Structural events in the caries process in enamel, cementum, and dentin.

Structural Events in the Caries Process in Enamel, Cementum, and Dentin R.M. FRANK Centre de Recherches (Unite mixte CNRS-INSERM), Faculte de Chirurgie Dentaire, Universite Louis Pasteur, 4 Rue Kirschleger, 67000 Strasbourg, France The structural events observed in enamel, cementum, and dentin during the caries process have been reviewed. In incipient enamel lesions, the prevailing concept of an almost intact surface layer has been seriously challenged by SEM and TEM observations demonstrating structural pathways (such as enlarged prism junctions or sheaths) from the enamel surface to the sub-surface lesion. The destruction in this latter location consisted of (1) enlarged prism junctions, (2) dif[use mineral destruction in the prism cores, and (3) destruction of the interprismatic substance. In root caries, the destruction oj cementum started along junctions between calcified layers of extrinsic (Sharpey) and intrinsic collagen fibers as well as along incremental lines. Invasion oj Gram-positive micro-organisms followed these enlarged junctions. Dentin caries was similar in coronal and root caries. It consisted of sclerosis of the lumens oj the dentinal tubules, followed by an important gradient of demineralization oj intertubular dentin and destruction oj occluded tubular lumens and pcritubular dentin. Bacterial penetration occurred initially in the dentinal tubules and was followed by bacterial invasion and destruction oj the intertubular dentin. Various phenomena oj crystalline remineralization were described in enamel and dentin. Whereas in enamel and dentin caries, an important gradient oj demineralization was observed before bacterial invasion, a simultaneous destruction oj the mineral and organic components seemed to occur in cementum.

J Dent Res 69(Spec 155):559-566, February, 1990

Introduction. It is now generally recognized that dental caries is an infectious disease which causes localized destruction of the coronal and the radicular dental hard tissues induced mainly by acids produced in dental plaque (Nikiforuk, 1985; Thylstrup and Fejerskov, 1985). The structural events observed in enamel, cementum, and dentin during the caries process will be described in this report.

Technical remarks. For scanning electron microscopy (SEM), white and brown enamel lesions on approximal surfaces of 15 permanent molars and premolars of patients aged 12-15 years were fixed in 70% alcohol. After 24 h of treatment in 10% NaOCI, the specimens were washed in distilled water, and plano-parallel l-rnm-thick sections were cut in a mesio-distal plane through the caries lesions with a Gilling-Bronwill microtome (Rochester) equipped with a special diamond disk (Chung Ming Grinding Materials, Yaumati, Kowloon, Hong Kong), allowing undamaged enamel surfaces to be prepared without being previously embedded. After use of a critical-point apparatus and being coated with a thin Au-Pd layer in a Hummer-Juniorcathodic evaporator (Siemens, Karlsruhe), the surfaces and the depths of the lesions were studied in a JEOL (Tokyo) lSM 35 C scanning electron microscope under 10-15 kV. For transmission electron microscopy (TEM), white spots Presented at a Joint IADR/ORCA International Symposium on Fluorides: Mechanisms of Action and Recommendations for Usc, held March 21-24, 1989, Callaway Gardens Conference Center, Pine Mountain, Georgia

on approximal enamel surfaces of eight permanent premolars of patients aged 12-20 years were fixed for four h in a 2% paraformaldehyde/2% glutaraldehyde solution buffered at pH 7.4, followed by a one-hour 2% osmium tetroxide post-fixation in the same buffer. After being embedded in Epon 812, nondecalcified thin sections were prepared with a diamond knife and stained with uranyl acetate and lead citrate. For the TEM study of caries in human coronal dentin, 12 molars from 20- to 35-year-old patients were used, with occlusal fissure caries having reached the enamel-dentin junction as judged by intra-oral x-ray. Carious coronal dentin was fixed and prepared as previously described. Typical superficial root caries, with and without cavitations, of incisors and premolars of 18 patients, aged 52 to 60 years, were prepared for TEM with the same technical procedures as those described for enamel and dentin caries. Thin, non-decalcified sections of carious cementum as well as carious coronal and root dentin were decalcified with 0.4 mol/L EDTA solutions (pH = 7.4) for 24 h. All TEM observations were made in a lEOL (Tokyo) 100B electron microscope.

Results and discussion. The incipient enamel lesion. - Following the initial observation of Applebaum (1932), it is well-recognized (by polarized light microscopy, microradiography, and microdensitometry) that the incipient lesion in enamel consisted of a relatively intact surface layer overlying a demineralized sub-surface zone (Thewlis, 1940; Gustafson, 1957; Darling, 1958; Schmidt and Keil, 1971; Silverstone, 1973; Silverstone et al., 1988). By use of polarizing microscopy, four different regions have been described in the early enamel lesion (Silverstone, 1973). Moving from the surface inward, these are the surface zone, the lesion body, the dark (or positive) zone, and the translucent zone. Silverstone (1973) and Silverstone et al. (1988) considered that the surface zone and the dark zone are formed as a result of remineralization phenomena, whereas the body of the lesion and the translucent zone were produced as a result of demineralization. In SEM (Figs. 1-4) and TEM (Figs. 5-8), similar convergent ultrastructural observations were made on incipient white and brown spots. As already noted by Haikel et al. (1983), it appearedthat, after treatment with sodium hypochlorite, the brownspot lesions were bleached and became macroscopically similar to white-spot lesions. Only two zones consisting of a surface layer and a sub-surface lesion or lesion body were clearly identified in SE\1 on mesio-distal sections in both types of incipient enamel caries (Figs. 1 and 2). The surface layershowed less mineral destruction than the body lesion, and, for a prismatic surface layer, a striking broadening of the prism sheath areas was apparent (Fig. 2, arrows). These enlarged sheaths or prism junctions could be followed in SEM from the enamel surface (S) to the body of the lesion (CP) through the whole prismatic layer (Fig. 2). Such an observation confirmed earlier findings of Haikel e/ al. (1983) and evidenced minute pathways from the enamel surface, where the dental plaque was located, to the sub-surface lesion. These pathways, following 559

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Fig. I - Scanning electron microscopic observation of a mesio-distal section of an approximal white-spot lesion of a permanent upper premolar. The small lesion consists of an apparently intact surface layer (L) overlying a demineralized sub-surface area (8). E = intact enamel; S = enamel surface. Fig. 2 - Higher magnification of the apparently intact surface layer (L) overlying a dem ineralized sub-surface area in scanning electron microscopy. A striking broa deni ng o f the prism j unc tion s or prism sheath spaces (black arrows) can be foll owed fr om the enamel surface (S) to the demineralized subsurface. where the prism cores (CP) arc destroyed. Fig. 3 - Diagram of an ea rly ena mel caries lesion with an apparently intact surface layer overlying a demineralized subsurface area (8). In the apparently intact layer. miner al dissolution was preferentially observed along the prism junct ions or pri sm sheaths (arrows). In the sub-surface area (8), the prism cores (P) are destroyed initially. SI = imcrprismatic substance. Fig. 4 - Scanning electron microscopic observa tion of an unetched enamel surface of an approximal brown spot having lost the surface layer. The prism cores (P) have been destr oyed, whereas the interprismatic substance (SI) is less affected.


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Fig. 5 - Non-decalcified thin section of normal human adult enamel seen by transmission electron microscopy. Regular distribution of apatite crystals over prisms (P) and intcrprismatic substance with no apparent prism sheath regions. Fig. 6 - Initial mineral dissolution in human enamel at the interfaces between prism (P) and interprismatic substance (SI), with appearance of enlarged prism sheaths (white lines). TEM. Fig. 7 - In carious human enamel, minerai dissolution is more advanced in prisms (P) than in interprismaric substance (SI). TEM. Fig. 8 - Transverse section of carious human enamel showing bacterial invasion (M) in irregular areas where prisms and interprismatic substance are cornpletely destroyed. TEM.


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Fig. 9 - R ics. Irregular oot cartion of dcstrucwith cementum (C) out any a gradient of pparent destruction ~~neral stroycd t"' Issue ie deplaced b s rc-

Gram_po~li~~me:ous

organisms. D ~ICro dentin. TEM - root Fig. 10 :... caries B . Root vasio~ actcrial in(C) al of ccmentum ong enl longitudinal vo·~rged gradient of ~. s. No destruction' ineral mainin In the rcTEM. g cementum. Fig. 11 caries - Root . . Inl'rmate I tionship of G re aitiv . ram-pose micro-or . with a garusms m pparently hoogeneously . mmeralized TEM. cementum. Fig. 12 caries I Root rerial' i~portant bacsom aston with e remnants enscly calcific of d mentum (C).

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Fig. 13 - Transverse section of a sclerosed dentinal tuhule of transparent carious dentin. The dentin lumen (5) is calcified by a regular deposition of tiny crystals. PD = peritubular dentin. ID = intertubular dentin. TEM. Fig. 14 - Transverse section of several dentinal tubules of opaque carious dentin. Bacterial invasion of the tuhules with diffuse mineral destruction of intertubular dentin (10). TEM. Fig. 15 - Numerous Gram-positive micro-organisms in transverse-sect ioned dentinal tuhules with bacterial infiltration of demineralized intcrruhular dentin (ID). TEM. Fig. 16 - Presence of Gram-positive hacteria (8) in intertubular carious dentin. Mineral destruction with unmasked crossstriated collagen fihrils (arrows). TEM.


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the interface between prisms and interprismatic substance (Fig. 3), were due to limited dissolution of minerals. In TEM, the prismatic surface layers of normal permanent human enamel appeared to be composed of a dense grouping of apatite crystals oriented differently in prisms and interprismatic substance, without any apparent prism sheath regions (Fig. 5). In prismatic surface layers of incipient caries lesions, examined in TEM, initial mineral dissolutions occurred along the interfaces between prisms and interprismatic substance, with the appearance of enlarged prism sheaths (Fig. 6). In the body of the lesion, convergent observations could be made in SEM and TEM concerning the carious destruction of prismatic enamel. The earliest observed event at these levels consisted of an enlargement of the sheath regions or prism junctions very similar to the one observed in the surface layer. This was followed by a progressive and diffuse destruction of the apatite crystals located in the prism cores, whereas the interprismatic substance was relatively unaffected (Fig. 7). This observation was confirmed by the fact that the prism cores were destroyed before the interprismatic substance (Fig. 4), in agreement with the SEM study of Lustman et al. (1974). Bacterial invasion followed the destruction of the prism cores and the interprismatic substance (Fig. 8) and occurred (in depth) along the longitudinal axis of the prisms. It involved (in width) several groups of destroyed prisms with very irregular outlines (Fig. 8). Numerous Gram-positive micro-organisms filled the destroyed enamel areas. We were not able to confirm the observations of Brannstrorn et al, (1980) and Seppa et al. (1985), who found bacteria in sub-surface enamel under white-spot lesions without any apparent cavitation on their surfaces. The penetration of the caries process through the surface layer has been the subject of controversial opinions, in view of the prevailing concept of an almost intact surface layer, originating from the microradiographic study of natural lesions. The intact surface layer was explained by a specific biochemical composition consisting of a higher level of mineralization and trace elements - such as fluorides, zinc, lead, and chlorides, etc. - and a lower water and carbonate content (Brudevold et al., 1965; Hallsworth et al., 1973). Another prevailing concept, already suggested by Andresen (1926), considered the apparently intact surface layer as a result of remineralization of dissolved minerals (Silverstone, 1988; Aoha et al., 1981). In fact, it now seems that minute and limited structural pathways of mineral dissolution, such as enlarged prism sheaths or junctions, through the enamel surface layer could be the possible explanation for the demineralized sub-surface lesion, as already suggested by Thylstrup and Fejerskov (1981) and Haikcl et al. (1983). Using a special replica technique, Shellis and Hallsworth (1987) confirmed in the SEM that the principal event at the advancing front of early caries lesions is an opening of the prism junctions, reinforcing the concept that these interfaces are more susceptible to the initial exposure to acids than are the prisms. At later stages of caries evolution, these enlarged interfaces were able to undergo remineralization phenomena consisting of the presence of large rhombohedral crystals (Johnson, 1967), identified as whitlockite by Vahl and Plackova (1967) and Nonomura (1980). Similarly, Palarnara ct al. (1986) described an occlusion of prism boundary spaces and formation of needle-shaped crystals as remineralization phenomena. Extensive investigations were made on the carious dissolution of individual enamel apatite crystals. Holmen et al. (1985) suggested in a SEM study that the carious apatite crystals were destroyed through dissolution of the peripheries of individual crystals which were reduced in thickness and width, with resuiting enlargements of intercrystalline spaces. However, most

of the numerous published studies suggest that the initial crystal dissolution occurred as a central core lesion developing anisotropically along the c axis (Johnson, 1967; Swancar et al., 1971; Scott et al., 1974; Voegel and Frank, 1974, 1977; Arends and Jongeb1oed, 1977; Kerebel et al., 1978; Simrnelink and Nygaard, 1982; Lee and LeGeros, 1985). The development of the central core dissolution has been related to a strain field generated in the crystal by a screw dislocation of the Burger vector parallel 10 the [OOUl] direction (Arends, 1973) or to edge dislocations (Takuma et al., 1987). Bres et al. (1984) have identified three distinct types of disorders that may act as dissolution sites: (1) regions of lattice buckling with departure from hexagonal symmetry, (2) dislocations, and (3) grain and twin boundaries. Since the initial crystal dissolution occurred at the same place where a central dark line has been described (Ronnholrn, 1962; Nylen et a/., 1963; Frazier, 1968; Voegel, 1968; Marshall and Lawless, 1981; Nakahara, 1982; Nakahara and Kakei, 1984), it was related to the crystal dissolution phenomenon. Using theoretical image calculation procedures, Bres et al. (1986) found that this dark line was due to the presence of a twin houndary, whereas for Nelson et al. (1986) the central planar defect found in certain apatite crystallites represents a thin plate probably consisting of a single-unit cell thickness of octocalcium phosphate embedded in the apatite matrix. It is clear that further investigations are necessary to elucidate this major and fundamental problem of the exact nature of the initial dissolution sites of the apatite crystal. The cementum caries lesions. - In human root caries, observed generally in patients of advanced age, thick plaque accumulations, rich in typical corncob configurations, were located at the surface and depth of the caries lesion. In TEM, these micro-organisms were predominantly Gram-positive cocci, rodshaped, and filamentous bacteria with homogeneous cell walls (Figs. 9-12). The caries process involved the acellular fibrillar cementum considered by Jones (1981) and Schroeder (1986) as being formed almost exclusively hy Sharpey fibers originating from the periodontal ligament and penetrating the cementum perpendicularly. We found calcified collagen fibrils with orientations ohlique or parallel to the cementum surface corresponding to intrinsic matrix fibers, in agreement with Selvig (1965) and Furseth (1974). Bacterial invasion into cementum seemed to occur at a much earlier stage than in coronal enamel caries, as already noted by Nyvad and Fejerskov (1983). This bacterial invasion developed without any gradient of demineralization (Figs. 9-12). Our ultrastructural observations did not confirm the microradiographic studies of Kruger and Rakuttis (1952), Furseth and Johansen (1968), Westbrook et al. (1974), and Hals and Selvig (1977), who found a demineralized sub-surface zone under a relatively intact surface layer somewhat akin to the early enamel lesion. Initially, single rows of Gram-positive hacteria followed junctions between homogeneously calcified cementum layers, 5 or 6 urn wide, in which the collagen fibrils had a similar orientation, but this orientation often differed from one layer to the other. The junctions between these calcified layers were either almost indistinct and well-calcified (Fig. 9) or more often were poorly mineralized, with irregular outlines, and filled with some uncalcified cross-striated collagen fihrils. These junctions (preferably invaded hy bacteria) corresponded to boundaries between extrinsic Sharpey fiber bundles, as already noted by Jones and Boyde (1987), or islands of intrinsic fibers as well as incremental lines. In enlarged junctions, several rows of Gram-positive micro-organisms were seen along wellcalcified layers of cementum (Fig. 10). These bacteria were found in intimate contact with the densely calcified cementum


TOOTH STRUCTURE AND THE CARIES PROn'SS


in true microlacunae (Fig. 11). The abrupt destruction of collagen fibrils in close contact with invading Gram-positive bacteria was confirmed by the observation of similar sections decalcified for 24 h in 0.4 rnol/L EDTA solution. This simultaneous destructive process of the mineral and organic components of cementum continued progressively, and numerous Gram-positive bacteria could be shown between small but still well-calcified cementum remnants of very irregular shapes (Fig. 12). Caries lesions in coronal and radicular dentin. - Carious coronal and root dentin showed great ultrastructuralsimilarities and followed the involvement of enamel or cementum, respectively. In some cases, however, related to absence of the thin acellular cementum due to abrasion or iatrogenic factors, root caries started directly in root dentin. Two layers were usually found in dentin caries. They consisted of a deep transparent zone and a superficial opaque dentin. The deep transparent zone, adjacent to normal inner dentin, presented mainly more or less calcified lumens of sclerosed dentinal tubules (Fig. 13). Intratubular calcification occurred either by an initial mineralization of the peri-odontoblastic space followed by calcification of the odontoblast process, or by an initial intracytoplasmic calcification followed by a secondary peri-odontoblastic mineralization (Frank and Voegel, 1980). In addition to the presence of intratubular hydroxyapatite crystals, large rhombohedral crystals were often observed in sclerosed dentinal tubules, which were identified as whitlockite crystals (Frank et al., 1964; Vahl et al., 1964; Takuma et al., 1969; Daculsi et al., 1979, 1987; Frank and Voegel, 1980). Daculsi et al. (1987) suggested that these large Mg-substituted 13-tricalcium phosphate (TCP) crystals were due to initial dissolution of the dentin mineral followed by reprecipitation of Mg-substitutcd 13-TCP. The superficial opaque dentin is a fuchsin-stainable (Ogushi and Fusayama, 1975), demineralized layer rich in micro-organisms (Figs. 14-16) and can be divided into two zones. The deepest zone, adjacent to the transparent sclerotic dentin, was mainly characterizedby the presence of Gram-positivebacteria in previously sclerosed dentinal tubules, as could be judged by the frequent interbacterial presence of calcified remnants of the tubular plug. On transverse sections of invaded dentinal tubules (Fig. 14), it was obvious that the peritubular dentin was also destroyed. In addition, an important gradient of demineralization well in advance of bacterial invasion was noticed in the remaining intertubular dentin. The most superficial layer of the opaque dentin was a zone of advanced destruction which was progressively formed through different mechanisms. Dentinal tubules filled with bacteria (Fig. 14) enlarged progressively through peripheral resorption of their walls, and their confluence gave rise to large caverns rich in micro-organisms. Such zones of complete destruction could also be formed by progressive invasion of previously demineralized intertubular dentin by bacteria (Figs. 15 and 16). Micro-organisms often followed the lateral branches of the dentinal tubules before infiltrating the intertubular dentin. After dissolution of the hydroxyapatite crystals of intertubular dentin, typical cross-striated collagen fibrils were still observed in close contact with invading Gram-positive micro-organisms (Fig. 16).

Concluding remarks. The structural and ultrastructural events of enamel, cementum, and dentin caries are now well-documented. However, the consequences of fluoride use at the apatite monocrystal level need still further investigation. Visualizationof ionic sites and crystal defects at the unit cell level of normal and carious

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apatite crystals by lise of powerful high-resolution electron microscopes, coupled with image calculation procedures, will probably contribute to a better understanding not only of the precise dissolution mechanisms of the apatite monocrystal but also of fluoride action on tooth minerals. REFERENCES ANDRESEi', Y. (1926): The Physiological and Artificial Mineralization of the Enamel, Copenhagen: Hoffensbergske. AOBA, T.; MORIWAKI, Y.; DOl, Y.; OKAZAKI, M.; TAKAHASHI, J.; and YAGI, T. (1981): The Intact Surface Layer in Natural Enamel Caries and Acid Dissolved Hydroxyapatite Pellets, J Oral Pathol 10:32-39. APPLEBAUM, E. (1932): Incipient Dental Caries,J Dent Res 12:619-

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J Oral Pathol 15:28-35. RON:"HOLM, E. (1962): The Amelogenesis of Human Teeth as Revealed by Electron Microscopy. I\. The Development of the Enamel Crystallites, J Ultrastruct Res 6:249-303. SCHMIDT, W.J. and KEIL, A. (1971): Polarizing Microscopy of Dental Tissues, New York: Pergamon Press. SCHROEDER, H. (1986): The Periodontium. In: Handbook of Microscopic Anatomy, A. Oksche and L. Vollrath, Eds., Berlin: Springer, Vol. 5, p. 5.

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The inorganic components of cementum- and enamel-related dentin in the rat incisor.

Enamel organic matrix: potential structural role in enamel and relationship to residual basement membrane constituents at the dentin enamel junction.

Human age estimation from tooth cementum and dentin.

Adaptive properties of human cementum and cementum dentin junction with age.

Caries-resistant bonding layer in dentin.

Optical analysis of enamel and dentin caries in relation to mineral density using swept-source optical coherence tomography.

Identification of O-phosphoserine, O-phosphothreonine and gamma-carboxyglutamic acid in the non-collagenous proteins of bovine cementum; comparison with dentin, enamel and bone.

cementum thickness after hand or ultrasonic instrumentation.

Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

Patterns of morphological variation in enamel-dentin junction and outer enamel surface of human molars.

Role of dentin MMPs in caries progression and bond stability.

Discussion: Effects of fresh mineralized dentin and cementum on socket healing: a preliminary study in dogs.

In vitro determination and quantification of 30% hydrogen peroxide penetration through dentin and cementum during bleaching.

Nanoindentation hardness of intertubular dentin in sound, demineralized and natural caries-affected dentin.

Concomitant caries and calculus formation from in situ dentin caries model.

Effects of fresh mineralized dentin and cementum on socket healing: a preliminary study in dogs.

Retention of composite inlays in enamel dentin cavities.

Enamel hypoplasia in relation to caries in Guatemalan children.

Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals.

Abrasion of enamel and cementum in human teeth due to toothbrushing estimated by SEM.

The extent of the odontoblast process in human dentin.

8DSS-promoted remineralization of initial enamel caries in vitro.

dentin in mesial roots of mandibular first molars.

Lead in enamel and saliva, dental caries and the use of enamel biopsies for measuring past exposure to lead.