J. Feridontal Res. 11: 331-338,, 1976.

Cellular, afibrillar coronal cementum in human teeth J. StLNESS, F. GusTAVSEN, O. EEJERSKOV, T . KARRING AND H . LOE

Department of Prosthodontics, University of Bergen, Norway, Department of Dental Pathology and Operative Dentistry, Department of Periodontology, Royal Dentai College, Aarbus, Denmark and School of Dental Medicine, Farmington, Conn., U.S.A. Mineralized tissue has been observed to be contained in the fissures of unerupted human third molars. The tissue had morphological features characteristic of afibrillar coronal cementum. In addition, lacunae with canaiiculi were contained in the tissue. It is suggested that the tissue is a cellular, afihrillar variety of coronal cementum produced by connective tissue cells which have transformed into cementoblasts after the disappearance of the reduced enamel epithelium from tbe fissures of the unerupted teeth. (Received for publication Jan. 25, 1976; accepted April 10, 1976}

Introduction

Dental cementum which covers the enamel surface to varying degrees is generally referred to as coronal cetnentum. The presence of coronal cementum has been known for a long time (Owen 1840-1845). Various amouttts of coronal cementum have been reported to occur io a large number of animals and in man. The literature on coronal cementum in different species has recently beett comprehensively reviewed by Listgarten (1968), Ainamo (1970), aod Gustavseti (1972). Like root cementum coronal cementum may either be of the cellular, fibrillar variety (Listgarten & Kamin 1969, Ainamo 1970, Furseth 1970, Gustavsen 1972) or of the acellular fibrillar diversity (Glimcher, Fiiberg & Levine 1964, Listgarten 1968, Listgarten & Kamin 1969). It has also been shown that an acellular, afibrillar t)/pe of coronal cementum may occur both in man and anitnals (Listgarteo 1966 a,b, Listgarfen 1968, Listgarten &

Kamin 1969, Schroeder & Listgarten 1971, Gustavsen 1972). The present paper reports on the presence of a lacunar mitieralized tisstje with morphological characteristics of coronal cementum contained within the fissures of unerupted human teeth. Materials and Methods

Completely unerupted third molars were collected at the surgical departments of the dental schools in Aarhus, Denmark, and in Bergen, Norway. For the present study 24 teeth were used. The material consisted of 13 matidibular tbird molars and 11 maxillary third molars. All teeth had fully formed or nearly fully formed roots. After removal the teeth were immediately fixed either in 10 per cent neutralized formalin or in 3 per cent glutaraldehyde buffered to pH 7.2 with 0.02 M sodium cacodylate. Ten teeth were dehydrated and embedded in Araldife under negative pressure in order

332

S i L L N E S S ,

G U S T A V S E N ,

F E J E R S K O V ,

to Stabilize possible fissure contents. The remaining 14 teeth were not embedded. Bucco-lingual sections, approximately 300 [im thick, were cut with "Gilling's Hamco Thin Sectioning Machine" under waterspray. Unembedded sections were ground and polished to thicknesses from 20 to 50 j,im as described by Silness (1967). Embedded sections, ground by hand, varied in thickness between 80 and 100 pim. In all, 85 ground sections were prepared and examined optically and microradiographicaliy. Microradiographs were obtained using Vfiltered Cr-radiation and Ni-filtered Cu-radiation from a Philips X-ray tube energized at 15 kV and 10 mA, and 35 kV and 20 mA, respectively. The target to plate distance was 25 cm. Contact between specimen and emulsion was maintained by means of a vacuum holder (Scott, Njden & Pagh 1962). The images were recorded on Kodak Spectroscopic Plates (type 649-0) which were developed in Kodak D-11 under constant agitation. After fixation, washing and drying, the emulsion was protected by means of Eukitt and a coverslip. Following the light microscopic and microradiographic examinations, ground sections were placed in aluminium foil containers on the top of a polymerized layer of Araldite and covered with Araldite syrup. After polymerization small resin blocks containing the fissore atid adjacent hard tissue were prepared. The blocks were re-embedded in gelatin capsules in such a way that thin sections could be cut perpendicular or parallel to the fissure walls. Ground sections were also decalcified according to the Sundstrom chromium sulphate technique (Sundstrom 1966, Sundstrom & Zelander 1968) as used by Silness and Gustavsen (1969). The decalcified sections were handled and embedded in capsules in the same way as the undecalcified sections. An LKB Ultrotome equipped either with a diamond or glass knife was used for sec-

K A R R I N G

A N D L O E

tioning. The sections were picked up on copper grids coated with formvar-carbon membranes. The grids were examined in a JEM 100-B electron microscope operated at 80 kV. Results

In the ground sections a mineralized tissue contained within the fissures appeared as plugs both in light microscopy (Fig. 1) and in the microradiographs (Fig. 2). These plugs were found in one or more ground sections of 16 of the 24 teeth. Microradiographically they were more radiolucent than the enamel (Fig. 2). The plugs appeared in several variations (e.g. Figs. 3, 4 & 5). They were usually located at the bottom of the fissure, but were also seen to occur at the fissure entrance (Fig. 4). In sections from two teeth the plugs seemed to rest upon dentin with apparently no separating enamel tissue between the plug and the dentin (Fig. 5). The plugs were not microradiographicaliy homogeneous. Radiolucent lacunae of varying size, which occurred frequently, were irregularljr distributed withio the tissue (Figs. 2 & 3). In some cases the occlusal layer (Fig. 3) or the apical portion

•

- •

-••

1

" >

;

" " • •

.

'

Fig. 1. Fissure plug. Ground section. Ligiit micrograph. ApproK. X 1:40.

CELLULAR,

A F I B R I L L A R

C O R O N A L

C E M E N T U M

333

Fig. 2. Microradjograph of the same area as shown m Fig. 1. Note Ihe iacunar appearaince of the piug. Approx. X 14Q.

Fig. 4. Microradiograph showing piug at the entrance of the fissure. Approx. x 140,

of the plug (Fig. 4) showed increased density. Lateral extensions from the plug occasionally continued in occlusa! direction along the fissure walls (Fig. 3). In such cases the occlusal, bucco-lingual contour of the plug was concave. The lacunae which were radioluceot in the microradiographs appeared in the electron microscope either completely electron lucent (Fig. 6) or contained varying atnounts of stainable matter in decalcified sections (Fig. 7). The contents appeared as

a finely granular, vacuolated material with a reduced density as compared to that of the surrounding tissue. From the lacunae canaiiculi of variable length extended into the surrounding matrix (Fig. 8). Electron microscopic observations on the mineralized plugs revealed needle-shaped crystals considerably smaller than the enamel crystals (Fig. 9). The small crystals were randomly oriented. The needles were approximately 6 to 7 nm thick. When viewed from the broad-side these structures were

Fig. 3. Microradiograph Note the laconar appearance of the fissure plug. Arrow indicaies lateral extension of the plug. Approx. X 140.

Fag. 5.. Microradiograph showiing plug at the bottom of the fissure with apparently no enamel between the pilug and the deotine., Approx. X 140.

334

S ILLNESS.

GUSTAVSEN,

FEJERSKOV,

K A R R I NIG

AND

L O E

iJFig, 6. Lacuna within the plug tissue. Mote apposjtional lines. Elsctron micrograph of decalcified tissue, Approx. X 21.000.

p!ate-like and showed reduced density (Fig. 9). They varied in length from 30 to 50 nm. Decalcified plugs displayed a fairly uniform, granular matrix (Fig. 10). No striated collagen fibrils or any other fibrillar component were observed within the organic matrix of the plug. The decalcified matrix showed appositional lines which were more or less parallel to the fissure walls and to the walls of the iacanae (Fig. 6). The lateral extensions of the

Fig. 7. Lacuna containing vacuoiated materiai. Eiectron micrograph of decaicified tissue. ApproK. x 60.000,

Fig. 8. Lacuna with canaliculi, EJeclron micrograph of decalcified tissue. Approx. x 21,000.

plugs appeared to contain crystals of tfie same shape and size as those present elsewhere in the plug. In the superficial layer the minerals occasionally occurred as aggregates of crystalline matter (Fig. 11). The extensions showed distinct appositiona! lines. The enamel crystals of the superficial layer of the fissure walls were usually not densely packed (Figs. 9 & 11). As a conse-

H I , '1 I ., - ll ' u, J tusus ''Jote fr? d.fforr.Tce L- rrys_:l size between tiie plug tis,sue {top) and the enamej (bottomj. Arrow indicates piate-like crystals. Approx. X 60,000.

CELLULAR.

AFJBRILLAR

Fjg. 10. EileclrO'n miciroradiograph of decalcified plug tissue. Note the lack of fibriSlar structures and the granular appearance of the matrix. Approx x 180.000.

C O R O N A L

C E M E N T U M

335

Fig. 12. Selected area diffraction pattern of plug tissue.

Discussion

quence the plug tissue did not lie against a sharply delineated enamel wall Instead, the small crystals of the plug tissue and the larger enamel crystals intermingled to form a transitional zone consisting of an intimate mixture of small and larger crystals. Electron diffraction of selected areas of undecalcified plug sections revealed an apatite pattern (Fig. 12).

Fig. 11. Electron miorograph of undecalcified, laterai extension of plug (top). Note the crystal stze, ttie appositional lines and the aggregation of minerai matter in the superficial layer. Approx. X 45.000.

The present study of completely unerupted third molars has shown that the fissures may contain mineralized structures. Based on the light microscopic and microradiographic appearance of these structures in individual ground sections they have here been termed plugs. These plugs resembled "the rod-like extensions directly underneath the grooves" described by Awazawa (1969). This author examined human first and second molars as weli as premolars which, as far as can be judged, were erupted teeth. In his material the rod-like extensions occurred in approximately 80 per cent of the molars and 73 per cent of the premolars. In the present study plugs were observed to occur in one or more sections in approximately 65 per cent of the teeth. Since piugs were occasionally present and occasionally missing in sections from the same tooth it might be that plugs or part of plugs were lost during the preparation of ground sections. Observations on sections of the Araldite embedded teeth showed, however, that in one or more sections of individual fissures the resin was in contact with the bottom of the fissures and there was no intervening plug material. Si-

336

SI L L N E S S ,

GUSTAVSEN,

FEJERSKOV,

multaaeously, in one or more sections of the same fissures plugs occurred between the bottom and the resin. The results of the present study, therefore, indicate that plug material may occur in spots along the fissures of unerupted third molars. The mineralized "rod-like extensions" described and diagrammatized by Awazawa (1969) apparently showed a similar patcby distributional pattern in first and second molars as well as premolars. Awazawa (1969) who studied replicas with the electron microscopy was of the opinion that the "rod-like extensions" underneath the grooves consisted of hypocalcified enamel. As judged by the qualitative microradiographic method used in the present work the plug tissue was less dense than the enamel. However, the mineral component of the plugs consisted of apatite crystals considerably smaller than the enamel crystals. The size and shape of these crystals make them resemble the crystals of human root cementum (Furseth 1967, Selvig 1969, Furseth & Johansei) 1970) and the crystals of coronal cementum of the kangaroo (Gustavsen 1972). Decalcified sections showed that the organic matrix of the plugs was a granular substance devoid of striated collagen fibrils and any other fibrillar component. The absence of fibrils is probably not related to the use of the chromium sulphate decalcification and staining procedures since it has been shown that banded collagen fibrils in dentin (Sundstrom, Takuma & Nagai 1970) and in coronal cementum (Gustavsen 1972) may be demonstrated with the chromium suiphate method. Therefore, the piug tissue resembled the afibrillar coronal cementum, as described by Listgarten (1966 a,b), Listgarten (1967), Listgarten (1968), Listgarten & Kamin (1969), Schroeder & Listgarten (1971) and Gustavsen (1972). These authors also showed that the afibrillar coronal cementum may exhibit well defined appositional

KARRING

AND LOE

lines which were observed to occur also in decalcified and undecalcified plug tissue. The presence of apatite crystals with shape and size similar to coronal and root cementum crystals, the lack of fibrils, the granular nature of the organic matrix, and the frequent occurrence of appositional lines make the plug tissue resemble the afibrillar coronal cementum described by the authors referred to above. Moreover, the additional finding of lacunae in the plugs makes it reasonable to look upon the plug tissue as being a cellular, afibrillar coronal cementum. This tissue is different from the cartilage-like coronal cementum in the grooves of guinea-pig molars (Listgarten & Shapiro 1974). Unlike the cementum described in the present paper the cartilage-like cementum contains collagen fibrils as well as vascular channels with blood vessels, and, in addition, it has an organic matrix consisting mainly of granular, spherical globules. Up to now afibrillar cementum has been shown to be of the acellular variety containing no recognizable cellular elements (Listgarten & Kamin 1969, Schroeder & Listgarten 1971). It is well established that in some animals the reduced enamei epithelium must disappear to allow the onset of coronal cementogenesis (Kronfeld 1927, Mills & Irving 1967, Listgarten 1968, Listgarten & Kamin 1969, Ainamo 1970). In man the reduced enamel epithelium usually remains intact until the teeth erupt into the orai cavity (Waerhaug 1952, McHugh & Zander 1965). It has been shown, however, that the reduced enamel epithelium may disappear on impacted human teeth (Kotanyi 1924) and on developing human teeth (Listfarten 1966 b). Kotanyi (1924) found that occlusal enamel surfaces which were unprotected by the reduced enamel epithelium had often been covered with cementum. Likewise, the disappearance of the reduced enamel epithelium and the subsequent contact of

C E L L U L A R ,

A F I B R I L L A R

C O R O N A L

C E M E N T U M

337

of a calcified layer of coronal cementum in erupted bovine teeth. /. Ultrastruct. Res. 10: 7fr-88. Gustavsen, F,, 1972. The fine structure of dental enamel and coronal cementum of the kangaroo (Macropus giganteus). Thesis. University of Bergen, Norway. Kotanyi, E. 1924. Histologlsche Befunde an retinierten Zahnen. Z. Stomatol. 22: 747-790. Kronfcid, R. 1927. Einige histologische Befunde an Schafzahnen. Schweiz Mschr. Zahnheilk. 37: 392-397. Listgarten, M, A. 1966 a. Electron microscopic study of the gingivo-dentai junction of man. Amer. J. Anat. 119: 147-178, Listgarten, M. A. 1966 b. Phase-contrast and electron microscopic study of the junction between reduced enamel epithelium and enamei in unerapted human teeth. Arch Oral Biol. 11: 999-1016. Listgarten, M. A. 1967. A mineralized cuticular structure with connective tissue characteristics on the crown of unerupted teeth in amelogenesis imperfecta. A light and electron microscopic study. Arch. Oral Biol. 12: 877890. Listgarten, M. A. 1968. A light and electron microscopic study of coronal cementogenesis. Arch. Oral Biol. 13: 93-114. Listgarten, M. A. & Kamin, A. 1969. The development of a cementum layer over the enamel surface of rabbit molars - a light and electron microscopic study.. Arch. Oral References Biol. 14: 961-985. Ainamo, J. 1970, Morphogenetic and func- Listgarten, M. A. & Shapiro,, I. M. 1974. Fine structure and composition of coronal cetional characteristics of coronal cementum in mentum in guinea-pjg molars. Arch. Oral bovine molars, Scand. J. Dent. Res. 78: 378Biol. 19: 679-696. 386. Awazawa, Y. 1969. Re-examination of the McHugh, W. D, & Zander, H,, A. 1965. Cell division in the periodontium of developing morphology of caries susceptible grooves in and erupted teeth. Dent. Pract. Dent. Rec. the premolar and molar. J. Nikon Univ. 15: 451-457. Sch. Dent. 11: 1-15. Furseth, R. 1967. A microradiographic and Mills, P. B. & Irving, J. T. 1967. Coronal cementogenesis in cattle. Arch. Oral Biol. 12: electron microscopic study of the cementum 929-932. of human deciduous teeth. Acta Odont. Scand. 25: 613-64.5. Schroeder, H. E. & Listgarten, M. A. 1971. Fine structure of the developing epithelial Furseth, R. 1970. A microradiographic, light attachment of human teeth. In: Monographs microscopic and electron microscopic studj' in developmental biology. Vol. 2 (edited by of the cementum from decidtious teeth of Wolsky, A.) Karger, Basel. pigs. Acta Odont. Scand. 28: 305-322. Scott, D. B., Nylen, M. U. & Pugh, M. 1962. Furseth, R. & lohansen, E. 1970. The mineral Some technical aspects of microradiography. phase of sound and carious human dental Norelco Reporter 9: 103-109. cementum studied by electron microscopy. Acta Odont. Scand. 28: 305-322. Selvig, K. A. 1969. Biological changes at the tooth-saliva interface in periodonta! disease. Glimcher, M. J., Friberg, V. A. & Levine, P. J. Dent. Res. 48: 846-855. T. 1964. Identification and characterization enamel with connective tissue facilitated the formation of afibrillar cementum in the cervical region of erupting humao teeth (Listgarten 1966 b). It is possible, therefore, that disappearance of the reduced enamel epithelium from the fissures of unerupted third molars frequently brings the connective tissue cells into contact with the enamel. Under these conditions the nnesenchyme may transform into cementum producing celis, cetnentoblasts, which after having been surrounded by the cementum matrix are transformed to cementocytes residing in the lacunae of the fissure plug tissue. Schroeder & Listgarten (1971) also felt that the afibrillar cementum originates as a product of connective tissue ceils, probably cementoblasts. If the interpretation of our observations is correct, the present findings may be taken to support the idea {Schroeder & Listgarten 1971) that in fibrillar cementum the matrix has been produced by the cementoblasts to anchor the collagen fibrils which themselves are produced by fibroblasts.

338

SILLNESS.

GUSTAVSEN,

FEJERSKOV,

Silness, J. 1967. Elements of the organic framework of dental enamel of the hedgehog (Erinaceus europaeus L.) Thesis. Universitetsforlaget, Oslo. SilBess, J. & Gustavsen, F. 1969. Structure of the organic matrix of dental enamel of the hedgehog (Erinaceus europaeus L.) Odontol. Revy 20: Suppl. 19. Sundstrom, B. 1966. A new technique for decalcifying thin ground specimens of adult human enamel. Arch. Oral Biol. 11: 12211231. Sundstrom, B. & Zelander, T. 1968. On the morphological organization of the organic matrix of adult human enamel after decaK cification by means of a basic chronium

KARRING

A N D L d E

(III) sulphate solution. Odontol. Revy 3: 249-263. Sundstrom, B., Takuma, S. & Nagai, N. 1970. Uitrastructural aspects of human dentine decalcified with chromium sulphate. Calc. Tissue Res. 4: 305-313. Waerhaug, J. 1952. The gingival pocket, anatomy, pathology, deepening and elimination. Odont. T. m: Suppl. 1. Address: Department of Prosthodontics University of Bergen Bergen Norway