ORIGINAL ARTICLE

H. Kierdorf Æ U. Kierdorf Æ C. Witzel

Deposition of cellular cementum onto hypoplastic enamelof fluorotic teeth in wild boars (Sus scrofa L.)

Accepted: 21 October 2004 / Published online: 23 December 2004� Springer-Verlag 2004

Abstract The nature of deposits present in hypoplasticdefects of fluorotic enamel of wild boar teeth wasstudied by light microscopy and scanning electronmicroscopy. The fluorotic enamel showed differentdevelopmental abnormalities, denoting a severe dis-turbance of ameloblast function during the secretorystage of amelogenesis. These abnormalities includedthe occurrence of grossly accentuated incremental lineswith associated zones of aprismatic enamel and thepresence of different forms of hypoplastic defects. Twotypes of deposits were present on the hypoplastic en-amel: cellular cementum and posteruptively acquired,presumably partially mineralized dental plaque.Coronal cementum is not normally formed in pigteeth. Presence of this tissue in fluorotic teeth of wildboars is seen as indicative of a premature disintegra-tion of the enamel epithelium prior to the completionof amelogenesis. This was supposed to have resulted ina contact of mesenchymal cells of the dental folliclewith the surface of the immature enamel and, inconsequence, in a differentiation of these cells intocementoblasts. To our knowledge, this is the firststudy reporting the formation of coronal cementum aspart of the spectrum of pathological changes influorotic teeth in a species whose tooth crowns arenormally free of cementum.

Keywords Coronal cementum Æ Dental enamel ÆEnamel hypoplasia Æ Fluorosis Æ Wild boar (Sus scrofa)

Introduction

Dental cementum is a mineralized tissue generallypresent in the roots of mammalian teeth. Prior to theonset of cementogenesis, Hertwig’s epithelial root sheathdisintegrates, and mesenchymal cells of the dental folli-cle come into contact with the root surface. These cellsthen differentiate into cementoblasts and start to formroot cementum (Diekwisch 2001). In humans and manyother mammals with low-crowned teeth (brachydontcondition), the tooth crowns are generally free ofcementum, except for the frequent occurrence of smalltongues or spurs of cementum that extend over a shortdistance from the root cementum onto the cervicalcrown portion (Listgarten 1967; Freeman 1985; Sch-roeder 1991, 1992; Kierdorf and Kierdorf 1992). Incontrast, in mammals with high-crowned cheek teeth(hypsodont condition), including cattle, sheep, goats,horses, and rabbits, the crowns of the cheek teeth and, inseveral species, also those of the front teeth are coveredby a well-developed layer of cementum to a varyingdegree (Weinreb and Sharav 1964; Mills and Irving1967; Listgarten 1968; Listgarten and Kamin 1969;Jones and Boyde 1974; Kilic et al. 1997; Mitchell et al.2003). Several studies have demonstrated that a pre-requisite for the formation of this coronal cementum is adisintegration of the reduced enamel epithelium prior tothe completion of enamel maturation in the teeth (We-inreb and Sharav 1964; Mills and Irving 1967; Listgarten1968; Listgarten and Kamin 1969; Jones and Boyde1974), thereby enabling the contact of mesenchymal cellsof the dental follicle with the surface of the formingenamel.

Results of experimental studies support the view thatthe deposition of coronal cementum is triggered by theexposure of mesenchymal cells of the dental follicle tothe surface of the forming enamel. Thus, removal of theenamel epithelium from molar tooth germs of mice wasfollowed by the formation of cementum onto the ex-posed surface of the immature enamel (Heritier 1982).

H. Kierdorf (&) Æ C. WitzelDepartment of Biology, University of Hildesheim,Marienburger Platz 22, 31141 Hildesheim, GermanyE-mail: kierdorf@rz.uni-hildesheim.de

U. KierdorfInstitute of General and Systematic Zoology,Justus-Liebig-University of Giessen,Heinrich-Buff-Ring 26–32, 35392 Giessen, Germany

Anat Embryol (2005) 209: 281–286DOI 10.1007/s00429-004-0442-x

Initially an acellular cementum was formed; however,with increasing cementum thickness, cells became in-cluded in the tissue. Based on more recent experimentalevidence, it has been suggested that the differentiation ofmesenchymal cells of the dental follicle into cemento-blasts is induced by exposure of the cells to enamelmatrix proteins (Hammarstrom 1997; Spahr and Ham-marstrom 1999; Handa et al. 2002).

The teeth of pigs (both domestic pigs and wild boars)are normally free of coronal cementum (Zietschmannet al. 1943; Briedermann 1990). However, in a previousstudy of wild boars, we observed the presence of con-spicuous deposits that partly occluded hypoplastic de-fects in the enamel of fluorotic teeth (Kierdorf et al.2000). Thus far, the exact nature of these deposits hasnot been studied. The aim of the present study, there-fore, was to characterize the nature of the deposits, usinglight and scanning electron microscopy.

Materials and methods

Three mandibular first incisors (I1), one mandibularpremolar second (P2), and three mandibular molars (oneM2 and two M3) showing fluorotic changes of their en-amel, including severe enamel hypoplasia, were obtainedfrom five wild boars. The animals had lived in areas ofGermany and the Czech Republic exposed to atmo-spheric fluoride pollution and were killed during normalhunting operations. After extraction, the teeth were firstinspected macroscopically and photographed. Forscanning electron microscopic inspection of untreatedenamel surfaces, teeth were mounted on aluminum stubsand sputter-coated with gold. The specimens were thenviewed in a Hitachi S 520 scanning electron microscopeoperated at 10 kV. For inspection of cut surfaces, theteeth were sectioned longitudinally in either a labiolin-gual (incisors) or a buccolingual (premolar and molars)plane and the cut surfaces polished wet with siliconecarbide sandpaper (grades 600–4000). The polished cutsurfaces of the specimens (tooth-halves) were thenetched with 34% (v/v) phosphoric acid for 3–6 s, fol-lowed by a thorough rinse in distilled water, and furtherprocessed for scanning electron microscopy as describedabove.

For light microscopy, tooth halves were vacuum-embedded in epoxy resin (Epofix, Struers, Copenhagen,Denmark) and longitudinal, labiolingually-oriented(incisors), or buccolingually-oriented (molars) slices ofapproximately 200-lm thickness were cut from theblocks using an electric saw with a water-cooled dia-mond blade (WOKO 50, Conrad, Clausthal-Zellerfeld,Germany). Ground sections of approximately 40-lmthickness were prepared from these specimens, cover-slipped, and viewed in transmitted light in an Axioskop2 Plus microscope (Zeiss, Jena, Germany) equipped witha Canon Powershot G2 digital camera. The acquireddigital images were further processed using AdobePhotoshop 7.0 (Adobe, San Jose, USA).

Results

Macroscopically, the inspected teeth were characterizedby severe enamel hypoplasia affecting larger parts of thetooth crown or almost the entire crown (Fig. 1a). Thehypoplastic defects were often of a pit type; however,more extended defects were also encountered. Furthermacroscopic alterations regularly observed in the stud-ied teeth were enamel opacity, brown to black discol-oration of the enamel, and pathologically increased wear(Fig. 1a).

Scanning electron microscopy of untreated enamelsurfaces revealed that the hypoplastic defects were oftenfilled with deposits of a coarse and sometimes reticularstructure (Fig. 1b). Inspection of sectioned teeth, how-ever, showed that the deeper portions of the defectsfrequently contained a denser and more homogeneouslystructured material, which was overlaid by the morecoarsely structured deposits (Figs. 2a, 3a). The moredense and homogeneously structured material, whichhad been laid down directly onto the enamel surface,exhibited numerous lacunar spaces of varying shape anda width of about 8–12 lm (Fig. 2). The lacunar wallsshowed small orifices, which apparently were the en-trances of canalicular structures (Fig. 2b). In addition tothe lacunae, a few larger cavities with diameters of up to

Fig. 1a,b Severe enamel hypoplasia in fluorotic teeth of wild boars.a Buccal view of a mandibular third molar whose crown showspronounced pit-type enamel hypoplasia. Also note increased wearof the tooth. b Labial enamel surface of a mandibular first incisor.Hypoplastic defects are filled with deposits of a coarse and reticularstructure (asterisks). (Scanning electron micrograph; E enamelsurface)

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50–60 lm, probably representing vascular spaces, werealso observed in this material (Fig. 2a). Histologically,the structure of the more densely and homogeneouslystructured material corresponded to that of cellulardental cementum (Figs. 4, 5). Thus, numerous ce-mentocyte lacunae were present that were intercon-nected by a system of canaliculi (Fig. 4b). Based on itsstructural characteristics, the material in question wastherefore identified as cellular cementum.

Presence of this cellular coronal cementum was notrestricted to the deep and often narrow (pit type)

hypoplastic defects. Instead, this tissue was occasionallyalso observed in areas where the enamel showed moreextended hypoplastic defects (Figs. 4c, 5). In theselocations, the cementum was not or was only in placescovered by the coarser, reticular material that hadapparently been acquired posteruptively. Generally, thepresence of coronal cementum was limited to areas ofthe tooth crowns that were to some extent protectedagainst wear (Figs. 3–6). This finding could be taken asan indication that prior to eruption, larger parts of thetooth crowns had been covered by cementum that hadbeen lost posteruptively in areas exposed to more pro-nounced attrition.

The enamel underlying the bottom of the hypoplasticdefects showed various structural abnormalities. Typi-cally, a defect was underlain by a grossly accentuated,broad incremental line (Figs. 3, 4a, 5, 6). The presence ofthese lines was associated with a disruption of enamelmicromorphology, in that enamel with a normal prism/interprism structure was replaced by aprismatic enamelshowing a layered structure (Fig. 3). Numerous short,canal-like structures, which apparently did not reach upto the surface of the enamel and thus were hypoplastic

Fig. 2a,b Longitudinal section through a mandibular first incisor;same specimen as in Fig. 1b. a Large hypoplastic defect in theenamel (E) filled with cementum (C) that shows numerouscementocyte lacunae (arrowheads) and larger cavities presumablyrepresenting vascular spaces (white arrow). The cementum iscovered by posteruptively acquired deposits (asterisk). Notecanal-like structures (black arrows) in the enamel underlying thebottom of the hypoplastic defect. (Scanning electron micrograph;CS cementum surface, D dentin, ES enamel surface). b Highermagnification of a cementocyte lacuna with openings of canaliculi(arrowheads). (Scanning electron micrograph)

Fig. 3a,b Longitudinal section through buccal enamel of a man-dibular second premolar. a Narrow hypoplastic defect whosedeeper portion contains a plug of cellular cementum (C) that iscovered by posteruptively acquired deposits (asterisk). Noteaccentuated, cleft-like incremental line (arrows) underneath thebase of the hypoplastic defect. Arrowhead indicates canal-likestructure (internal hypoplastic defect) in the enamel (E). Scanningelectron micrograph. b Higher magnification of part of Fig. 3a.Note zone of aprismatic enamel (AE) internal to the accentuatedincremental line (arrows) and internal hypoplastic defects (arrow-head) originating at this line. (Scanning electron micrograph; Ccellular cementum)

Fig. 4a–c Longitudinal ground section through a mandibular thirdmolar, viewed in plain, transmitted light; same specimen as inFig. 1a. a Buccal enamel (E) showing numerous hypoplastic defectsmostly filled with cellular cementum (black arrows). Two severelyaccentuated incremental lines are indicated (white arrows). Thehypoplastic defects originate along these lines. b Higher magnifi-cation of a hypoplastic defect in the lingual enamel (E). The defectis filled with cementum (C) containing numerous cementocytelacunae (arrow). c Infundibular region of the tooth with severelyhypoplastic enamel (E). Most of the infundibulum is filled withcellular cementum (C). Arrow indicates structure probably repre-senting impacted food. (D dentin)

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defects located within the enamel, originated along theseverely enhanced incremental lines. (Figs. 3, 6).

Discussion

Our findings indicate that two types of deposits werepresent in the hypoplastic defects of fluorotic enamelfrom the wild boar teeth. The denser and more homo-geneously structured material, which was found deeperwithin the pit-type hypoplastic defects and which inplaces also covered more extended enamel areas, wascharacterized by the occurrence of numerous lacunarspaces, previously occupied by cementocytes, and asystem of canaliculi connecting these cementocyte lacu-nae. Because of its typical histological structure (Ismailand Weber 1988; Kagayama et al. 1997; Yamamotoet al. 2000), the material was identified as cellularcementum. The distribution of this coronal cementumon the tooth crowns suggests that prior to eruption,larger parts of the tooth crowns were covered by thistissue. The larger cavities within the coronal cementummost likely constitute vascular spaces, which are alsoregularly present in the coronal cementum of cattle andhorses (Listgarten 1968; Kilic et al. 1997; Mitchell et al.2003). In our view, the more superficially depositedcoarse and reticular material represents posteruptivelyacquired and presumably partially mineralized dentalplaque.

In species in which the cheek teeth are regularlycovered by coronal cementum, the formation of thistissue onto the enamel takes place following a disin-

tegration of the reduced enamel epithelium. This oc-curs before completion of enamel maturation anderuption of the tooth into the oral cavity (Weinreb andSharav 1964; Mills and Irving 1967; Listgarten 1968;Listgarten and Kamin 1969). Human teeth are gener-ally free of coronal cementum. However, under certainpathological conditions, coronal cementogenesis hasbeen observed also in human teeth. Thus, coronalcementum has been found in impacted teeth withhypoplastic enamel (Kotanyi 1924; Arwill 1974) and incases of amelogenesis imperfecta (Weinmann et al.1945; Listgarten 1967), as well as in enamel pits ofunknown causation and in enamel fissures of une-rupted and erupted teeth (Silness et al., 1976; Kodakaet al., 2002). Both the acellular and the cellular types

Fig. 6 Longitudinal ground section through a mandibular secondmolar showing enamel hypoplasia (phase-contrast microscopy). Apit-type hypoplastic defect in the buccal enamel (E) is partly filledwith cellular cementum (C). A grossly accentuated incremental line(arrows) is located internal to the base of this defect. Notenumerous internal hypoplastic defects (arrowheads) that alsooriginate along the accentuated incremental line. The dentin (D)shows pronounced interglobular spaces

Fig. 5 Longitudinal ground section through a mandibular firstincisor, viewed in plain, transmitted light. The enamel showsnumerous hypoplastic defects, located external to a grosslyaccentuated incremental line (indicated by arrowhead in the labialenamel). The lingual enamel surface is partly covered by cellularcementum (C). Arrow indicates partly detached cementum tongue,and asterisks indicate posteruptively acquired deposits. (D dentin,E enamel)

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of coronal cementum have been described in humanteeth; however, the identification of the latter type bySilness et al. (1976) was doubted by Kodaka et al.(2002). In each case, formation of coronal cementumoccurred as a sequela of a severe disturbance ofamelogenesis. Based on the available experimentalevidence (Heritier 1982; Hammarstrom 1997; Spahrand Hammarstrom 1999; Handa et al. 2002), it may beassumed that in all these cases, the formation ofcoronal cementum was induced by exposure of mes-enchymal cells of the dental follicle to the surface ofthe immature enamel following a premature disinte-gration of the enamel epithelium.

A severe disturbance of amelogenesis was also evi-dent in the fluorotic teeth of the wild boars, and wesuppose that the sequence of events outlined abovehad also occurred in these cases. The presence of dif-ferent types of hypoplastic defects denotes a markedimpairment of secretory ameloblast function. Thegrossly enhanced incremental lines in the enamelindicate the location of the surface of the formingenamel at the time of a severe impact on the secretoryameloblasts that caused a permanent or transient ces-sation of matrix secretion (Kierdorf et al. 2000, 2004).Externally visible hypoplastic defects formed whengroups of ameloblasts failed to resume their normalsecretory function. Presence of the canal-like struc-tures, which also originated along the grossly accen-tuated incremental lines but did not reach up to theenamel surface, is hypothetically ascribed to a tempo-rary arrest of enamel matrix secretion by groups ofameloblasts that, however, remain attached to theameloblast row and therefore move in centrifugaldirection along with neighboring secretory active cells(Kierdorf et al. 2000). As a consequence, hypoplasticdefects located deep within the enamel are formed.This type of developmental defect, which is not visibleat the enamel surface but can only be seen in sectionedteeth, is termed internal hypoplasia to distinguish itfrom the externally visible surface hypoplasia of fluo-rotic enamel.

In conclusion, the occurrence of coronal cementum influorotic teeth of wild boars indicates a partial or com-plete premature disintegration of the enamel epitheliumin these teeth. It cannot be determined whether thispremature breakdown is solely attributable to an excessfluoride exposure of the developing teeth or whetheradditional factors caused an exacerbation of the fluorideimpact. Possible such factors could be heavy parasiticinfestation and severe undernutrition (Tonge andMcCance 1965; Suckling et al, 1986; Goodman andRose 1990). In cattle, an intensification of coronal ce-mentogenesis in developing incisors has been reported asa result of increased fluoride exposure (Shearer et al.1978). However, to our knowledge, this is the first studyreporting coronal cementogenesis as part of the spec-trum of pathological changes of dental fluorosis in aspecies in which the teeth are normally free of coronalcementum.

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