Optical Microscopic, Microradiographic and Scanning Electron Microscopic Observations of Some Dens Invaginatus

Original J. Showa Univ. Dent. Soc. 15: 7-16, 1995

Tetsuo KODAKAand Shohei HIGASHI

SecondDepartment of Oral Anatomy,Showa University School of Dentistry 1-5-8 Hatanodai,Shinagawa-ku, Tokyo, 142 Japan (Chief:Prof. ShoheiHigashi)

Abstract: The simple or branched invaginations of dens invaginatus were histologically observed by using 6 ground sections (2 molars and 4 incisors). In all the teeth, the invaginations have hypoplastic enamel showing a variable thickness and an irregular outline. The relatively thick enamel irregularly arranged the Retzius lines and prismless structures were present in the innermost layers besides the surface layers. In 4 teeth, enamel-free areas were partially found on the dentin surfaces, and afibrillar cementum occasionally covered the enamel-free areas as well as the enamel surfaces. The dentin of a molar tooth had giant tubules between the dichotomously branched invaginations and other giant tubules opened into the invagination floor. Some dentinal tubules in the terminal regions had abnormal structures similar to the Tomes' granules adjacent to the invaginations of the 2 molar teeth. In all the incisor teeth, a seam line of dentin fusion or a slit line succeeding to the dental pulp cavity was present in the dentin under the linguogingival ridge. Thus, the gross formation of dens invaginatus also causes the invagination to form locally abnormal structures, especially in the enamel regions; although some findings have been previously reported. Key words: dens in dente, invagination, prismless enamel, afibrillar cementum, enamel-free area

It is strongly suggested that most of the dens lar cementum25) including cementicle-like globular in dente, recently named dens invaginatus, are structures26) on the floors, although the dentin formed by the abnormal infolding of an enamel under the fissures generally shows a normal organ towards the dental papilla during a tooth structure. In human teeth, prismless structures germ stage'-8). The gross morphology of dens often occupy the thin cervical ename123,27), and invaginatus has been reported with intra-oral afibrillar cementum showing a periodic lamina- radiographs1-3,5-18), radiographs of thick-section frequently attaches to the cervical region26,28). tioned') or intact extracted teeth3,5-8,12,15,16,19), The cervical enamel is formed by ameloblasts decalcified sections3,4,6,7,11,13,20), ground sec- differentiated in the last stage of amelogenesis21). tions1-3,5,7,8,11,18,19), and contact microradiographs In this preliminary report of the invaginations of ground sections?). On the other hand, there of some dens invaginatus, the fine structures were are only a few histological observations of the observed by microradiography and several micro- invaginations',5-7). scopies. As is well known, the deep fissures of human molar teeth, somewhat similar to the invagina- Materials and Methods tions, are the native structures formed by ameloblasts differentiated in the later to the last stage Six human teeth possessing a simple or of amelogenesis21). The fissure enamel has usu- branched invaginations, namely dens invaginatus, ally prismless structures in the surface layer22-24) were collected from the ground sections of 440 and the deep fissures occasionally deposit afibril- teeth with a transmitted-light microscope (Nikon, 8 昭和歯学会雑誌第15巻第1号

20 kV after coating with an about 15-nm-thick platinum-palladium layer in an ion sputtering apparatus (IB-S, Eiko, Japan).

Results

Figure 1 shows the ground section of a small invagination of a tooth, probably the third molar. The invaginated enamel was very thin and partially free on the dentine surface. Several cementicle-like globular structures (AFC) were seen on the enamel surface. No caries areas were seen in the invagination, In the dentin surrounding the floor of the invagination, some dentinal tubules in the terminal regions had abnormal structures similar to the Tomes' granules in the root surface dentin. Figure 2a shows the ground section of a molar tooth possessing dichotomously branched invaginations. Both the branches showed an irregular 1 outline and enamel-free areas were partially observed on the dentin surfaces (Figs. 2b, c). No Fig. 1 A small invagination of a molar tooth. caries areas were seen in the invaginations. The Transmitted-light micrograph. V: invagina- relatively thick enamel occasionally showed prism- tion. E: enamel. D: dentin. EFA: less structures in the innermost layers as well as enamel-free area. AFC: cementicle-like in the surface layers. In Fig. 2b, these prismless globular structure (afibrillar cementum in layers gradually proceeded to the thinner enamel Fig. 2f). AT: abnormal structures of itself. In some parts of the periperal regions of dentinal tubules. the invaginations, unidentified material and cementicle-like globular structures were seen (AFC in Japan), which had been made out in the routine Figs. 2b, c). The dentin between the branched dissection course at the School of Dentistry, invaginations had giant tubules, and other giant Showa University, Tokyo. These 6 teeth (2 tubules opened into the enamel-free floor of molars and 4 incisors) were ground again with the left invagination (Figs. 2d, e). Some dentinal grindstones up to about 100 pm in thickness, The tubules in the terminal regions had abnormal ground sections were observed with several structures similar to the Tomes' granules under optical microscopes including transmitted light the left invagination (Fig. 2d). (Nikon), polarized light (Olympus, Japan), and Figures 2e and 2f show the SEM images of the differential interference contrast (Nikon). After left invagination shown in Fig. 2a after EDTA the contact microradiographs of the ground etching. The SEM image (Fig. 2e) revealed the sections were prepared with a soft X-ray irradia- complex area of the enamel and dentin as com- tion apparatus (Softex, Japan), the ground planes pared with the transmitted-light micrograph (Fig. were polished with 5- and 0.3-pm alumina on 2c). The unidentified material and cementicle- polishing cloths and then cleaned ultrasonically like globular structures as shown in Figs. 1, 2b, in distilled water. This was followed by etching and 2c were clearly distinguishable from the with 2 % EDTA at pH 7.2 for 15 to 30 min, rinsing enamel by SEM (Fig. 2f). The material showing in running water, dehydrating with ethanol, and a periodic lamination on the ground plane etched drying with a critical point of CO, in a critical with EDTA, evaluated as afibrillar cementum, point dryer (HCP; Hitachi, Japan). The EDTA- covered the enamel-free dentin as well as the etched planes were observed with a Hitachi S-430 enamel surface (Fig. 2f). scanning electron microscope (SEM) operated at Figure 3a shows the microradiograph of a main 92acbd 平成7年 3月

Fig. 2 a-d Dichotomously branched invaginations of a molar tooth. Transmitted-light micrographs. LV and RV: left and right invaginations. E: enamel. D: dentin. EFA: enamel-free area. PLE: prismless enamel. AFC: unidentified material or cementicle-like globular structure (afibrillar cementum in Fig. 2f). AT: abnormal structures of dentinal tubules. GT: giant tubule of the dentin. 10 昭和歯学会雑誌第15巻第1号

Fig. 2 e, f The SEM images after EDTA etching in part of Fig. 2a. The region of Fig . 2f is shown with the arrow f in Fig. 2e. LV: left invagination. E: enamel. D: dentin. GT: giant tubule . AFC: afibrillar cementum.

invagination with a small branch in an incisor had a seam line under the linguogingival ridge. tooth. These surfaces were attached with dental The innermost enamel besides the surface enamel calculus showing a poreous structure compared occasionally showed prismless structures (Fig. 4b). with the enamel, whereas no caries areas were A similar simple invagination was observed in seen. The dentin under the linguogingival ridge another incisor tooth, although the enamel was had a low mineralized line. Figures 3b—d show partially free on the dentin surface. the SEM images of the invaginations after EDTA Figures 5a and 5b, respectively, show the ground etching. The enamel was partially interrupted on section and the microradiograph of a typical dens the dentin surface of the main invagination when invaginatus in an incisor tooth. In the invagi- dental calculus was removed (Figs. 3b, c). In nation, the dentin-enamel junction, especially the the left side of the main invagination (Fig. 3c), lingual region, showed an irregular line and the afibrillar cementum covered the enamel-free dentin caries-free enamel showed variable thickness. A as well as the enamel surface. Prismless struc- linear small gap was seen in the enamel. In the tures were observed in the innermost enamel. dentin under the linguogingival ridge, a slit line Figure 3d shows the floor of the small branched succeeding to the dental pulp cavity was clearly invagination attached with dental calculus. The seen (Fig. 5b). Figure 5c shows the polarized- abnormal Retzius line was embedded with the both- light micrograph of part of the left-side invaginated side prisms arranged almost at right angles to the enamel. The orientation of the Retzius lines prism directions from each other. The prismless was distinguishable between the enamel regions layer with a fine lamination was seen in the surface divided with the linear gap. Figure 5d shows enamel. the differential-interference-contrast micrograph Figure 4a shows the SEM image of a simple of part of the right-side invaginated enamel. The invagination in an incisor tooth. The caries-free innermost and surface prismless layers were invagination was entirely covered with the enamel observed. Such prismless layers are also seen in although the thickness was irregular. The dentin the SEM image (Fig. 5e). The prismless enamel

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Fig. 3 a-d An invagination with a small branch of an incisor tooth. a: microradiograph. b-d: SEM images after EDTA etching. The regions of Figs. 3b-d are shown with the arrows b to d in Fig. 3a, respectively. V: invagination. E: enamel. D: dentin. DC: dental calculus. SL: seam line of the dentin under the linguogingival ridge. EFA: enamel-free area. AFC: afibrillar cementum. PLE: prismless enamel. RL: Retzius line or the first formed surface of the enamel. The left hands indicate the lingual side.

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4a b

Fig.4 a, b A simple invagination of an incisor tooth. SEM images after EDTA etching. The region of Fig. 4b is shown with the arrow b in Fig. 4a. V: invagination. E: enamel. D: dentin. SL: seam line of the dentin under the linguogingival ridge. PLE: prismless enamel. The left hands indicate the lingual side. surface attached with afibrillar cementum showed orientation of the prisms surrounding the Retzius a fine irregular outline. Figure 6 shows a sche- line was found (Fig. 3d). Such a Retzius line is matic illustration of the dens invaginatus based probably formed as follows : the first formed on a microradiograph, and several optical and enamel surface is covered with the enamel which SEM micrographs shown in Fig. 5. is later formed with ameloblasts arose at a quite different region, and finally the first formed Discussion enamel surface becomes the Retzius line. Prismless structures of human teeth are com- Though numerous observations on dens invagi- monly distributed in the surface ename123,24,27). natus or dens in dente have been reported by However, the innermost layer of the coronal radiography"--3,5-19), there are only a few obser- enamel shows an initial prism structure29), al- vations on fine structure1,6,7). In gross morphol- though several investigators21,30) suggested that ogy, the invaginated enamel shows an irregular prismless enamel was present there. On the other outline1,5,7) and a variable thickness1-3,5-8). His- hand, prismless structures often occupy the thin tologically, the enamel has the irregular Retzius cervical enamel24'27). In the invaginated enamel, lines') and the invaginations have defective no prismless structures have been reported, but enamel') with enamel-free areas on the dentin we found prismless areas in the innermost enamel surfaces1,5,7). We also obtained the same results as well as in the surface enamel. as the previous reports1,3,5-8) moreover, the At the floor of the deep invaginations, un- abnormal Retzius line showing a quite different identified material') and material showing an Fig.5 a-e A typical invagination of an incisor tooth. a: transmitted-light micrograph. b: microradiograph. c: polarized-light micrograph. d: differential-interference-contrast micrograph. e: SEM image after EDTA etching. The regions of Figs. 4c-e are shown with the arrows c to e in Fig. 5b, respectively. V: invagination. E: enamel. D: dentin. SL: seam and slit lines succeeding to the dental pulp cavity (P) under the linguogingival ridge. LG: linear gap of the enamel. RL: Retzius line. PLE: prismless enamel. The left hands indicate the lingual side. 平成7年3月 13

b 5a

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afibrillar cementum25,26). From above-mentioned results and discussion, the invaginated region has more or less enamel hypoplasia6,8,10,13,18,20,31,32).Though the invagination is not a native structure, the invaginated enamel is somewhat similar to the deep fissure enamel and the cervical enamel, since prismless enamel and afibrillar cementum are frequently present in all of them; in addition, they were formed by ameloblasts differentiated in the later or the last stage of amelogenesis2"). The dentin surrounding the invagination has often meandered dentinal tubules' and the seam line of dentin fusion5.14,19)or the slit line succeeding to the dental pulp1,4-6,11,14). In this study, the abnormal orientation of dentinal tubules was not observed, but the seam or the slit line was always seen in the dentin under the linguogingival ridge of each of all the incisors. It is known that holes33) or irregular structures") running along dentinal tubules are occasionally observed in the central streak33) of the incisal or cuspal dentin. Therefore, the seam and slit lines are probably homologous to the central streak33) and the holes33) or irregular structures"), respectively. Pindoborg6) reported vascular inclusions in the dentin under the invagination. We found giant tubules similar to the holes or irregular struc- Fig. 6 A schematic illustration of the dens in- tures33,34) and the vascular inclusions') in the vaginatus shown in Fig. 5. The curved dentin between the branched invaginations of a lines in the enamel show the Retzius lines. The thick arrows indicate the innermost molar tooth and at the invagination floor (Figs. and surface prismless enamel. V : invagina- 2d, 2e). tion. E: enamel. D: dentin. P: dental We also found abnormal structures in the pulp cavity. The left hand indicates lingual terminal regions of dentinal tubules similar to the side. Tomes' granules in the dentin under the invaginations (Figs. 1, 2d). In addition, the Tomes' irregular outline with an intermediate mineral- granules are usually present in the surface layer ization between the enamel and the dentin') have of the root dentin34). These structures suggest been reported. We also found similar material that the dentin adjacent to the invagination shows and cementicle-like globular structures that were some abnormality, although the abnormality may identified as afibrillar cementum25,26,28) in the SEM be less than that of the invaginated hypoplastic image (Fig. 2f). The afibrillar cementum was enamel. present on the enamel surface and on the enamel- It has been reported that the invaginations of free dentin surface in the deep invagination. dens invaginatus are caries-susceptible regions7,17). Previously, cementum1,5) and bone6) were reported However, Onodera7) and Gotoh et al.17) reported on the invaginations in ground or decalcified that the frequency of the caries in the invagina- sections at low magnifications. Some of these tions was less than previously reported. We tissues may be afibrillar cementum. Afibrillar observed caries-free areas in all the invaginations cementum frequently attaches to the cervical used in this study. Onodera7) reported that the enamel and cementum surfaces28), while the deep entrance areas of the invaginations might show fissure enamel occasionally is attached with enamel caries rather than the interiors, and sug- 平成7年 3月 15

gested that the appearance was similar to the Japanese) fissure enamel in clinical observations. Certainly, 8) Ishikawa G, Akiyoshi M: Oral Pathology, Vol. I. Nakagawa35) revealed that caries-unsusceptible Kyoto, 1976, Nagasue Book, pp 17-20, 58-108 fissures were not wide and shallow V-shaped but (in Japanese) 9) Amos ER: Incidence of the small dens in dente rather narrow and deep slit and kolben-shaped . J Am Dent Assoc, 51: 31-33, 1955 by comparison with caries-free premolar teeth 10) Boyle PE: Kronfeld's Histopathology of the extracted from young orthodontic and old peri- Teeth, 4th Edn. Philadelphia, 1955, Lea & Febiger odontic patients. Therefore, the invaginations , pp 17-20, 43-57 themselves may apt to be unsusceptible to caries, 11) Shafer WG : Dens in dente. New York State Dent when the entrance areas of the invaginations are J, 19: 220-225, 1963 relatively narrow. 12) Ulmansky M, Hermer J: Double dens in dente According to many previous studies2,3,"'12,15,19, in single tooth; report of a case and radiographic 36), most of the incisor teeth used in this study study of the incidence of small dens in dente . Oral are probably the maxillary incisors, especially the Surg, 17: 92-97, 1964 lateral incisors showing the high frequency of 13) Spouge JD: Oral Pathology. St Louis, 1973, CV Mosby, pp 132-134, 148-156 dense invaginatus. In the future prospects, we 14) Thomas JG: A study of dens in dente. Oral Surg, will focus the three-dimensional and the fine 38: 653-655, 1974 structures of the invaginations of the maxillary 15) Conklin WW: Double bilateral dens invaginatus incisor teeth. in the maxillary incisor region. Oral Surg, 39: In conclusion, the invaginated enamel showed 949-952, 1975 a more abnormal structure than the dentin ad- 16) Conklin WW: Bilateral dens invaginatus in the jacent to the invagination. Histologically, the mandibular incisor region. Oral Surg, 45: 905- invaginations possessing prismless enamel and 908, 1978 afibrillar cementum may be somewhat similar to 17) Gotoh T, Kawahara K, Imai K, Kishi K , Fujiki the deep fissures and the cervical regions . Y: Clinical and radiographic study of dens invaginatus. Oral Sung, 48: 88-91, 1979 18) Bhaskar SN: Synopsis of Oral Pathology, 6th Ed. Acknowledgments St Louis, 1981, CV Mosby, pp 109-112, 116-118 19) Miyoshi S, Fujiwara J, Nakata T, Yamamoto K, We thank the numerous dentists, who had made the Deguchi K: Dens invaginatus in Japanese incisors. ground sections of human teeth when they were dental Jpn J Oral Biol, 13: 539-543, 1971 (in Japanese) students, School of Dentistry, Showa University. 20) Rushton MA, Cooke BED, Duckworth R: Oral Histopathology, 2nd Ed. Edinburgh, 1970, E & S References Livingstone, pp 1-8, 24-27 21) Fejerskov 0, Thylstrup A: Dental enamel. In 1) Gustafson G, Sundoberg 5: Dens in dente. Brit MjOr IA, Fejerskov 0 (eds): Human Oral Embry- Dent J, 88: 83-88, 111-122, 144-146, 1950 ology and Histology, Copenhagen, 1986, Munks- 2) Hallett GEM: The incidence, nature and clinical gaard, pp 50-89 significance of palatal invaginations in the maxil- 22) Haikel Y, Frank RM: Microscopie electronique lary incisor teeth. Proc Roy Soc Med, 46: 491- a balayage de la surface d'amail aprismatique 499, 1953 normal et carie de dents temporaires humaines. 3) Oehlers FAC: Dens invaginatus (dilated com- J Biol Buccale, 10: 111-124, 1982 posite odontomes). Oral Surg, 10: 1204-1218, 23) Kuroiwa M: Acid resistance of surface `prismless' 1302-1316, 1957 enamel in human deciduous and permanent teeth. 4) Rushton MA: Invaginated teeth (dens in dente): Showa Univ J Med Sci, 2: 31-44, 1990 contents of the invagination. Oral Surg, 11: 1378- 24) Kodaka T, Kuroiwa M, Higashi S: Structural and 1387, 1958 distribution patterns of surface `prismless' enamel 5) Oehlers FAC: The radicular variety of dens in human permanent teeth. Caries Res, 25: 7-20 , invaginatus. Oral Surg, 11: 1251-1260, 1958 1991 6) Pindoborg JJ: Pathology of the Dental Hard 25) Silnes J, Gustafson F, Fejerskov 0, Karring T, Tissues. Copenhagen, 1970, Munksgaard, pp 58- LOe H: Cellular, afibrillar coronal cementum in 64, 88-95 human teeth. J Periodontal Res, 11: 331-338, 7) Onodera A: Histopathological study of the dens in 1976 dente. Jpn J Oral Biol, 13: 428-464, 1971 (in 26) Kodaka T, Nakagawa T: Cementum-like struc- 16 昭和歯学会雑誌第15巻第1号

tures observed in the enamel fissures and the 31) Hals E: Hereditary enamel hypoplasia, investiga- bifurcations with enamel drops. Jpn J Oral Biol, tions of two families. Odontolgisk Tidskrift, 66: 25: 17, 1983 (in Japanese) 562-582, 1958 27) Kodaka T, Nakajima F, Kuroiwa M: Distribu- 32) Miller J, Forrester RM: Neonatal enamel hypo- tion patterns of the surface `prismless' enamel in plasia. Brit Dent J, 106: 93-104, 1959 human deciduous incisors. Bull Tokyo Dent Coll, 33) Tronstad L: Optical and microradiographic ap- 30: 9-19, 1989 pearance of intact and worn human corona] 28) Schroeder HE: The periodontium. In Oksche A, dentine. Arch Oral Biol, 25: 481-502, 1983 Vollrath L (eds): Handbook of Microscopic Anat- 34) MjEir IA: Dentin and pulp. In MjOr IA, Fejerskov omy, Vol V/5. Berlin, 1986, Springer-Verlag, pp O (eds): Human Oral Embryology and Histology, 33-46 Copenhagen, 1986, Munksgaard, pp 90-130 29) Kodaka T, Kuroiwa M, Abe M: Fine structure 35) Nakagawa T: Morphological study of unsus- of the inner enamel in human permanent teeth. eptible occlusal pits and fissures against dental Scanning Microsc, 4: 975-985, 1990 caries in old premolars. Jpn J Oral Biol, 25: 481- 30) Osborn JW: Variations in structure and develop- 502, 1983 (in Japanese) ment of enamel. In Melcher AH, Zarb GA (eds): 36) Fujita T: Anatomy of Teeth. Tokyo, 1949, Ka- Oral Science Reviews, Vol 6. Copenhagen, 1973, nehara Pub, pp 33-36 (in Japanese) Munksgaard, pp 3-83 (Received November 22, 1994)