Maiko Yamada et al.: Hypomineralized Enamel of the Dens Invaginatus

OriginalHypomineralized Enamel of Dens Invaginatus:

Its distinct images and pathogenesis of the Type III invagination usingmicro-focusing computed tomography

Maiko Yamada1), Motohiko Nagayama2), Akitoshi Katsumata3), Satoshi Kawano1), Keika Gen4),Michiko Ehara2), Juna Nakao2), Jun-ichi Tanuma2) and Takakazu Yoshida1)

1) Department of Endodontics, Asahi University School of Dentistry, Mizuho, Gifu, Japan2) Department of Oral Pathology, Asahi University School of Dentistry, Mizuho, Gifu, Japan3) Department of Oral Radiology, Asahi University School of Dentistry, Mizuho, Gifu, Japan4) Department of Dentistry for the Disability and Oral Health, Asahi University School of Dentistry, Mizuho, Gifu, Japan(Accepted for publication, September 11, 2014)

Abstract: Dens invaginatus (DI) is one of the rare developmental tooth anomaly, and generally resulting frominvagination of the inner enamel epithelium into the dental papilla before calcification. However, it remainsunclear whether the invaginated enamel mineralizaion is lower than outer enamel, the association with theresistance to invaginated enamel caries and the inflammatory lesions mediated their invagination. Because ofthe difficulties of treating and the tendency of their inflammatory lesions mediated their malformed structures,teeth extracted from the patients with DI, were collected in our hospital and analyzed by three-dimensionalmicro-focusing computed tomography (µCT) to clarify the differences of the invaginated enamel in mineraldensity (MD), and to find their pathogenesis of tooth anomaly and associated inflammatory lesions by histologicalanalysis. The teeth were fixed with 10% formaldehyde and analyzed by µCT, decalcified specimens wereperformed to histological (HE) and immunohistochemistry for cytokeratin AE1/AE3. All invaginated teethenamel showed extremely lower MD than did the outer enamel. Interestingly there were no carious lesions inthe invaginated enamel. HE showed thin reduced enamel epithelium on the surface of invaginated enamel andcytokeratin AE1/AE3 markedly showed positive their cellular matrix. The persistent reduced enamel epitheliumof the invaginated enamel indicates delayed or inhibited maturation after tooth eruption. This failure of maturationresults in lower MD but resistance to enamel caries. However, the structural abnormalities of DI teeth mayallow bacteria to enter the interior of the tooth through the invagination, causing pulpitis or apical periodontitis.(243 words)

Key words: Dens in dente, Dens invaginatus, Mineral density, Three-dimensional micro-focusing computedtomography, Cytokeratin immunohistochemistry

Correspondence to: Dr. Motohiko Nagayama, Department of Oral Pa-thology, Division of Oral Pathogenesis and Disease Control, Asahi Uni-versity School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu, 501-0296Japan; Tel/Fax: +81-58-329-1427; E-mail: moto@dent.asahi-u.ac.jp

IntroductionDental anomalies are associated with developmental disorders

before or after tooth eruption. Dens invaginatus (DI) is a rareanomaly resulting from infolding of the inner enamel epitheliumor enamel organ into the dental papilla during toothdevelopment1,2). The prevalence of DI ranges from 0.04 % to 10%3); however, reports vary by study design, sample size andcomposition, and diagnostic criteria2). Possible etiologies of DIhave been reported in the literature, including focal growthretardation4), growth pressure of the dental arch5), localizedexternal pressure in focal areas of the tooth bud1), infection, andtrauma6). The most commonly affected teeth are the maxillary

lateral incisors (43 % of all DI cases)7). Teeth with DI exhibitmorphologic variations in enamel infolding, and are classified byOehlers into three types based on invagination severity (Fig. 1).Type I is an enamel-lined minor form occurring within the confinesof the crown and not extending beyond the cementoenameljunction. Type II is an enamel-lined form that invades the root butremains confined as a blind sac that might or might notcommunicate with the dental pulp. Type III penetrates the rootand perforates the apical area, with a second foramen in the apicalor periodontal region8). DI may allow irritants such as oral bacteriato enter the invaginated space. Invagination may predispose teethto the development of dental caries and to irritant invasion,resulting in pulpitis or apical periodontitis. However, it remainsunclear whether the invaginated enamel is associated withinvaginated enamel caries. We have experienced the opportunity

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Journal of Hard Tissue Biology 23[4] (2014) 449-454 © 2014 The Hard Tissue Biology Network Association

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J.Hard Tissue Biology Vol. 23(4):449-454, 2014

to collect type III DI associated with lateral and apical periodontitisin the maxillary lateral incisor and canine tooth of a patient. Tocompare with other type of DI, we analyzed these teeth usingthree-dimensional micro-focusing computed tomography ( CT),and proceeded to histological and immunohistological studies toanalyze of their pathogenesis of type III DI and associated lesions.This is the first study of the fine structures and mineralization ofDI teeth using CT reconstructed three-dimensional imaging. Wealso discuss invaginated enamel mineralization and its associationwith apical periodontitis.

Fig. 1. Classification of dens invaginatus (DI) by Oehlers (1957)8),with modification.Type I: A minor form occurring within the tooth crown and notextending beyond the cementoenamel junction; Type II: A formextending beyond the cementoenamel junction; Type III: A formpenetrating the root, perforating at the apical or the lateral areathrough a second foramen. Scale bar: 5 mm

Materials and MethodsDI teeth were collected from patients suffered from their

symptoms such as painful or cosmetics reasons. Because of thedifficulty of their maintenance, these affected teeth weresurgically removed, and ex vivo examinations were performedafter obtaining informed consent. Two of type III DI teeth(maxillary right lateral incisor, 12 and canine, 13, indicated intwo-digit FDI system) were picked up and analyzed by μCTanalysis to compare with other types of DI (type I and type II)teeth previously extracted. All clinical data have been allowedthe ethics committee at Asahi University Hospital (#23116).

All the teeth were fixed in 10 % neutral buffered formalinbefore evaluation using a μCT apparatus (ScanX mate RX-series,Comscan Techno, Yokohama, Japan) with settings as follows:90 kV; 89 μA; magnification, 4.3-fold (23.15 μm/pixel);accumulation, 1; accumulation rate, 0.6 frame/sec; binning mode,1 (matrix 1220 × 1216); projection, 600. To avoid beam hardeningartifact findings, we used a tin foil before capture. To analize thefine structures and mineral density (MD) of the teeth, the regionof interest (ROI) of the invaginated enamel was designed as acolumn in three-dimensional configuration aligned longitudinallyin the invaginated area. For MD calibration, a series of mineralreference phantoms (Phantom, Ratoc System Engineering, Tokyo,Japan) was scanned prior to specimen scanning (TriBon 3D,Ratoc System Engineering, Tokyo, Japan). To determine thedegree of hypomineralization, volume of MD values (VMD) werethen analyzed with Student’s t test statistical analysis (p < 0.01).

After MD analysis type III DI teeth were demineralized in10% ethylene diamine tetra-acetic acid in Tris buffer, pH 7.3 fortwo weeks with shaking. Demineralized specimens weredehydrated by graded ethanol series, embedded in paraffin, and4 µm slices were made for hematoxylin-eosin staining and

Fig. 2. Macro photographs, μCT, and MD images of type III DI teeth.Extracted teeth associated with lateral (12) or apical (13) periodontitis(arrows) are shown in A and D. μCT reveals that the invaginated enamel(ie) is lined with a thin enamel layer (B and E) showing lower MD thanouter enamels in the MD images (C and F). Scale bars: 2 mm in A-F

Fig. 3. Volume of mineral density (VMD) values derived from MDimages.** represents the VMD value differences by Student’s t test statisticalanalysis (p < 0.01).Both (12 and 13) VMD values of invaginated enamel representextremely lower than outer crown enamel.

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Maiko Yamada et al.: Hypomineralized Enamel of the Dens Invaginatus

Fig. 4. Histopathological findings of type III DI teeth.Individual DI tooth shows a lateral inflammatory periodontal cyst lined with epithelium in the lateral incisor (12, A-D) and an apical periodontitiswith pulp necrosis in the canine (13, E-F). Boxed areas in A and E are shown at higher magnification in B, C and F, G. Both invaginated teethinteriors are filled with bacterial flora (ba) and calcification deposits (ca) (C and G). Boxed area is shown at higher magnification in D. Theinterior of the lateral incisor tooth is covered by a thin layer of material with underlying prismless enamel (arrow), and without carious lesions onthe invaginated enamel (ie) surface (D). In the canine tooth, the thin layers on the surface of the invaginated enamel (ie) shown immunopositivefor AE1/AE3, are indicated the characteristic of immature enamel or reduced enamel epithelium (arrowheads in H).Scale bars: 4 mm in A, E; 200 m in B, F; 100 m in C; 50 m in D, G, H

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immunohistochemistry.To clarify the mechanism of resistance to enamel caries, we

performed immunohistochemistry for cytokeratin AE1/AE3 (AE1/AE3; Dako Cytomation, Glostrup, Denmark) according to themanufacturer’s protocol.

ResultsMacroscopically, both the type III DI teeth exhibited cone-

shaped crowns with red soft tissue lesions on the side or apex ofthe root (Fig. 2A, D). The μCT images revealed that eachinvagination was entirely lined with hypomineralized enamel (Fig.

J.Hard Tissue Biology Vol. 23(4):449-454, 20142B, E). However in MD images these findings obviously revealedtheir lower mineralization corresponded with the μCT images (Fig.2C, F). Each VMD of the type III DI invaginated enamel was alsolower than that of the outer enamel in order of lateral incisor andcanine, 2782.7±38.7 and 2757.6±72.1 in outer enamel;2334.6±17.0 and 2063.0±179.7 in invaginated enamel;1303.0±32.9 and 13437±49.2 in dentin, respectively (Fig. 3). Thisfinding was found equally in both the type I and type II DI teethextracted previously in a similar manner (data not shown).

Histologically there was no evidence of bacterial infection orinflammatory cell infiltration in the dental pulp of the lateralincisor. The lateral root lesion was diagnosed as a lateralinflammatory periodontal cyst, lined with odontogenic epithelium.The dental pulp of the canine tooth was necrotic and the apicalforamen was wide. Apical periodontitis with pulp necrosis wasdiagnosed. These findings clearly showed that the lateral and apicalperiodontitis were caused by oral bacteria entering theperiodontium via the foramen with the severe invagination of typeIII DI. Interestingly, the invaginated surface was immature, withprismless (rodless) enamel and irregular enamel rods, and theinvaginated space was filled with bacteria and calcified deposits.However, there were no apparent carious enamel lesions in theinvagination. Immunohistochemistry showed immunopositivestaining for AE1/AE3 localized on the surface of the invaginatedinner enamel (Fig. 4).

DiscussionMore than 200 reports have been published on cases of DI,

presenting etiological, microscopic, ultrastructural, andmicroradiographic investigations with a wide range of findings7).However, descriptions of the structure and thickness of the enamellining of the invaginations are varied2). Most authors consider DIas a deep folding of the foramen coecum during tooth development,may result in a second apical foramen in some cases2).

Stud ies o f the microscopic , u l t ras t ructural , andmicroradiographic analysis of DI malformation report a wide rangeof findings. Dentin under the invaginated enamel may be normalwithout irregularities9,10). However, some authors have reportedhypomineralized or irregularly structured dentin9,11,12). In the DIenamel, large differences in the structure and composition of theouter and inner enamel have been found. The inner invaginatedenamel exhibits irregular, atypical, and more complex rod shapes,while its surface contains the typical honeycomb pattern, butwithout the perikymata normally observed on the toothsurface5,10,12,13). Beynon reported hypomineralization of the innerenamel at the base of the invagination, whereas Morfis et al.detected up to eight times more phosphate and calcium ininvaginated enamel compared with outer enamel, based onchemical analysis12,14). Recently Mishra et al. stated the hyperdenseCT number at the invaginated enamel surface in a case report of

type III DI. However the ground section of the invaginated enamelshowed hypomineralized15). The μCT analysis of the DI teeth inour study revealed extremely low MD in the invaginated enamel.Thus the inner enamel was hypomineralized compared with theouter enamel.

Enamel invagination sometimes allows irritants such as oralbacteria and food to enter the invaginated area, which is separatedfrom the dental pulp tissue by a thin layer of enamel and dentin,predisposing to the development of dental caries. Because theenamel lining is incomplete in some cases, channels may alsoexist between the dentin and the dental pulp. Therefore, pulpnecrosis or periapical inflammatory lesions, such as apicalgranulomas or radicular cysts, often occur within a few years oftooth eruption2).

Although there are different classifications to describe thedegree of enamel invagination, for purposes of clinical treatmentDI should generally be described either as a coronal invaginationwithout a second foramen in the periodontal area, or asinvagination with a second foramen penetrating and perforatingthe root.

In μCT analysis, these DI teeth showed no carious lesions ofthe invaginated enamel, but dentin caries were recognized. Thereis no report stated about the type III DI invaginated enamel cariesand correlation with the pathogenesis of periapical lesions. Ourfindings suggest that the invaginated enamel has thin layerspreventing bacterial demineralization even though it has a lowerMD than normal enamel. Because invaginated enamel is adevelopmental abnormality of the enamel organ, we expected tofind the reduced enamel epithelium, which may explain resistanceto enamel caries in invagination. Mishra et al. emphasized theirreport about the unusual hyperdense enamel of the invagination,however there was no histologic correlation for it15). We first reportthis interested phenomenon to verify in detail. Our performanceof immunohistochemistry for AE1/AE3 revealed that theinvaginated enamel surface was covered with a thin layer ofreduced enamel epithelium. In the normal outer enamel, thereduced enamel epithelium will be eroded by occlusion after tootheruption. However, once the crown has invaginated during DItooth development, there is no longer maturational activity in theinvaginated enamel.

The above findings indicate that the invagination of DI leadsto delayed or inhibited enamel maturation, resulting in lower MDbecause of the presence of reduced enamel epithelium andprismless enamel. These features of the invaginated epitheliumindicate that the anomaly develops in the post-differentiation stageof amelogenesis, rather than during the time of normal enamelmaturation at tooth eruption. The delayed enamel maturation mayresult in resistance to bacterial demineralization on the invaginatedenamel surfaces. However, DI leads to excessive accumulation ofbacteria and the formation of dental calculus, which may induce

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Maiko Yamada et al.: Hypomineralized Enamel of the Dens Invaginatus

dentin caries and result in more severe pulpitis or apicalperiodontitis via the invagination.

AcknowledgementsThe authors thank Mr. Nobuhito Nango, President of Ratoc

System Engineering, for assistance with the analysis ofmicrofocusing CT data, and Dr. Yukiko Nakano for the drawingillustration of Fig. 1.

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