs at all ages butincreases both in prevalence and intensity with age,and is reported to be seen more often in males than infemales (106).

Similar to tertiary dentin formation, sclerosis is anon-specific reaction and physiological dentin sclerosisespecially occurs in older teeth (106–108). It is typi-cally observed to decrease in the direction of the pulp

(107), while most tubules in the translucent zone maybe totally occluded (108). It has been proposed thatphysiological sclerosis should be regarded as beingdifferent from the tubular sclerosis seen in relation tocaries (100,109). Physiological dentin sclerosis mayeven reduce the formation of tertiary dentin, possiblyby reducing the permeability of dentin before the irri-tation occurs, whereas reactive dentin sclerosis doesnot prevent tertiary dentin formation. Physiologicaldentin sclerosis also develops in areas without cariouslesions or irritation, for example on the floor of thepulp chamber and root canals. In roots, dentin sclero-sis progresses with age from the apex toward the cer-vical portion (Fig. 10) (106,110,111).

Tertiary dentinTertiary dentin forms as a response to externalirritation—attrition, abrasion, erosion, trauma, caries,or cavity preparation—in order to increase the thick-ness of the mineralized tissue barrier between the oralmicrobes and the pulp tissue. It has also been calledirritation dentin, irregular dentin, irregular secondarydentin, etc. (14). Defensive reactions can be observed

a b

Fig. 9. (a) Field emission scanning electron microscope (FE-SEM) image of a cross-section of a mouse second lowermolar. Pulp tissue with odontoblasts, odontoblast processes, and predentin have been mechanically removed. Densecollagen network covers the tubular walls immediately below the orifice. In most intertubular dentin ridges, theindividual collagen fibrils cannot be seen, presumably due to mineralization (arrows). The ridges thus represent themineralization front. Collagenous mesh is apparent just below the tubule orifice. Slightly deeper, the collagen fibrilsare masked with more homogenous structure, representing either non-collagenous proteins or the initial formation ofperitubular dentin. Magnification = 10,000 ¥; bar = 1 mm. (b) Higher magnification of the area marked with a squarein (a). Relatively sharp borderline between visible fibrils and homogenous surface of non-collagenous proteins orperitubular dentin indicates highly regulated process in this area. The fibril diameter varies between approximately 20and 100 nm. Globular structures in connection with fibrils and in the homogenous surface—either uniform layer ofnon-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 peritubulardentin porosity suggested by Gotliv & Veis (84,94). Magnification = 35,000 ¥; bar = 1 mm.

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very early, with enzymatic changes being seen duringthe earliest stage of enamel caries both in the dentin–pulp border (104,112–115) and in dentin itself(104,116–119), but not in pulpal tissues (120,121).

The dentinogenic potential of the dental pulp,including different ex vivo and in vivo experimentalmodels and the chemical and biomolecular agentsused in these experiments, has very recently beenextensively reviewed by Tziafas (122). Therefore, ter-tiary dentin is only briefly reviewed here.

The aim of tertiary dentin is to protect pulpal tissueby increasing the thickness of dentin between the pulpand external wear or irritation (Fig. 11). Attrition issuggested to be more prone to induce tertiary dentinformation than caries (106). From a physiologicalpoint of view, this is acceptable, since caries is more or

less related to the modern diet containing excessrefined sugars, which favors caries formation. Morenatural, coarse diets are prone to cause abrasion andattrition, and it is tempting to speculate that tertiary

Fig. 10. The classical image demonstrating, for the firsttime, the age-related root dentinal sclerosis advancingfrom the apical part toward the crown. Longitudinalcentral 0.5 mm section of an adult tooth photographedby transmitted light shows the transition between trans-parent apical root dentin (dark) and opaque dentin(white). Reproduced with permission from Nalbandianet al., 1960 (110).

a

b

c

�Fig. 11. (a) Pulp chamber obliteration in a lower firstmolar by tertiary dentin formation, as seen in the apicalradiograph. The radio-opaque area around the calcifica-tion in the second molar (black arrows), as well asirregular calcification in the distal wall of the secondpremolar pulp chamber (white arrow), indicate the pres-ence of pulp stones rather than tertiary dentin formationin these teeth. (b) Removal of the filling from the firstmolar 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 ofthe demarcation line, exposed the pulp chamber floorand allowed instrumentation of the root canals.

Overview of dentin structure

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dentin formation—as a defensive reaction to increasethe dentinal width between the pulp and the oralcavity—is “designed” to protect the pulp from exten-sive wear. According to Stanley et al. (106), the preva-lence and intensity of tertiary dentin formation undercaries is proposed to be independent of age in humans.However, this conclusion can be criticized because thegroup of young teeth in that particular study con-tained only three specimens and the age scale was wide(11 to 19 years). In young (3 to 8 weeks) rat molarswith ongoing primary dentinogenesis, a non-linearrelationship (123) or even a negative correlation (124)between dentin formation and extension of dentinalcaries has been observed, indicating that the tertiarydentin-like response does not occur in teeth withprimary dentinogenesis.

The form and regularity of tertiary dentin dependson the intensity and duration of the stimulus. Gener-ally, two forms of tertiary dentin are recognized:reactionary dentin (produced by original primaryodontoblasts) and reparative dentin (produced bynewly differentiated replacement odontoblasts) (109,125–128). In addition, it has been suggested that athird type of mineralization be distinguished as amerely defensive non-specific production of mineral-ized matrix. This would be produced by so-calledfibrodentinoblasts, and the product would be calledfibro- or osteodentin (122). In fact, Taintor et al.(129) already challenged the term “reparative” dentinin 1981. They argued that since repair, by definition,consists of the replacement of damaged tissue, theirregular dentin formed in response to external irre-versible damage would be comparable to scar tissueformation (129,130), and should rather be called“irritation” or “irritational” dentin (129). While thiswould be, strictly speaking, a more exact term, thepresent terminology (tertiary dentin and reactionaryor reparative dentin) is commonly accepted and istherefore also used in this review.

In clinical situations, tertiary dentin usually containsatypical fibrodentin, reparative dentin, and reactionarydentin. Since the criteria for the assessment of differ-entiated odontoblast-like cells (or the level of differ-entiation) are not well defined (131), the presence ofdifferent types of tertiary dentin at the same site mayreflect the process of odontoblast-like cell differentia-tion from non-specific, hard-tissue forming cells intofully differentiated odontoblast-like cells. In general,reactionary dentin has a more or less tubular continu-

ity with secondary dentin, while the structure, organi-zation, and mineralization of reparative dentin varysignificantly. Since reparative dentin is generally atu-bular, it may form a relatively impermeable barrierbetween tubular dentin and pulp tissue. The regularityof reparative dentin is supposed to be inversely relatedto the degree of irritation (122).

The goal of pulpal treatment procedures aimed atpreserving pulp vitality could be the reduction ofdentin permeability beneath the injury, thus isolatingthe pulp from further irritation. The junction betweenprimary and reparative dentin is considered to act as aprotective barrier against carious stimuli (109,132). Itcan, however, also be argued that the complete isola-tion of the pulp by non-tubular reparative dentin isnot necessarily desirable. Pulpal sensory nerve fibersundergo extensive sprouting in response to injury, andit is commonly accepted that pulpal nerves are pro-tective in nature and are involved in the recruitmentof inflammatory and immunocompetent cells to theinjured pulp (133). The degree and state of theresponse seem to be highly dependent on the changesin dentin permeability. Interestingly, the number ofdentinal tubules close to the DEJ is significantlyhigher in the cuspal area than in other parts of dentin(44). Under the cusps, the dentinal tubules are alsostraighter and the odontoblast processes penetratedeeper to the dentin–pulp border (134,135) or evento the DEJ (136). Since the cusps are the first area tobe worn due to abrasion or attrition, it is tempting tospeculate that the reason may be related to the regu-lation of defensive mechanisms in the dentin–pulpcomplex. The dentinal tubules may be more directand odontoblast processes may penetrate more deeplyin order to deliver the message of dental wear andinduce tertiary dentin formation so as to maintain thehard tissue barrier between dental pulp and the oralcavity. The coronal dentin–pulp border has other dis-tinctive histological features: a dense innervation ofinner dentin and the odontoblast layer (137), pulpcells producing nerve growth factor and its receptor(137), and an extremely dense capillary network(138,139). The co-localization of these tissue compo-nents together with the straight tubules and longodontoblast processes may indicate a role in sensingthe external irritation and controlling defensive reac-tions. The connection of pulpal histological featureswith odontoblasts and their processes is at leastindirectly supported by the finding that the dense

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innervations normally seen under the odontoblastlayer (the subodontoblastic plexus of Raschkow) withsome axons passing into dentinal tubules (7) is notseen in the case of reparative dentin (140). Since thenociceptive fibers contribute to normal homeostaticregulation and vasoregulation and to healing afterinjury (133,137), this significant reduction in inner-vations may affect the inflammatory and immuneresponses under reparative dentin. As well, the initialimmunodefensive reaction (measured as the accumu-lation of antigen-presenting cells) occurs beneath thedentinal tubules communicating with superficial carieslesions (141,142). However, after substantial forma-tion of sound reparative dentin, the inflammatoryresponse to the microbial burden subsides (142,143).The re-accumulation of antigen-presenting cellsoccurs only after bacterial invasion of reparativedentin, close to the pulp (142). We conclude that thissupports the concept that the junction between theprimary and reparative dentin may act as a barrier toprevent carious stimuli (109,132). However, even ifreparative dentin prevents microbial component pen-etration into the pulp, it also allows caries progressionclose to the pulp without inducing normal defensivereactions occurring in the dentin–pulp complex(142). Therefore, the formation of reactionary den-tin with tubular continuity is preferable (122). Insummary, it must be emphasized that the understand-ing of the mechanisms regulating reparative dentinformation is still limited. Excessive dentin repair mayalso be detrimental when the response is not limitedto the site immediately below the dentinal injury butresults in generalized root canal system calcification(129) (Fig. 11). The decrease of vital pulp tissue mayreduce the defensive potential of the pulp (for details,see next paragraph), and obliteration of the root canalsystem certainly makes endodontic procedures morechallenging.

The dentin matrix contains biologically activemolecules such as growth factors (for details, see thearticle on dentin non-collagenous proteins in the nextissue), which can induce dentinogenic events both invivo and in vitro. These dentin matrix bioactive factorsare supposed to possess a similar inductive potentialfor tertiary dentin formation as seen by the enamelepithelium and basement membrane during physi-ological dentinogenesis in embryonic conditions(122,128). An appropriate pulpal environment (suchas the absence of severe inflammation and adequate

vascularity) and mechanical support with a favorablesurface for cell attachment (such as dentin, or suffi-ciently solid calcium hydroxide, or—perhaps evenbetter—MTA in the case of pulp capping) are con-sidered absolute requirements for appropriate tertiarydentin formation (122). The dentin–pulp complexmay possess a remarkable capacity to survive evenintensive dentinal damage, with reactionary andreparative dentin formation occurring under a re-maining dentin thickness of as thin as 0.5 mm(144,145). In the absence of microbial infection,injuries to the pulp coincident with dentinal injuriesare presumed to be reversible. After experimentalexposure of dentinal tubules, repair and healing of thepulp occurs in spite of continuous exposure of cutdentinal cavities to the salivary microflora (143), sug-gesting that dentin is able to oppose bacterial threatseven when a small rim (�1.5 mm) remains. Clinically,the dentin–pulp complex is a target for repeatedmicrobial, mechanical, and chemical insults such asprimary and secondary caries, replacement of restora-tions, and attrition. Empirical clinical experience hasindicated that the pulpal healing potential is reducedby a repeated series of stimuli. For example, thetendency for teeth restored with full crowns (146–150) or traumatized teeth with pulp canal oblitera-tions (151–155) to develop pulpal necrosis has beenobserved in numerous studies. It is likely thatrepeated dentinal irritation affects the mechanisms forreactionary dentin synthesis and replacement of odon-toblasts as well as causing pulpal scarring and loss ofperivascular stem cells. While there is ample evidencedemonstrating increased pulpal cell proliferation, col-lagenous protein deposition, and reparative dentinformation after a single dentinal injury (cavity prepa-ration) (156–163), after a double injury, the increasein cell proliferation and collagenous protein deposi-tion is significantly less than after the single injury(163). Although the response may have beendecreased by pulp–dentin defensive reactions to thefirst injury (such as occlusion of dentinal tubules orimpermeable reactionary dentin formation), the studystill supports the decreased ability of the dentin–pulpcomplex to respond to repeated insults. The authorsuggests that the timing of sequential episodes of den-tinal irritation could be used to minimize pulpaldamage after extensive restorative dental treatment(163). However, more research is needed to validatethis suggestion.

Overview of dentin structure

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Root dentinThe epithelial cells of Hertwig’s epithelial root sheath(HERS) initiate odontoblast differentiation very similarto that in the coronal area (7,12). In principle, rootdentin 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 basallamina, not perpendicular as often seen in the crown(25). The terminology and even the structure of rootmantle dentin are also controversial: it has been calledthe “hyaline layer” and reported to be absent in someanimals and to vary in thickness in others (25). Struc-turally, the human CDJ represents a region of inter-spaced 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 thedentin intermingle with collagen fibers parallel to theroot surface, ensuring a tight attachment of cementumto the dentin (Fig. 12). The CDJ also contains pores,possibly representing HERS remnants (164). Thegranular layer of Tomes is located in the outermost partof the root dentin. It is believed to represent the mantledentin in the root surface or be located immediatelybelow the root mantle dentin (25). Similar to coronalmantle dentin, it also displays thin canaliculi and poorlyfused globules (12,25). However, the granular struc-tures can only be seen in ground sections, not inhistological staining or electron microradiographs (7).Most likely they represent the mineralization pattern inthe initial phases of root dentin and cementum forma-tion. The density of tubules in root dentin is still asomewhat controversial issue. Some studies have indi-cated a rather moderate decrease in tubular densityfrom the cemento–enamel junction (CEJ) toward theapex (44,166) while others have indicated a morepronounced decrease (167). The reason for the differ-ences may be the different methods or different teethused for the analyses, as Schellenberg et al. (167) foundmarkedly fewer tubules in mesio-distal than bucco-lingual surfaces of premolars, but not in third molars.The main part of the root dentin is rich with both finetubular branches and microbranches, with occasionalmajor branching (Fig. 13) (44).

Apical dentin

It has been suggested that the dentin in the mostapical part of the roots differs from the rest of the

a

b

c

Fig. 12. FE-SEM images of human cement–dentinaljunction (CDJ). (a) 10 to 15 mm cementum layer inintimate contact with dentin. Magnification = 500 ¥;bar = 10 mm. (b,c) Higher magnification demonstratesthe mineralized collagen fiber continuity from cemen-tum to underlying dentin. Magnifications = 2,500 ¥ (b)and 5,000 ¥ (c); Bars = 10 mm (b) and 1 mm (c).

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root dentin in many respects. The number andregularity of tubules markedly decreases in the apicalroot (44,166,168,169). Apical primary dentinaltubules 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 causingthe formation of reparative dentin, the dentin formedafter primary dentinogenesis should, by definition,be secondary dentin. However, a distinct borderbetween primary and “secondary” dentin, with dis-continuity of dentinal tubules, has been demon-strated. The apical portion of human teeth also showsother marked variations in structure, such as acces-sory root canals, areas of resorption and repairedresorption, and cementum-like tissue lining the apicalroot canal wall. The causative factors for these kindsof dentin formation could be, for example, pulpinflammation or a response to occlusal loading. Inaddition, age-related root tubular sclerosis thatstarts in the third decade of life from the apical regionand advances coronally (111) has recently beensuggested to be the main factor influencing thepermeability of root dentin (170,171) (Fig. 14).Root dentin seems to have regional differences inits permeability: buccal and lingual curvatures ofroot canals show patent tubules that take up dyes,while the mesial and distal pulpal borders seem to beoccluded with minerals (170,171) (Fig. 15). Onewonders how this pattern of tubule patency corre-sponds to local stress distributions in the functioningof these roots, and whether these stress distributionstranslate into regional differences in dentinal fluidshifts (172).

Pulp stonesPulp stones are discrete or diffuse pulp calcificationsthat can be classified structurally as well as based onlocation (173). Structurally, there are “true” and“false” pulp stones, the distinction being morphologi-cal. A third type, “diffuse” or “amorphous” pulpstones, are more irregular in shape than false pulpstones 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 arelined with odontoblasts (or rather odontoblast-likecells), while “false” pulp stones with atubular calcifi-cation have been considered to be formed from degen-erating cells of the pulp that mineralize. Thedistinction between the “true” and “false” pulp stonesmay be somewhat artificial, as both tubular and atu-bular dentin can be found in a single pulp stone(Fig. 16).

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 numerousfine branches, giving it the appearance of an interdentaltoothbrush. H-E staining of a premolar from a 12-year-old. (c) Dentinal tubules in cervical area showing varia-tions of fine branches from the same tooth as in (b). (d)Variation in branching of dentinal tubules in rootdentin. Hematoxylin-eosin (H-E) staining (a–c); Massonstaining (d). Reproduced with permission from Mjör &Nordahl, 1996 (44).

Overview of dentin structure

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Fig. 14. Relative mean dye penetration (in percentage of complete dentin area) after extensive (60 day) incubation ofMethylene 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 barbellshape and dye penetration pattern of Patent blue. Magnification = 16 ¥. (b–d) Back-scattered electron micrograph ofareas with (b) and without (c,d) dye penetration, demonstrating patent dentinal tubules in (b) and tubular sclerosisin (c,d). Magnifications = 1,000 ¥ (b,c); 3,000 ¥ (d). Reproduced with permission from Paqué et al., 2006 (170).

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a b

c d

e f

Fig. 16. Heterogenous structure of pulp stone. (a) Pulp stone in human dental pulp, stained with hematoxylin-eosinstaining. The pulp stone appears as a solid calcified mass. The width of the stone is approximately 3 mm. Originalmagnification = 50 ¥. (b) Adjacent section stained with Toluidine blue. The pulp stone appears much more heterog-enous. Original magnification = 50 ¥. (c) Higher magnification of the lower left corner of (a). Odontoblast-like cellsline the lower border of the pulp stone, while the right side is devoid of odontoblast-like cells. Original magnifica-tion = 100 ¥. (d) Same area stained with Toluidine blue demonstrates tubular structure in the pulp stone at the siteof 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 theright side are cut across the tubule direction and appear less organized. Original magnification = 200 ¥. (f) In anotherpart of the pulp stone, tubules appear more sparse and with numerous fine branches and microbranches. Originalmagnification = 400 ¥.

Overview of dentin structure

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A single tooth may contain one or several pulpstones of varying size, most often found in coronalpulp but sometimes also present in radicular pulp.Despite a number of studies, the exact cause of suchpulp calcifications remains largely unknown. Externalirritation (caries, attrition) has been suggested as acause, but pulp stones also appear in teeth with noapparent cause (e.g. impacted third molars). Pulpstones have also been noted in relation to systemic orgenetic diseases, including dentin dysplasia [especiallydentin dysplasia type II (175,176)], and certain syn-dromes (176). Large pulp stones in the pulp chambermay obstruct the canal orifices (Fig. 17), and in theroot canal they may complicate access to the apicalcanal. For a comprehensive description of pulp stones,the reader is referred to a recent review article by Gogaet al. (176).

Words of caution: dentindifferences between species

It is important to realize that differences in the struc-ture and chemistry of dentin between differentspecies have long been recognized (12). Forexample, the cellular junctions between odontoblasts,the amount of peritubular matrix, the structure andmechanism of formation of peritubular dentin,and the presence and thickness of root mantle dentinvary between species (25). These differences mayaffect the usability of certain animal models. Becausemany (maybe most) biochemical studies have beenperformed on bovine and rat dentin, available dataoften directly concern only these species. In general,in terms of protein composition of the organicmatrix, dentin species can be divided into two main

a b

c d

Fig. 17. Pulp stones. (a) Pulp stone obliterating most of the coronal pulp chamber in a lower molar. (b) Highermagnification of (a). The pulp chamber contains both loose pulp stone “a” originally surrounded by pulpal tissue, andpulp stone attached to the pulp chamber wall “b”. (c) Pulp stone filling a premolar pulp chamber. The uneven formof 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. Itmust be noted that some material was lost as the pulp stone had to be drilled out of the pulp chamber. Therefore, theoriginal size of the stone may have been even larger.

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groups: (a) continuously growing rodent teeth;and (b) teeth with limited primary dentinogenesis(such as bovine, porcine, rat molar, and humandentin). Therefore, care must be exercised whenextrapolating findings from one species to another(12,25).

Mouse models (both in vivo and developing toothorgan cultures) have been used to study tooth devel-opment and abnormalities [reviewed by Fleischman-nova et al. (177)], and transgenic and gene knockouttechniques have allowed more sophisticated methodsof studying the roles of single gene products on toothmorphogenesis and development. Even though thesemodels have occasionally led to breakthrough discov-eries of the role and interference of a single protein intooth development, in many cases the effects of asingle gene deletion are minor or non-existent. Thisdemonstrates the presence of “back-up systems” thatare able to compensate for the function of a defectivegene.

In research on mineralized tissues, the rat has longbeen considered as a particularly good experimentalmodel (178). The same mechanisms control gains andlosses in bone mass in young and aged rats andhumans. The rat model is not as time-consuming asthose of larger animals. Osteopenic and osteoporoticchanges in rat bones copy the human forms. Mechani-cal usage and drug effects as well as bone healing caneasily be studied on rats (178). Rodent incisors growcontinuously and therefore the entire sequence ofenamel and dentin development, including the lifecy-cle of the amelo- and odontoblasts, may be studied inone tooth. Continuously growing teeth, however, aredisadvantageous when extrinsic factors affecting thetissues are studied. In rat incisors, only intertubularbut not peritubular dentin is present (12). The mainregulatory factor in dentinogenesis and odontogenesisin rat incisors is the attrition rate (179). Because of theconstant wear of incisors, enamel and dentin are con-stantly renewed and therefore incisors do not providea permanent record of mineral metabolism in dentin(53). Also, caries does not occur in rodent incisorsand therefore caries experiments are impossible in ratincisors.

Rodent molars, however, are in many ways similar tohuman molars. The crown is multi-cuspal and fissuresbear a great similarity to those in human molars. Therat molar has a limited growth and enamel and dentinformation resembles that in humans, even though sec-

ondary cementogenesis continues throughout life inorder to keep the tooth functional in spite of attrition(180). Therefore, rodent (especially rat) molars havebeen extensively used for caries research (181), includ-ing the response of the dentin–pulp complex to exter-nal irritation.

Bovine teeth have been frequently used in in vitroand in situ experiments to study mechanical proper-ties and to simulate human tooth behavior. Bovineteeth have been extensively used in studies involvingdental erosion and abrasion (182–185). Bovinecoronal dentin is similar to human dentin withrespect to the number and diameter of tubules (186)and hardness (187), but has a slightly higher radi-odensity, possibly due to the greater amount of peri-tubular dentin in bovine teeth in age-related samples(187). The use of bovine incisors as substitutes forhuman teeth in studies involving erosion/abrasion iswell accepted because dentin wear in human thirdmolars and bovine incisors is quite similar (188).Bovine dentin close to the enamel–cement junction isa viable substitute for human dentin in in vitro den-tin permeability experiments (189). Bovine incisorscan also be used as substitutes for human teeth inimmediate composite adhesive bond strength tests(190,191).

Concluding remarksUnlike bone, dentin does not turn over. That is, itsexternal surface is far removed from the cells whichproduced it developmentally, precluding coronaldentin repair. Unlike bone, dentin functions in aseptic, hostile environment with great temperatureand chemical challenges. It has evolved into a dynamictissue that is able, within biological limits, to react toenvironmental stresses in a protective, reactive manner.The microscopic nature of the dentin–enamel andcemento–dentin junctions has delayed the study oftheir mechanical and fracture properties as well as theirbiochemical composition. The recent application of,for example, atomic force microscopy, nanoDynamicMechanical Analysis (nanoDMA), patch-clamp elec-trophysiology, and molecular biology probes holdsgreat promise for a new understanding of how dentinresponds to various stimuli. Hopefully this will lead totherapeutic advances in Operative Dentistry andEndodontics.

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