/. Periodontal Res. 11: 313-330, 1976.
Ultrastructure of the dento-epithelial junction K E N I C H I KOEAYASHI, GEORGE G . ROSE AND CHARLES J. M A H A N
Departments of Periodontics and Internal Medicine, The University of Texas Health Science Center, Dental Branch, Houston, Texas. U.S.A. The detito-epithelial janctions (DEJ) of Rhesus monkeys were examined by electron microscopy. Various fixation procedures were employed in this study. It was revealed that the basal lamina between the junctionai epithelium and the tooth surfaces was composed of three layers: the lamina lucida, the lamina densa and the sub-lamina lucida. Fine filaments often coursed through the lamina densa,, lamina lucida and hemidesmosomes and combined them into a stroctural unit. An electron-dense linear border, presumably derived from tissue fluid, was generally observed on the tooth surface at the DEJ except that cotnposed of the fibrillar cementum. This border was helpful in distinguishing the afibrillar cementtim from the dental cuticle. The existence of a sub-lamina lucida hetween Hit lamina densa and the tooth surfaces is a newly defined layer. It is hypothetically CODsidered to be an area of competetive electrostatic forces of repulsion and London Van der Waals forces of attraction. (Received for publication Dec. 12, 1975; accepted Feb. 26, 1976)
In addition, a dental cuticle which possesses a high electron density and a relatively During the past decade, the ultrastructure homogeneous property is frequently interof the normal human dento-epithelial juiic- posed between the basal lamina and the tioti was described by several investigators tooth surfaces. As no reports to the con(Listgarten 1966, Ito, Enotnoto & Kobaya- trary have appeared, it is assumed that the shi 1967, Kobayashi 1968, Sehroeder 1969, junctional epitheliutn adheres in a similar Schroeder & Listgarten 1971). Additional manner to the various dental surfaces with studies dealt with the ultrastructure of the which it comes in contact, viz., the enamel, gingival reattachment after surgical inter- the coronal cementum (afibrillar cevention in both human and other mamma- mentum), the root cementum (fibrillar celian subjects (Listgarten 1967, 1972, Hirose mentum), and the dental cuticle. 1970,, Thilander & Hugoson 1970, Frank et In the present study, we fotmd new de,al. 1972, Taylor & Campbell 1972). lineations of the dento-epithelial junction in Based oti these reports, it is generally Rhesus monkeys. conceded that the adhesion of the juncThis new data coupled with the reports fional epitheliutn to tooth surfaces is me- on cell-to-cell and cell-to-solid substrate addiated by multiple hemidesmosomes and an hesions reviewed in the Discussion have exunderlying structure very similar to the tended our perspectives on the organization basal lamina overlying the connective tissue. of the attachment mechanism. Introduction
314
KOBAYASHI, ROSE AND MAHAN Materials and Methods
The investigations were carried out on five young adult Rhesus monkeys. Under Nembutal® (Abbott, Chicago, Illinois) anesthesia, tissues were surgically obtained and fixed in accordance with the schedule in Table I. Fixation By Immersion After making gingival incisions, teeth with intact gingiva, including the margin of alveolar bone, were excised en bloc with mallet and chisel. The specimens were immersed immediately in the fixatives described in Table I, groups 1-4 and 7—9. Fixation By Perfusion The external carotid artery was cannulated with a polyethylene catheter, and the internal juglar vein was exposed and cannulated for drainage. The blood was briefly washed out of the vessels by means of a rinse with
normal saline, and then the glutaraldehyde fixative solution was perfused for about 45 minutes until the tissues of the mandibular region showed a marked yellowish coloring and stiffening. The specimens were excised as for the immersion procedure and placed in the same perfusing fixative for an additional 4-5 hoors (see Table I, groups 5, 6). Decalcification The excess dental tissues vcere trimmed from all specimens with a dental air turbine and then decalcified in 0.1 M disodium EDTA solution containing 0.1 M sucrose and adjusted to pH 7.2 (Warshawsky & Moore 1967). When the decalcification vi^as completed (av. 5 days, 20°C; av. 25 days, 4°C), the specimens were cut into smaller blocks and some were post-fixed with osmium solution according to the schedule in Table I. Finally, all specimens were embedded in Epon (Luft 1961).
Table I Schedule of fixation procedures Group
Pre-fixation* 2"/o Giut.. 0.1M S-coll, or 0.1M Phos. (2hr)
Decalciftcalion D,1M disodium EDTA with 0,1M Sucrose "
Post-fixation* 2 7o Os,, 0,1M S-coli,
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Abbreviations Used: Glut, - glutaraldehyde; S-coll, = s-coJIidine buffer; Phos, = phosphate buffer; EDTA ; ethyJenediaminetetraacetio acid; Os, = osmium; Para, = paraformaldehyde; Aero, — acrolein, * pH of fixatives: 7,4; temperature during fixation; 0-4°C, Fixation with ,2'/o Os,, 0,1M S-ooll, phr) was interposed between pre-fixation and decaloificatio;i, *** -f4-5, hr after excising tissues. ""** Modified Karnovsky's (1965) fixative
THE
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Sections studied in this report were cut on a Porter-Blum MT-2 ultramicrotome, mounted on naked copper grids, routinely stained with aqueous uranyi acetate and lead citrate (Reynolds 1963) and examined in an RCA EMU 4B electron microscope operated at 75 KV.
Otisenrations
The general appearance of the dento-epithelial junction in the Rhesus monkeys conformed to that described in studies of human and other mammalian specimens (see Introduction). With low-power electron microscopy the junctional epithelium contacted the enamel, the afibrillar cementum covering the enamel surface (Fig. 1), and the root cementum (Fig. 2). Multiple hemidesmosomes were easily recognized along the cell membrane facing the tooth surfaces. The existence of the dental cuticle varied from specimen-to-specimen and area-to-area, and early stages appeared over the enamel or root cementum (Eig. 2) as the major density between the junctional epithelium and the tooth. Attachments Coronal to the CemenloEnamel Junction (Figs. 3—14, 16-19) Moderate-power electron micrographs obtained from various fixations revealed the many relationships between the junctional epithelium and the enamel (Eigs. 3-12). Well-developed dental cuticles were seen between the afibrillar cementum (overlying the enamel) and the junctional epithelium (Figs. 3 and 4). In other areas, well-developed portions of the dental cuticle were transformed into irregular portions of diminished mass (Figs. 5 and 6). The ultrastructure of the dental cuticle at moderate magnifications appeared to be equivalent in specimens prepared in different fixatives
J U N C T I O N
315
(Figs. 3.-6). Less well-defined dental cuticles were atso observed as continuous units (Fig. 7). Some areas had no evident dental cuticle (Figs. 8-12). Where the junctional epithelium apposed the afibrillar cementum without an intervening dental cuticle, there was, between the basal lamina and the afibrillar cementum, a thin dense line, the linear border (Figs. 6, 8, 10-12). This linear border was helpful in distinguishing the afibritIar cementum from the dental cuticle. For instance, where the outer lamination of the afibrillar cementum was dense and structurally similar to a dental cuticle, it was separated from the basal lamina by the linear border (Fig. 8). Such a narrow sharp line was never seen on the epithelial surface of the dental cuticle; if it were observed at all, it appeared between the dental cuticle and the enamel or afibrillar cementum (Fig. 6). In cases in which the junctional epithelium apposed the enamel surface directly, i.e., without an intervening dental cuticle or afibrillar cementum, a sharp linear border was visible at the location corresponding to the enamel surface (Fig. 9). Between the lamina densa and the linear border, there was a narrow clear zone we have designated the sub-lamina lucida. This was seen in varying degrees of clarity (Figures 4-6, 8, 10-12). High-power electron microscopy denoted more clearly the various structures of the epithelial attachment. The basal lamina was finely divided into an upper lamina lucida, a mid lamina densa, and a lower sublamina lucida (Fig. 13). In some cases the sub-lamina lucida was partially obscured by the dental cuticle (Fig. 14). Similarly, the linear border was usually obscured by the dental cuticle (Figs. 13 and 14). Although there was variation from specimen-to-specimen and fixation-to-fixation, this basic 3part design of the basal lamina remained
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constant (see line drawings. Fig. 15, a-d). The higher-power electron micrographs were used to determine the measurements of these layers. The lamina lucida** (140 ± 30 A wide) occupied the narrow space between the peripheral density and the lamina densa; the lamina densa*** (400' ± 100 A wide) appeared suspended between the clear lamina lucida and the clear sublamina lucida. The sub-lamina lucida (95 ± 20 A wide) lay between the lamina densa and the linear border (120 ± 20' A wide. Figs. 16, 18) or the dental cuticle (Figs. 13, 17, and 19). The dental cuticle was finely granular and of a high electron density. The border between the dental cuticle and the sub-
** Other authors define the lamina lucida as the space betweeo the basai or junctional cell membrane and the lamina densa. *** Other authors perforce include thiC sublamina ldcida in their measurements of the lamina deusa.
AC BL cf DC ES ff HD il IS JE LB LD
— afibrjllar cementtjm — basal lamina — collagen fibriis — deiHital cuticle = enamel space =: fine rilaments — hemidesmosame = inner leaflet = intercelluilar space = jonciional epithelium — linear border = lamina dense
J U N C T I O N
317
jacent afibrillar cementum was highly irregular and unclear (Fig. 17). In such areas the linear border was not defined as was the case in most areas in which the dental cuticle was well-developed. However, the linear border was sharply evident between the enamel and the sub-lamina lucida (upper part of Fig. 16), between the enamel and dental cuticle '(lower part of Fig. 16), and between the afibrillar cementum and the sub-lamina lucida (Fig. 18). The dental cuticle overlying the enamel was extremely variable. In some areas it was absent and within a few micra was evident again, tisually reappearing in a zone of transition (Fig. 16). Rarely, between the dental cuticle and the linear border there was a narrow clear zone (Fig. 16). The cell membrane of the junctional epithelium was not visible in those specimens not postfixed in osmium (Figs. 16 and 17), although the attachment plaques and the peripheral densities of the hemidesmosomes were clearly visible. In specimens postfixed
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Ftg. 1. Jynctional epitheiium (JE) adijacent to the afibrtlJar cementum (AC) coveripg the enamel surface. Fixatjoo: acrolein-osmium (Group 9). x 12,1:&Q. Rg. 2,. Junctional epithelium (JE) adjacent to the root cementum (RC). Fixation: acrolein-osmium (Group 9). X 16,500. Fig. 3. Well-developed dental cuticle (DC) between the afibrilfar cementum (AC) overlying the enamel and the junctional eptthefium (JE). Fixation; paraformaldehyde-glutaraldehyde miixture-osmium (Group 7). x 24,750. Fig. 4. Dental cuticle (DC] interposed between afibrillar cementum (AC) and iunctional epithelium (JE). Fixatioioi; glutaraldehyde-osmium-osmium (Group 3). x 24,750, Fig. 5. Denta! cuticle (DC) over enamel, iin the lower half of this figure, the dental cuticle is well-developed' in the upper half, the dental cuticle is thininer ain.d irregular {aggregation or disaggregatJon process, Fixation: paraformaldehyde-gtutaraidehyde mixture (Group 8). x 35,550.
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Fig, 15. Schema of complete untt of adhesion between junctjonai epithelium [JEJ and tooth developed Irom electron micrographs: a) enamel space (HS) covered with dental cuticie (DC); b} enamel space {ES) covered with Sinear border (LB): c) enamel space (ES) covered with afibrfllar cementum (AC) and dental cuticje {DC}; d) enamei space (ES) covered with afibriliar cementum (AC) and iineer border (LB); e) root cementum (RC) interspersed with patches of afibriilar cementum (AC) and covered with dental cuticle (DC); f) root cementum (RCJ interspersed with patches of afibrillar cemenium [AC) covered with the tinear border (LB).
Fig. 6. Irregular dentai cuticie (DC) .in Lfpper 2/5ths of this figure, diminishing m width at its ioweir extremity. The afibrillar cementum (AC) covers the entire enamei surface in this figure. The boundary between the afjbriiar cemenium and the basai lamina (BL) is a clear sharp dense line, the linear border (slanting arrows), Fixat'ion: glutraldehyde perfusion (Group 6). x 35,550. Fig.. 7. Dentai cuticle (DC) 5s less well-defiaed than in- preceding Itgures. Linear border is obscured by the dental cutjcle, Fixation:: acrolesn-osmium (Group 9). x 35,550.. Fig., B. Junction^al epIthiBliam (JE) apposing the atibriiMar cementum (AC) without an intervening dental cuticle. The outer lamination of the afibrilJar cementum is dense and almost indistingui-shabJe from a dental cuticle. The thin linear border (slanting arrows), separates the apposing surfaces of the basal iamina (BL) and the afibriilar cementum. Fixation: paraformaldBhyde-gtutaraldehyde mJxture-osmium (Group 7}. x 35,550. Fig.. 9.. Junctional epithelium (JE.) apposing enamei without an interveniing dental cuticie or afibriliar cemen•tum. A distinct linear border is on the enamel surface (slanting arrows). The three parts of the basal iamiina: sub-lamina lucida (SLL), iamina densa (LD), and lamina lucida (LL) are indicated. Fixation: paraformaldehydeglutaraldehyde miixiure (Group 8), x 35,550. Fig. 10. Basal lamina (BL) sharpiy separated from afsbrillar cementum (ACJ by the linear border (slanting arrows), No dental cuticle is visible: glutaraidehyde perfusion-osmium (Group 5), x 24,750.
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by an osmium solution, the outer leaflet of the unit membrane of the juBCtional epithelial cell was clearly observed (Figs. 18 and 19). Attachments Apical to the CementoEnamel Junctions (Figs. 20-28) At moderate magnifications of electron microscopy, the basal lamina was seen to maintain a relatively uniform width (Figs. 20-22). The lamina densa also could be identified at these magnifications. In some cases, the surfaces of tbe root cementum were covered with a dental cuticle of great thickness variation (Figs. 20-22). In the root cementum, especially near the surface area, there were portions lacking coiiagen fibrils, as well as portions packed with typicaliy periodic collagen fibrils (Fig. 21, see also line drawing in Fig, 15, e,f). The former areas were morphologically similar to the afibrillar cementum overlying the enamel. Higher-power electron micrographs (Figs. 23-27) showed the variations in the attachments between the root cementum and tbe junctional epithelium. The dental cuticle was not always present over the root cementum (Figs. 24, 25, and 27). The
J U N C T I O N
321
desmosomes were generally larger than those apposing the basal lamina of the oral epithelium, and they were sharply outlined along the surfaces of the juactional epithelium. In all specimens the attachment of the lamina densa to the root cementum or the dental cuticle was mediated by a thin clear space, the sub-lamina lucida (Figs. 23 and 28). The dental cuticle adapted to the uneven surface of the root cementum and was generally even on the surface facing the basal lamina. Accordingly, the lamina densa maintained a relatively uniform thickness and straight appearance (Fig. 23). However, the space between the lamina densa and the dental cuticle had occasional dilatations where the dental cuticle was not homogeneous. The linear border was especially evident in areas devoid of the dental cuticle, and where the root cementom lacked collagen fibrils, i.e., afibrillar cementum (Fig. 25). It was never observed between fibrillar cementum and the basal lamina or dental cuticle. Where the dental cuticle came in contact with afibrillar cementum, this line could not be detected (Figs. 23 and 26). Layers covering the root cementum and covered by the linear border, by our defini-
Fig. 11. Linear iborder (slanting arrows) between afibriltar cementum (AC) and basai lamina (BL). No dentai culicie is present. Fixation: glutaraldehyde periusion (Group 6). x 35,550. Fig:. 12. Linear border (sianting arrows) between afibrijiar cementum (AC) and basal iamina (BL). iNo dentai cuticle is present. Fixation: paraformaidehyde-osmium (Group 4). x 24,750. Fig. 13. Basai iamina (BL) is fmeiy divided into upper Samina iucida (LL), miid lamina dense fLD), and lower sublamina iucida (SLL). Linear border obscured by dental cuticle (DC). Fixation: glutaraidehyde-osmium-osmium (Group 3). x 58,500. Fig. M. Dentai cuticle (DC) is weli-deveioped and obscures linear border and parts ot sub-lamina lucJda. Fixation: paraformaWehyde-giutaraldehyde iTiixture-osmium (Group 7), x 58,500. Fig. 16. Epithelial attachment to the enamel. Three parts of basal iamina: sub-laminia jucida (SLL), iamina densa (LD), and iamina lucida (LL) are easily discriminated. As osmium was omitted in fixation, the cell membrane is not visibie as a component of Xhe hemidesmosomes (HD). In the upper part of micrograph there is no dental cuticie or a1ibri!1ar cementtjm. In lower half, there is an irreguiar dental cuticle which may represent an ongoi^ng process of cuticular aggregation OT disaggregation. Linear border (sianting arrows) lies on the surface of enamel throughout micrograph. Fixation: paratormaldiehyde-glutaraidehyde mixture (Group 8). x 135,000.
K O B A Y A S H I , ROSE AND M A H A N
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tion Vifere not the dental cuticle but the afihrillar cementum (Fig. 27). Special Considerations of the Denial Cuticle and Hemidesmosomes Under high-power electron microscopy the dental cuticle may be described as a relatively homogeneous, somewhat granular and electron-dense layer. In some sections, less dense spots appeared within the dental cuticle, but the granularity of the Epon embedding media prevented clear details of these areas at high powers of magnification (Fig. 29). These spots, as well as a dense mesh-like pattern surrounding the spots, were seen all over the dental cuticle (Fig. 29, a, b). Throughout these observations, the hemidesmosomes were well-preserved regardless of the fixation procedure used. In highpower views of selected areas, details of the hemidesmosomes contained dense pyramidal particles along the inner surface of the peripheral density (Fig. 30, a-d). Fine filaments extended from the peripheral density into the lamina densa (Figs. 30, a, e-g). In specimens fixed by perfusioo but not post-
J U N C T I O N
323
fixed with osmium, the particles on the peripheral densities were less distinct although larger and extended to the outer leaflet (Fig. 30' f, g).
Discussion
Our investigation has once again confirmed the previously established concept that an attachment apparatus consisting of multiple hemidesmosomes and a basal lamina promotes the adhesion of the junctional epithelium to the tooth surfaces. Additionally, It has been revealed in this study that this attachment apparatus behaves as a unit which is maintained a short distance from the tooth surface or dental cuticle by an intervening sub-lamina lucida. Since this junction is situated against a relatively impenetrable surface, a great deal of care was required in effecting the tissue preservation of the specimens. In order to avoid misunderstandings which might be attributed to a single technical procedure, various fixation methods were employed. 'Our results showed a remarkable consistency of the
Fig:. 17. Epitheiial-enamel junction. Three parts of the basai iamina (BL) over dental cuticie (DC) are ciearly recognized, viz., the sub-lamina lucida (SLL), the iamina densa (LD), and the lamina iuoida (LL). Linear border is not evident. Fixation: paraformaidehyde-glutaraldehyde mixture (Group S), x 135,000, Fig. 1B. Afibriilar cementum (AC) intervening between junctional epitheiium (JE) and enamel; there is no dental cuticle. Distinct linear border (slanting arrows) is on outer surface of afibriiiar cementum. Three components of basal lamina are delineated. Fixation: aorolein-osmium (Group 9). x 135,000, Fig. 19. Outer and inner ieafiets (oi, ii) ot cell membrane and the peripheral' density (pd) of the hemidesmosome (HD) are defined. Three iayers of tiie basai iamina (BL) are evident as an eleotron-light-dark-iight triad overlying the electron-dense dentai cuticie (DC). Fixation: paraformaldehyde-giiitarafdehyde mixture-osmium (Group 7), X 135,000. Fig. 20. Broad, dense, and homogeneous dental cuticle (DC) interposed between root cementum, (RC) and junctional epitheiium (JE). Fixation: giytaraldehyde-asmiium-osmium (Group 3), x 24,750. Fig. 21. Surface of the root cementum (RC) covered by thin eiectron-dense dental cuticle (DC). Basai lamina (BL) maintains a uniform width and its division Into the three sublayers is easily detected. The areas (AC) in the root cementum iacking coiiagen fibriis (cf) are' morphologtcaily similar to the afibriiiar cementum overlying the enamei. Fixation: acroiein-osmlum (Group 9). x 35,550, Fig. 22. Dentai cuticle' (DC) interposed between root cementum (RC) and! junctional epitheiium (JE), Area lacking coiiagen fibriis in root cementum appears to have been cieared by decaicificatlon procedure (vertical arrows). Fixation: glutaraldehyde (Group 2), x 24,750,
324
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spatial relationships between the attachment apparatus and tooth surfaces or dental cuticle. Where the attachment apparatus directly contacted the enamel surface, i.e., without an intervening layer of dental cuticle and/ or afibrillar cemeotum, as shown in Figure 9, the enamel surface was marginated with a sharp dense line, the linear border. Similar dense lines were always found on the surfaces of the afibrillar cementum covering the enamel,, as well as that found in the root cementum. However, it was often obscured by the presence of an overlying dental cuticle. There was no evidence that the dental cuticle and the linear border were of common origin, but there were two types of arrangements that suggested their separate origin: 1) the linear border had a relatively constant width and appeared to be overlaid by the encroachment of a developing dental cuticle of varying widths (Figs. 6, and 16); and 2) below the cemento-enamel junction, the linear border appeared only on the surface of the afibrillar cementum (Figs. 24 and 27) and never on the fibrillar cementum, whereas the dental cuticle appeared uniformly on both (Figs,, 21—23 and 26; see also schema in
JUNCTION
325
Fig. 15). Although Schroeder and Listgarten (1971) reported ultrastructural differences between the dental cuticle and the afibrillar cementum, we found the linear border was helpful in distinguishing these two layers,, especially when the cuticle was poorly developed and its existence questioned. Dense lines similar to the linear border have been observed on both plastic and mica culture surfaces upon which human dermal and oral cells were cultivated (Flaxman et al. 1968, Ijuhin et al. 1976). Additionally, between this dense line and the apposing cell, there was a layer of extracellular material of moderate electron density about 450 A wide. Flaxman et al. (1968) postulated that the thinner dense layer represented a condensate of serum protein from the culture medium at the cell substratum interface, and the moderate dense layer might be synthesized by the overlying cells, as the so-called "microexudate" described by others (Rosenberg 1960, Maslow & Weiss 1972, Yaoi & Kaoaseki 1972; Poste et al. 1973, Revel & Wolken 1973, Culp 1974). From the positional and dimensional standpoints, the narrow dense line (linear
Fig. 23. BasaS tamtna (BL) between junctional epithelium (JE) and root cementum (RC) characteristically s,eparated into its three compo,nentE,: lamina lucida (LL), !ami,na deosa (LD), and sub-iamina iucida (SLL), Dental cuticle (DC) adapts to uneven surface ol the root cementum (RC) but is reiativeiy even on surface facing basal lamina. The occasional apparent dilatation of the sub-lannina lucida may represent an aggregation or disaggregation process in the dental cuticle. Fixation: paraformaldehyde-giutaraldehyde mixture (Group 8), x 58,500. Fig. 24. Three sub-iayers of the basal tamina (BL) overlying root cementum (RC). Upper pcrtion (AC) of the root ce,mentum (RC) lacks collagen fibrils and is structurally similar to the afibrillar cementum covering the enamel. Fixation: acroieln-osmium (Group 9). x 76.500, Fig.-25. Surface of roct cementum (RC) is not clear except in the lower portion, where the dense linear bord:er (slanting arrows} indicates its surface. Cementum located beneath this Wne lacks collagen fibrils and is, therefore, afibriilar cementum (AC), There is no dental cuticle. Fixation: gtutaral,dehyde-osmium (Group 1), x 58,500. Fig. 26. Dental cuticle (DC) over root cementum (RC), Three sub-layers of the basal lamina (BL) are easily denoted. Fixation;: glutaraldehyde perfusion (Group 6), x 76,500. Fig. 27. Basal lamina (BL) ciearly divided into three sublayers. Layer covering root ce,mentum {RC) is reticular, and since it is covered by the linear border [slanting arrows), it is by definition not a dental cuticle bu: the alibrillar cemehtum (AC). Fixaticn: aorolein-osmium (Group 9|. X 58,500.
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border) over the enamel or the afibrillar cementum was similar to tiie tiiin layer found apposing the hard surfaces of culture vessels. If Flaxman's et al. (1968) postulate is proved correct, then the in vivo and in vitro dense lines may have the same nature and origin, that is, be adsorption layers derived from the tissue or culture fluids and consist mainly of serum proteins. On the root cementura surface, the linear border was not detected except over those portions lacking collagen fibrils, i.e., the afibrillar cementum. This discrepancy may have depended largely on the divergent surface structures of these two types ol exposed cementum, i.e., their adsorptive qualities. The major question arising from this study regards the origin and function of the thin sub-lamina lucida lying between the basal lamina (lamina densa) and the tooth surface. It is conceivable that the sublamina lucida is a layer of "adhesion in the secondary minimum" (Curtis 1973) created by the interplay of electrostatic forces of repulsion and a force of attraction arising from the London-van der Waals dispersion force in the lamina densa and tooth surface (linear border, dental cuticle, and fibrillar cementum). On the other hand, Weiss and Neiders (1971) studied biophysically the adhesion of human gingival epithelium to tooth and glass surfaces. They
Fig, 31. Scihema. of the compJete unit of adhesiO'Si composed of the .basal lamma [lamina •lucid.a, .LL, ia'mina densa, LD, and sub-Eamsna lucida, SLL) and the hemidesmosome. The special componeints iintegrating this unit are the pyramidal particles (pp) attached to the peripheraS density (pd) and the tine filaments (ff) penetrating the ilamioa densa, lamina ilucida., and' the^ peripheral density.
indicated that isolated gingival cells and tooth particles carried a net negative charge as determined by particle electrophoresis. Since relatively high centrifugal forces were inadequate to bring the cells into close con-
Fjg. 2B. Junction between root cementum (RG) and junctionai epithelium (JE). The three layers of Ihe basai lamina are clearly shown. Ftxataorii: acrolein-osmfum (Group 9). x 135,000. F]g. 29. Dental cyticie (DC) covering both afibrillar cemenium (AC) acid the root cementum (RC) at the cemento-enamel junction. Less dense spots are scattered throughout the dental cuticle due to a dense mesh-Mke pattern (mserts). FtKation paraformaldehyde-glutaraldehyde mixture, x 37,000; x 136,000 (insert a); x 170,000 (insert b). Fig. 30a-g. High-power eiectroni micrographs ol hemidesmosomes. Dense pyramidal particles {pp) occur alon.g i.nner surface of peripheral density (a-d). Associated with these particles are fine filaments {ff} extending into the lamina densa (a, e-g). Magnification line = 0.1 micron. Fixations- a) acrolein-osmium fGroup 9), x 1.36,000; b) glytaraldehyde perfusion-csmium (Group 5} x 132,000; c) glutaraldehyde perfusion-osmium" (Group 5}, x 135,000; d) paraformaldehyde-glutaraldehyde mi.xture-osmSum (Group 7), x 135,000; e) acrolein-osmium (Group 9), X 135,000; f) glytaraidehyde perfusion (Group 6), x 130,000; g) glutaraldehyde perfusion JGroup 6}. x 170,000.
328
KOBAYASHI, ROSE AND MAHAN
tact with glass or enamel surfaces, they concluded that the cells must have other mechanisms which effected their adherence to these surfaces in defiance of this electrostatic repulsion. Cuip (1974) suggested from his experiments on living celis that Ca+ • ions may be the cross-linking mechanism between negatively charged substrate, attached material deposited on the substrate and the negatively charged glycocalyx of cell surface. Hypothetically, we have considered that the unusual pyramidal particles on the inner surface of the peripheral densities are related both to a cementing substance and to the termination of the fine fiiaments extending from the lamina densa (Figs. 30 and 31). These filaments were not seen to pass into the cell through the cell membrane. Since the presence or absence of the pyramidal particles were affected by the various fixatives, the viscous (cement) nature of this area was suggested. Therefore, the original concept of the attachment apparatus (Listgarten 1956) consisting of hemidesmosomes and the basement lamina (lamina densa and lamina iucida in our study) is strengthened by these findings, as the filaments and pyramidal particles appear to function as units of integrity. However, the epithelial attachment extends beyond the lamina densa to the tooth surface (linear border, dental cuticle, and fibrillar cementum) and involves the apparently filament-free sub-lamina lucida. Conceptually, then, both the lamina densa and the tooth surface are negatively charged, the electrostatic repulsion keeps them at an appropriate distance (the sub-lamina lucida), and their adhesion is mediated either by London-van der Waals forces, cationic ions (as Ca++) or other mechanisms. The attachment apparatus and the region of electrostatic repulsion appear then to be the complete unit of adhesion at the dento-epithelial junction. There were a few instances
in which a separation occurred through the sub-lamina lucida but in no instance was the lamina densa found separated from the junctional epithelium. Therefore, it was concluded that the attachment apparatus is a slightly stronger union than the sublamina lucida. The dental cuticle is a substance between the basal lamina and the linear border on the tooth surfaces. Occasionally the cuticle has a transitional appearance on its dental surface, whereas the opposite side facing the epithelium is separated by a short distance from the lamina densa, viz., the sublamina lucida. If the sub-lamina lucida is as postulated, an area of electrostatic repulsion, then the dental cuticle would be the negatively charged structure substituting for the linear border or fibrillar cementum in the concept of the complete unit of adhesion. One hypothesis is that the dental cuticle is produced by a condensation of the basal lamina material (Schroeder & Listgarten 1971). Accordingly, the uniqueness of the microenvironment does not permit an easy escape of any extra basal lamina substance, and consequently it could accumulate on the tooth surfaces as the dental cuticle. Although this may he a valid explanation, the discontinuity of the dental cuticle from the lamina densa effected by the intervening sub-lamina lucida complexes such a simple deduction. Alternatively, the sub-lamina lucida over the dental cuticle may eontain disaggregated components of the lamina densa which are reaggregated into the dental cuticle. There are many instances in which the existence of the dental cuticle is not necessary tO' maintain a dento-epithelial adhesion (Fig. 15, b, d, f). This fact and the ubiquitous presence of a sub-lamina lucida even over a dental cuticle diminishes the possibility that the dental cuticle is a single adhesive agent, acting as a mediator which overcomes the electrostatic repulsive force
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Kobayashi, K. 1968. Electron microscopic study of the human epithelial attachment. Jap. J. Oral Biol. 10: 101-118. Listgarten, M. A. 1966. Electron microscopic study of the dentogingival junction of man. Amer. J. Anat. 119: 147-177. Listgarten, M. A. 1967. Electron microscopic features of the newly formed epithelial attachment after gingival surgery. /. Periodontal Res. 2: 46-52. Listgarten, M. A. 1972. Electron microscopic study of the junction between surgically denuded root surfaces and regenerated periodontal tissues. J. Periodontal Res. 7: 68-90. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Acknowledgements Cytol. 9: 409-414. This investigation was supported by Maslow, D. E. & Weiss, L. 1972. Cell exudation and cell adhesion. Exp. Cell Res. 71: USPHS through NiH-NIDR-DE01547 and 204-208. NIH-GRS-RR05344. Poste, G., L. W. Greenham, L. Mallncci, P. Reeve & Alexander, D. 3. 1973. The study of cellular "microexudates" by cUipsometry References and their relationship to the cell coat. Exp. Cell Res. 78: 303-313. Gulp, L. A. 1974. Substrate-attached glycoproteins mediating adhesion of normal and Revel, J. P. & Wolken, K. 1973. Electronmivirus-transformed mouse fibroblasts. J. Cell croscope investigations of the underside of Biol. 63: 71-83. cells in culture. Exp. Cell Res. 78: 1-41. Curtis, A. S. G. 1973. General functions of Reynolds, E. L. 1963. The use of lead citrate the eel! surface. In "Cell Biology in Mediat high pH as an electroD-opaque stain in cine", Ed. E. E. Bittar, pp. 441-471, New electron microscopy. /. Cell Biol. 17: 208York, John Wiley & Sons. 212. Flaxman, B. A., M. A. Lutzner & Van Scott, Rosenberg, M. I960. Microexudates from cells E. J. 1968. Ultrastructure of cell attachment grown in tissue culture. Biophys. J. 1: 137to substratum in vitro. ]. Cell Biol. 36: 406159. 410. Schroeder, H. E. 1969. Ultrastructure of the Frank, R., G. Fiore-Donno, G. Cimason & junctional epithelium of the human gingiva. Ogilvie, A. 1972. Gingival reattachment after Helv. Odont. Acta. 13: 65-83. surgery in man: an electron microscopic Schroeder, H. E. & Listgarten, M. A. 1971. study. J. Periodontol. 43: 597-605. Eine structure of tbe developing epithelial Hirose, S. 1970. Electron microscopic study of attachment of human teetb. "Monographs in tbe epithelial reattachment of the human Developmental Biology", Ed. A. Wolsky, S. teeth. Jap. J. Oral Biol. 12: 327-345. Karger, Basel, Vol. 2. Ijuhin,, N., G. G. Rose & Mahan, C. J. 1976. Taylor, A. C. 1970. Adhesion of cells to surfaces. In "Adhesion of Biological Systems", Subcellular organization of human gingival Ed. R. S. Manly, pp. 51-71, New York, epithelium cultivated in circumfasion sysAcademic Press. tems. Arch. Oral Biol, in press. Ito, H., S. Enomoto & Kobayashi, K. 1967. Taylor, A. C & M. M. Campbell. 1972. Reattachment of gingival epithelium to the Electron microscopic study of the human teeth. /. Periodontol. 43: 281-293. epithelial attachment. Bull. Tokyo med. Thilander, H. & A. Hugoson. 1970. The border dent. Univ. 14: 267-277. zone too'b-enatnel and epithelium after peKarnovsky, M. J. 1965. A formaldehyde-gluriodontal treatment. An experimental electaraidehyde fixative of high osmolality for tron microscopic study in the cat. Acta use in electron microscopy. J. Cell Biol. 27: Odont. Scatid. 28: 147-155. 137A.
between negatively charged substances. The true significance of the cuticle then remains a mystery which begs for a more concerted effort of study than possible with conventional observations. Whatever the origin and role the dental cuticle might have, we found the complete unit of adhesion, i.e., the attachment apparatus plus the sub-lamina lucida, was a constant structure which united the junctional epithelium with either the dental cuticle, the enannel, the afibrillar cementum or fibrillar cementum.
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Warshawsky, H. & G. Moore. 1967. A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytoehem. IS: 542-549. Weiss, L. & M. E. Neiders. 1971. A biophysical approach to the adhesion of human
Address: The University of Te.xas, Dental Branch P. O. Box 20068 Houston, Texas 77025
gingiva! epithelial cells to tooth and glass surfaces. J. Periodont. Res. 6: 28-37. Yaoi, Y. & T. Kanaseki. 1972. Role of microexudate carpet in cell division. Nature (Land.) 237: 283, 285.
Ultrastructure of the dento-epithelial junction K E N I C H I KOEAYASHI, GEORGE G . ROSE AND CHARLES J. M A H A N
Departments of Periodontics and Internal Medicine, The University of Texas Health Science Center, Dental Branch, Houston, Texas. U.S.A. The detito-epithelial janctions (DEJ) of Rhesus monkeys were examined by electron microscopy. Various fixation procedures were employed in this study. It was revealed that the basal lamina between the junctionai epithelium and the tooth surfaces was composed of three layers: the lamina lucida, the lamina densa and the sub-lamina lucida. Fine filaments often coursed through the lamina densa,, lamina lucida and hemidesmosomes and combined them into a stroctural unit. An electron-dense linear border, presumably derived from tissue fluid, was generally observed on the tooth surface at the DEJ except that cotnposed of the fibrillar cementum. This border was helpful in distinguishing the afibrillar cementtim from the dental cuticle. The existence of a sub-lamina lucida hetween Hit lamina densa and the tooth surfaces is a newly defined layer. It is hypothetically CODsidered to be an area of competetive electrostatic forces of repulsion and London Van der Waals forces of attraction. (Received for publication Dec. 12, 1975; accepted Feb. 26, 1976)
In addition, a dental cuticle which possesses a high electron density and a relatively During the past decade, the ultrastructure homogeneous property is frequently interof the normal human dento-epithelial juiic- posed between the basal lamina and the tioti was described by several investigators tooth surfaces. As no reports to the con(Listgarten 1966, Ito, Enotnoto & Kobaya- trary have appeared, it is assumed that the shi 1967, Kobayashi 1968, Sehroeder 1969, junctional epitheliutn adheres in a similar Schroeder & Listgarten 1971). Additional manner to the various dental surfaces with studies dealt with the ultrastructure of the which it comes in contact, viz., the enamel, gingival reattachment after surgical inter- the coronal cementum (afibrillar cevention in both human and other mamma- mentum), the root cementum (fibrillar celian subjects (Listgarten 1967, 1972, Hirose mentum), and the dental cuticle. 1970,, Thilander & Hugoson 1970, Frank et In the present study, we fotmd new de,al. 1972, Taylor & Campbell 1972). lineations of the dento-epithelial junction in Based oti these reports, it is generally Rhesus monkeys. conceded that the adhesion of the juncThis new data coupled with the reports fional epitheliutn to tooth surfaces is me- on cell-to-cell and cell-to-solid substrate addiated by multiple hemidesmosomes and an hesions reviewed in the Discussion have exunderlying structure very similar to the tended our perspectives on the organization basal lamina overlying the connective tissue. of the attachment mechanism. Introduction
314
KOBAYASHI, ROSE AND MAHAN Materials and Methods
The investigations were carried out on five young adult Rhesus monkeys. Under Nembutal® (Abbott, Chicago, Illinois) anesthesia, tissues were surgically obtained and fixed in accordance with the schedule in Table I. Fixation By Immersion After making gingival incisions, teeth with intact gingiva, including the margin of alveolar bone, were excised en bloc with mallet and chisel. The specimens were immersed immediately in the fixatives described in Table I, groups 1-4 and 7—9. Fixation By Perfusion The external carotid artery was cannulated with a polyethylene catheter, and the internal juglar vein was exposed and cannulated for drainage. The blood was briefly washed out of the vessels by means of a rinse with
normal saline, and then the glutaraldehyde fixative solution was perfused for about 45 minutes until the tissues of the mandibular region showed a marked yellowish coloring and stiffening. The specimens were excised as for the immersion procedure and placed in the same perfusing fixative for an additional 4-5 hoors (see Table I, groups 5, 6). Decalcification The excess dental tissues vcere trimmed from all specimens with a dental air turbine and then decalcified in 0.1 M disodium EDTA solution containing 0.1 M sucrose and adjusted to pH 7.2 (Warshawsky & Moore 1967). When the decalcification vi^as completed (av. 5 days, 20°C; av. 25 days, 4°C), the specimens were cut into smaller blocks and some were post-fixed with osmium solution according to the schedule in Table I. Finally, all specimens were embedded in Epon (Luft 1961).
Table I Schedule of fixation procedures Group
Pre-fixation* 2"/o Giut.. 0.1M S-coll, or 0.1M Phos. (2hr)
Decalciftcalion D,1M disodium EDTA with 0,1M Sucrose "
Post-fixation* 2 7o Os,, 0,1M S-coli,
Figures 25
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Abbreviations Used: Glut, - glutaraldehyde; S-coll, = s-coJIidine buffer; Phos, = phosphate buffer; EDTA ; ethyJenediaminetetraacetio acid; Os, = osmium; Para, = paraformaldehyde; Aero, — acrolein, * pH of fixatives: 7,4; temperature during fixation; 0-4°C, Fixation with ,2'/o Os,, 0,1M S-ooll, phr) was interposed between pre-fixation and decaloificatio;i, *** -f4-5, hr after excising tissues. ""** Modified Karnovsky's (1965) fixative
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Sections studied in this report were cut on a Porter-Blum MT-2 ultramicrotome, mounted on naked copper grids, routinely stained with aqueous uranyi acetate and lead citrate (Reynolds 1963) and examined in an RCA EMU 4B electron microscope operated at 75 KV.
Otisenrations
The general appearance of the dento-epithelial junction in the Rhesus monkeys conformed to that described in studies of human and other mammalian specimens (see Introduction). With low-power electron microscopy the junctional epithelium contacted the enamel, the afibrillar cementum covering the enamel surface (Fig. 1), and the root cementum (Fig. 2). Multiple hemidesmosomes were easily recognized along the cell membrane facing the tooth surfaces. The existence of the dental cuticle varied from specimen-to-specimen and area-to-area, and early stages appeared over the enamel or root cementum (Eig. 2) as the major density between the junctional epithelium and the tooth. Attachments Coronal to the CemenloEnamel Junction (Figs. 3—14, 16-19) Moderate-power electron micrographs obtained from various fixations revealed the many relationships between the junctional epithelium and the enamel (Eigs. 3-12). Well-developed dental cuticles were seen between the afibrillar cementum (overlying the enamel) and the junctional epithelium (Figs. 3 and 4). In other areas, well-developed portions of the dental cuticle were transformed into irregular portions of diminished mass (Figs. 5 and 6). The ultrastructure of the dental cuticle at moderate magnifications appeared to be equivalent in specimens prepared in different fixatives
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(Figs. 3.-6). Less well-defined dental cuticles were atso observed as continuous units (Fig. 7). Some areas had no evident dental cuticle (Figs. 8-12). Where the junctional epithelium apposed the afibrillar cementum without an intervening dental cuticle, there was, between the basal lamina and the afibrillar cementum, a thin dense line, the linear border (Figs. 6, 8, 10-12). This linear border was helpful in distinguishing the afibritIar cementum from the dental cuticle. For instance, where the outer lamination of the afibrillar cementum was dense and structurally similar to a dental cuticle, it was separated from the basal lamina by the linear border (Fig. 8). Such a narrow sharp line was never seen on the epithelial surface of the dental cuticle; if it were observed at all, it appeared between the dental cuticle and the enamel or afibrillar cementum (Fig. 6). In cases in which the junctional epithelium apposed the enamel surface directly, i.e., without an intervening dental cuticle or afibrillar cementum, a sharp linear border was visible at the location corresponding to the enamel surface (Fig. 9). Between the lamina densa and the linear border, there was a narrow clear zone we have designated the sub-lamina lucida. This was seen in varying degrees of clarity (Figures 4-6, 8, 10-12). High-power electron microscopy denoted more clearly the various structures of the epithelial attachment. The basal lamina was finely divided into an upper lamina lucida, a mid lamina densa, and a lower sublamina lucida (Fig. 13). In some cases the sub-lamina lucida was partially obscured by the dental cuticle (Fig. 14). Similarly, the linear border was usually obscured by the dental cuticle (Figs. 13 and 14). Although there was variation from specimen-to-specimen and fixation-to-fixation, this basic 3part design of the basal lamina remained
316
KOBAVASHI, ROSE AND MAHAN
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constant (see line drawings. Fig. 15, a-d). The higher-power electron micrographs were used to determine the measurements of these layers. The lamina lucida** (140 ± 30 A wide) occupied the narrow space between the peripheral density and the lamina densa; the lamina densa*** (400' ± 100 A wide) appeared suspended between the clear lamina lucida and the clear sublamina lucida. The sub-lamina lucida (95 ± 20 A wide) lay between the lamina densa and the linear border (120 ± 20' A wide. Figs. 16, 18) or the dental cuticle (Figs. 13, 17, and 19). The dental cuticle was finely granular and of a high electron density. The border between the dental cuticle and the sub-
** Other authors define the lamina lucida as the space betweeo the basai or junctional cell membrane and the lamina densa. *** Other authors perforce include thiC sublamina ldcida in their measurements of the lamina deusa.
AC BL cf DC ES ff HD il IS JE LB LD
— afibrjllar cementtjm — basal lamina — collagen fibriis — deiHital cuticle = enamel space =: fine rilaments — hemidesmosame = inner leaflet = intercelluilar space = jonciional epithelium — linear border = lamina dense
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jacent afibrillar cementum was highly irregular and unclear (Fig. 17). In such areas the linear border was not defined as was the case in most areas in which the dental cuticle was well-developed. However, the linear border was sharply evident between the enamel and the sub-lamina lucida (upper part of Fig. 16), between the enamel and dental cuticle '(lower part of Fig. 16), and between the afibrillar cementum and the sub-lamina lucida (Fig. 18). The dental cuticle overlying the enamel was extremely variable. In some areas it was absent and within a few micra was evident again, tisually reappearing in a zone of transition (Fig. 16). Rarely, between the dental cuticle and the linear border there was a narrow clear zone (Fig. 16). The cell membrane of the junctional epithelium was not visible in those specimens not postfixed in osmium (Figs. 16 and 17), although the attachment plaques and the peripheral densities of the hemidesmosomes were clearly visible. In specimens postfixed
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Ftg. 1. Jynctional epitheiium (JE) adijacent to the afibrtlJar cementum (AC) coveripg the enamel surface. Fixatjoo: acrolein-osmium (Group 9). x 12,1:&Q. Rg. 2,. Junctional epithelium (JE) adjacent to the root cementum (RC). Fixation: acrolein-osmium (Group 9). X 16,500. Fig. 3. Well-developed dental cuticle (DC) between the afibrilfar cementum (AC) overlying the enamel and the junctional eptthefium (JE). Fixation; paraformaldehyde-glutaraldehyde miixture-osmium (Group 7). x 24,750. Fig. 4. Dental cuticle (DC] interposed between afibrillar cementum (AC) and iunctional epithelium (JE). Fixatioioi; glutaraldehyde-osmium-osmium (Group 3). x 24,750, Fig. 5. Denta! cuticle (DC) over enamel, iin the lower half of this figure, the dental cuticle is well-developed' in the upper half, the dental cuticle is thininer ain.d irregular {aggregation or disaggregatJon process, Fixation: paraformaldehyde-gtutaraidehyde mixture (Group 8). x 35,550.
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Fig, 15. Schema of complete untt of adhesion between junctjonai epithelium [JEJ and tooth developed Irom electron micrographs: a) enamel space (HS) covered with dental cuticie (DC); b} enamel space {ES) covered with Sinear border (LB): c) enamel space (ES) covered with afibrfllar cementum (AC) and dental cuticje {DC}; d) enamei space (ES) covered with afibriliar cementum (AC) and iineer border (LB); e) root cementum (RC) interspersed with patches of afibriilar cementum (AC) and covered with dental cuticle (DC); f) root cementum (RCJ interspersed with patches of afibrillar cemenium [AC) covered with the tinear border (LB).
Fig. 6. Irregular dentai cuticie (DC) .in Lfpper 2/5ths of this figure, diminishing m width at its ioweir extremity. The afibrillar cementum (AC) covers the entire enamei surface in this figure. The boundary between the afjbriiar cemenium and the basai lamina (BL) is a clear sharp dense line, the linear border (slanting arrows), Fixat'ion: glutraldehyde perfusion (Group 6). x 35,550. Fig.. 7. Dentai cuticle (DC) 5s less well-defiaed than in- preceding Itgures. Linear border is obscured by the dental cutjcle, Fixation:: acrolesn-osmium (Group 9). x 35,550.. Fig., B. Junction^al epIthiBliam (JE) apposing the atibriiMar cementum (AC) without an intervening dental cuticle. The outer lamination of the afibrilJar cementum is dense and almost indistingui-shabJe from a dental cuticle. The thin linear border (slanting arrows), separates the apposing surfaces of the basal iamina (BL) and the afibriilar cementum. Fixation: paraformaldBhyde-gtutaraldehyde mJxture-osmium (Group 7}. x 35,550. Fig.. 9.. Junctional epithelium (JE.) apposing enamei without an interveniing dental cuticie or afibriliar cemen•tum. A distinct linear border is on the enamel surface (slanting arrows). The three parts of the basal iamiina: sub-lamina lucida (SLL), iamina densa (LD), and lamina lucida (LL) are indicated. Fixation: paraformaldehydeglutaraldehyde miixiure (Group 8), x 35,550. Fig. 10. Basal lamina (BL) sharpiy separated from afsbrillar cementum (ACJ by the linear border (slanting arrows), No dental cuticle is visible: glutaraidehyde perfusion-osmium (Group 5), x 24,750.
320
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by an osmium solution, the outer leaflet of the unit membrane of the juBCtional epithelial cell was clearly observed (Figs. 18 and 19). Attachments Apical to the CementoEnamel Junctions (Figs. 20-28) At moderate magnifications of electron microscopy, the basal lamina was seen to maintain a relatively uniform width (Figs. 20-22). The lamina densa also could be identified at these magnifications. In some cases, the surfaces of tbe root cementum were covered with a dental cuticle of great thickness variation (Figs. 20-22). In the root cementum, especially near the surface area, there were portions lacking coiiagen fibrils, as well as portions packed with typicaliy periodic collagen fibrils (Fig. 21, see also line drawing in Fig, 15, e,f). The former areas were morphologically similar to the afibrillar cementum overlying the enamel. Higher-power electron micrographs (Figs. 23-27) showed the variations in the attachments between the root cementum and tbe junctional epithelium. The dental cuticle was not always present over the root cementum (Figs. 24, 25, and 27). The
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desmosomes were generally larger than those apposing the basal lamina of the oral epithelium, and they were sharply outlined along the surfaces of the juactional epithelium. In all specimens the attachment of the lamina densa to the root cementum or the dental cuticle was mediated by a thin clear space, the sub-lamina lucida (Figs. 23 and 28). The dental cuticle adapted to the uneven surface of the root cementum and was generally even on the surface facing the basal lamina. Accordingly, the lamina densa maintained a relatively uniform thickness and straight appearance (Fig. 23). However, the space between the lamina densa and the dental cuticle had occasional dilatations where the dental cuticle was not homogeneous. The linear border was especially evident in areas devoid of the dental cuticle, and where the root cementom lacked collagen fibrils, i.e., afibrillar cementum (Fig. 25). It was never observed between fibrillar cementum and the basal lamina or dental cuticle. Where the dental cuticle came in contact with afibrillar cementum, this line could not be detected (Figs. 23 and 26). Layers covering the root cementum and covered by the linear border, by our defini-
Fig. 11. Linear iborder (slanting arrows) between afibriltar cementum (AC) and basai lamina (BL). No dentai culicie is present. Fixation: glutaraldehyde periusion (Group 6). x 35,550. Fig:. 12. Linear border (sianting arrows) between afibrijiar cementum (AC) and basal iamina (BL). iNo dentai cuticle is present. Fixation: paraformaidehyde-osmium (Group 4). x 24,750. Fig. 13. Basai iamina (BL) is fmeiy divided into upper Samina iucida (LL), miid lamina dense fLD), and lower sublamina iucida (SLL). Linear border obscured by dental cuticle (DC). Fixation: glutaraidehyde-osmium-osmium (Group 3). x 58,500. Fig. M. Dentai cuticle (DC) is weli-deveioped and obscures linear border and parts ot sub-lamina lucJda. Fixation: paraformaWehyde-giutaraldehyde iTiixture-osmium (Group 7), x 58,500. Fig. 16. Epithelial attachment to the enamel. Three parts of basal iamina: sub-laminia jucida (SLL), iamina densa (LD), and iamina lucida (LL) are easily discriminated. As osmium was omitted in fixation, the cell membrane is not visibie as a component of Xhe hemidesmosomes (HD). In the upper part of micrograph there is no dental cuticie or a1ibri!1ar cementtjm. In lower half, there is an irreguiar dental cuticle which may represent an ongoi^ng process of cuticular aggregation OT disaggregation. Linear border (sianting arrows) lies on the surface of enamel throughout micrograph. Fixation: paratormaldiehyde-glutaraidehyde mixture (Group 8). x 135,000.
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tion Vifere not the dental cuticle but the afihrillar cementum (Fig. 27). Special Considerations of the Denial Cuticle and Hemidesmosomes Under high-power electron microscopy the dental cuticle may be described as a relatively homogeneous, somewhat granular and electron-dense layer. In some sections, less dense spots appeared within the dental cuticle, but the granularity of the Epon embedding media prevented clear details of these areas at high powers of magnification (Fig. 29). These spots, as well as a dense mesh-like pattern surrounding the spots, were seen all over the dental cuticle (Fig. 29, a, b). Throughout these observations, the hemidesmosomes were well-preserved regardless of the fixation procedure used. In highpower views of selected areas, details of the hemidesmosomes contained dense pyramidal particles along the inner surface of the peripheral density (Fig. 30, a-d). Fine filaments extended from the peripheral density into the lamina densa (Figs. 30, a, e-g). In specimens fixed by perfusioo but not post-
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fixed with osmium, the particles on the peripheral densities were less distinct although larger and extended to the outer leaflet (Fig. 30' f, g).
Discussion
Our investigation has once again confirmed the previously established concept that an attachment apparatus consisting of multiple hemidesmosomes and a basal lamina promotes the adhesion of the junctional epithelium to the tooth surfaces. Additionally, It has been revealed in this study that this attachment apparatus behaves as a unit which is maintained a short distance from the tooth surface or dental cuticle by an intervening sub-lamina lucida. Since this junction is situated against a relatively impenetrable surface, a great deal of care was required in effecting the tissue preservation of the specimens. In order to avoid misunderstandings which might be attributed to a single technical procedure, various fixation methods were employed. 'Our results showed a remarkable consistency of the
Fig:. 17. Epitheiial-enamel junction. Three parts of the basai iamina (BL) over dental cuticie (DC) are ciearly recognized, viz., the sub-lamina lucida (SLL), the iamina densa (LD), and the lamina iuoida (LL). Linear border is not evident. Fixation: paraformaidehyde-glutaraldehyde mixture (Group S), x 135,000, Fig. 1B. Afibriilar cementum (AC) intervening between junctional epitheiium (JE) and enamel; there is no dental cuticle. Distinct linear border (slanting arrows) is on outer surface of afibriiiar cementum. Three components of basal lamina are delineated. Fixation: aorolein-osmium (Group 9). x 135,000, Fig. 19. Outer and inner ieafiets (oi, ii) ot cell membrane and the peripheral' density (pd) of the hemidesmosome (HD) are defined. Three iayers of tiie basai iamina (BL) are evident as an eleotron-light-dark-iight triad overlying the electron-dense dentai cuticie (DC). Fixation: paraformaldehyde-giiitarafdehyde mixture-osmium (Group 7), X 135,000. Fig. 20. Broad, dense, and homogeneous dental cuticle (DC) interposed between root cementum, (RC) and junctional epitheiium (JE). Fixation: giytaraldehyde-asmiium-osmium (Group 3), x 24,750. Fig. 21. Surface of the root cementum (RC) covered by thin eiectron-dense dental cuticle (DC). Basai lamina (BL) maintains a uniform width and its division Into the three sublayers is easily detected. The areas (AC) in the root cementum iacking coiiagen fibriis (cf) are' morphologtcaily similar to the afibriiiar cementum overlying the enamei. Fixation: acroiein-osmlum (Group 9). x 35,550, Fig. 22. Dentai cuticle' (DC) interposed between root cementum (RC) and! junctional epitheiium (JE), Area lacking coiiagen fibriis in root cementum appears to have been cieared by decaicificatlon procedure (vertical arrows). Fixation: glutaraldehyde (Group 2), x 24,750,
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spatial relationships between the attachment apparatus and tooth surfaces or dental cuticle. Where the attachment apparatus directly contacted the enamel surface, i.e., without an intervening layer of dental cuticle and/ or afibrillar cemeotum, as shown in Figure 9, the enamel surface was marginated with a sharp dense line, the linear border. Similar dense lines were always found on the surfaces of the afibrillar cementum covering the enamel,, as well as that found in the root cementum. However, it was often obscured by the presence of an overlying dental cuticle. There was no evidence that the dental cuticle and the linear border were of common origin, but there were two types of arrangements that suggested their separate origin: 1) the linear border had a relatively constant width and appeared to be overlaid by the encroachment of a developing dental cuticle of varying widths (Figs. 6, and 16); and 2) below the cemento-enamel junction, the linear border appeared only on the surface of the afibrillar cementum (Figs. 24 and 27) and never on the fibrillar cementum, whereas the dental cuticle appeared uniformly on both (Figs,, 21—23 and 26; see also schema in
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Fig. 15). Although Schroeder and Listgarten (1971) reported ultrastructural differences between the dental cuticle and the afibrillar cementum, we found the linear border was helpful in distinguishing these two layers,, especially when the cuticle was poorly developed and its existence questioned. Dense lines similar to the linear border have been observed on both plastic and mica culture surfaces upon which human dermal and oral cells were cultivated (Flaxman et al. 1968, Ijuhin et al. 1976). Additionally, between this dense line and the apposing cell, there was a layer of extracellular material of moderate electron density about 450 A wide. Flaxman et al. (1968) postulated that the thinner dense layer represented a condensate of serum protein from the culture medium at the cell substratum interface, and the moderate dense layer might be synthesized by the overlying cells, as the so-called "microexudate" described by others (Rosenberg 1960, Maslow & Weiss 1972, Yaoi & Kaoaseki 1972; Poste et al. 1973, Revel & Wolken 1973, Culp 1974). From the positional and dimensional standpoints, the narrow dense line (linear
Fig. 23. BasaS tamtna (BL) between junctional epithelium (JE) and root cementum (RC) characteristically s,eparated into its three compo,nentE,: lamina lucida (LL), !ami,na deosa (LD), and sub-iamina iucida (SLL), Dental cuticle (DC) adapts to uneven surface ol the root cementum (RC) but is reiativeiy even on surface facing basal lamina. The occasional apparent dilatation of the sub-lannina lucida may represent an aggregation or disaggregation process in the dental cuticle. Fixation: paraformaldehyde-giutaraldehyde mixture (Group 8), x 58,500. Fig. 24. Three sub-iayers of the basal tamina (BL) overlying root cementum (RC). Upper pcrtion (AC) of the root ce,mentum (RC) lacks collagen fibrils and is structurally similar to the afibrillar cementum covering the enamel. Fixation: acroieln-osmium (Group 9). x 76.500, Fig.-25. Surface of roct cementum (RC) is not clear except in the lower portion, where the dense linear bord:er (slanting arrows} indicates its surface. Cementum located beneath this Wne lacks collagen fibrils and is, therefore, afibriilar cementum (AC), There is no dental cuticle. Fixation: gtutaral,dehyde-osmium (Group 1), x 58,500. Fig. 26. Dental cuticle (DC) over root cementum (RC), Three sub-layers of the basal lamina (BL) are easily denoted. Fixation;: glutaraldehyde perfusion (Group 6), x 76,500. Fig. 27. Basal lamina (BL) ciearly divided into three sublayers. Layer covering root ce,mentum {RC) is reticular, and since it is covered by the linear border [slanting arrows), it is by definition not a dental cuticle bu: the alibrillar cemehtum (AC). Fixaticn: aorolein-osmium (Group 9|. X 58,500.
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border) over the enamel or the afibrillar cementum was similar to tiie tiiin layer found apposing the hard surfaces of culture vessels. If Flaxman's et al. (1968) postulate is proved correct, then the in vivo and in vitro dense lines may have the same nature and origin, that is, be adsorption layers derived from the tissue or culture fluids and consist mainly of serum proteins. On the root cementura surface, the linear border was not detected except over those portions lacking collagen fibrils, i.e., the afibrillar cementum. This discrepancy may have depended largely on the divergent surface structures of these two types ol exposed cementum, i.e., their adsorptive qualities. The major question arising from this study regards the origin and function of the thin sub-lamina lucida lying between the basal lamina (lamina densa) and the tooth surface. It is conceivable that the sublamina lucida is a layer of "adhesion in the secondary minimum" (Curtis 1973) created by the interplay of electrostatic forces of repulsion and a force of attraction arising from the London-van der Waals dispersion force in the lamina densa and tooth surface (linear border, dental cuticle, and fibrillar cementum). On the other hand, Weiss and Neiders (1971) studied biophysically the adhesion of human gingival epithelium to tooth and glass surfaces. They
Fig, 31. Scihema. of the compJete unit of adhesiO'Si composed of the .basal lamma [lamina •lucid.a, .LL, ia'mina densa, LD, and sub-Eamsna lucida, SLL) and the hemidesmosome. The special componeints iintegrating this unit are the pyramidal particles (pp) attached to the peripheraS density (pd) and the tine filaments (ff) penetrating the ilamioa densa, lamina ilucida., and' the^ peripheral density.
indicated that isolated gingival cells and tooth particles carried a net negative charge as determined by particle electrophoresis. Since relatively high centrifugal forces were inadequate to bring the cells into close con-
Fjg. 2B. Junction between root cementum (RG) and junctionai epithelium (JE). The three layers of Ihe basai lamina are clearly shown. Ftxataorii: acrolein-osmfum (Group 9). x 135,000. F]g. 29. Dental cyticie (DC) covering both afibrillar cemenium (AC) acid the root cementum (RC) at the cemento-enamel junction. Less dense spots are scattered throughout the dental cuticle due to a dense mesh-Mke pattern (mserts). FtKation paraformaldehyde-glutaraldehyde mixture, x 37,000; x 136,000 (insert a); x 170,000 (insert b). Fig. 30a-g. High-power eiectroni micrographs ol hemidesmosomes. Dense pyramidal particles {pp) occur alon.g i.nner surface of peripheral density (a-d). Associated with these particles are fine filaments {ff} extending into the lamina densa (a, e-g). Magnification line = 0.1 micron. Fixations- a) acrolein-osmium fGroup 9), x 1.36,000; b) glytaraldehyde perfusion-csmium (Group 5} x 132,000; c) glutaraldehyde perfusion-osmium" (Group 5}, x 135,000; d) paraformaldehyde-glutaraldehyde mi.xture-osmSum (Group 7), x 135,000; e) acrolein-osmium (Group 9), X 135,000; f) glytaraidehyde perfusion (Group 6), x 130,000; g) glutaraldehyde perfusion JGroup 6}. x 170,000.
328
KOBAYASHI, ROSE AND MAHAN
tact with glass or enamel surfaces, they concluded that the cells must have other mechanisms which effected their adherence to these surfaces in defiance of this electrostatic repulsion. Cuip (1974) suggested from his experiments on living celis that Ca+ • ions may be the cross-linking mechanism between negatively charged substrate, attached material deposited on the substrate and the negatively charged glycocalyx of cell surface. Hypothetically, we have considered that the unusual pyramidal particles on the inner surface of the peripheral densities are related both to a cementing substance and to the termination of the fine fiiaments extending from the lamina densa (Figs. 30 and 31). These filaments were not seen to pass into the cell through the cell membrane. Since the presence or absence of the pyramidal particles were affected by the various fixatives, the viscous (cement) nature of this area was suggested. Therefore, the original concept of the attachment apparatus (Listgarten 1956) consisting of hemidesmosomes and the basement lamina (lamina densa and lamina iucida in our study) is strengthened by these findings, as the filaments and pyramidal particles appear to function as units of integrity. However, the epithelial attachment extends beyond the lamina densa to the tooth surface (linear border, dental cuticle, and fibrillar cementum) and involves the apparently filament-free sub-lamina lucida. Conceptually, then, both the lamina densa and the tooth surface are negatively charged, the electrostatic repulsion keeps them at an appropriate distance (the sub-lamina lucida), and their adhesion is mediated either by London-van der Waals forces, cationic ions (as Ca++) or other mechanisms. The attachment apparatus and the region of electrostatic repulsion appear then to be the complete unit of adhesion at the dento-epithelial junction. There were a few instances
in which a separation occurred through the sub-lamina lucida but in no instance was the lamina densa found separated from the junctional epithelium. Therefore, it was concluded that the attachment apparatus is a slightly stronger union than the sublamina lucida. The dental cuticle is a substance between the basal lamina and the linear border on the tooth surfaces. Occasionally the cuticle has a transitional appearance on its dental surface, whereas the opposite side facing the epithelium is separated by a short distance from the lamina densa, viz., the sublamina lucida. If the sub-lamina lucida is as postulated, an area of electrostatic repulsion, then the dental cuticle would be the negatively charged structure substituting for the linear border or fibrillar cementum in the concept of the complete unit of adhesion. One hypothesis is that the dental cuticle is produced by a condensation of the basal lamina material (Schroeder & Listgarten 1971). Accordingly, the uniqueness of the microenvironment does not permit an easy escape of any extra basal lamina substance, and consequently it could accumulate on the tooth surfaces as the dental cuticle. Although this may he a valid explanation, the discontinuity of the dental cuticle from the lamina densa effected by the intervening sub-lamina lucida complexes such a simple deduction. Alternatively, the sub-lamina lucida over the dental cuticle may eontain disaggregated components of the lamina densa which are reaggregated into the dental cuticle. There are many instances in which the existence of the dental cuticle is not necessary tO' maintain a dento-epithelial adhesion (Fig. 15, b, d, f). This fact and the ubiquitous presence of a sub-lamina lucida even over a dental cuticle diminishes the possibility that the dental cuticle is a single adhesive agent, acting as a mediator which overcomes the electrostatic repulsive force
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Kobayashi, K. 1968. Electron microscopic study of the human epithelial attachment. Jap. J. Oral Biol. 10: 101-118. Listgarten, M. A. 1966. Electron microscopic study of the dentogingival junction of man. Amer. J. Anat. 119: 147-177. Listgarten, M. A. 1967. Electron microscopic features of the newly formed epithelial attachment after gingival surgery. /. Periodontal Res. 2: 46-52. Listgarten, M. A. 1972. Electron microscopic study of the junction between surgically denuded root surfaces and regenerated periodontal tissues. J. Periodontal Res. 7: 68-90. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Acknowledgements Cytol. 9: 409-414. This investigation was supported by Maslow, D. E. & Weiss, L. 1972. Cell exudation and cell adhesion. Exp. Cell Res. 71: USPHS through NiH-NIDR-DE01547 and 204-208. NIH-GRS-RR05344. Poste, G., L. W. Greenham, L. Mallncci, P. Reeve & Alexander, D. 3. 1973. The study of cellular "microexudates" by cUipsometry References and their relationship to the cell coat. Exp. Cell Res. 78: 303-313. Gulp, L. A. 1974. Substrate-attached glycoproteins mediating adhesion of normal and Revel, J. P. & Wolken, K. 1973. Electronmivirus-transformed mouse fibroblasts. J. Cell croscope investigations of the underside of Biol. 63: 71-83. cells in culture. Exp. Cell Res. 78: 1-41. Curtis, A. S. G. 1973. General functions of Reynolds, E. L. 1963. The use of lead citrate the eel! surface. In "Cell Biology in Mediat high pH as an electroD-opaque stain in cine", Ed. E. E. Bittar, pp. 441-471, New electron microscopy. /. Cell Biol. 17: 208York, John Wiley & Sons. 212. Flaxman, B. A., M. A. Lutzner & Van Scott, Rosenberg, M. I960. Microexudates from cells E. J. 1968. Ultrastructure of cell attachment grown in tissue culture. Biophys. J. 1: 137to substratum in vitro. ]. Cell Biol. 36: 406159. 410. Schroeder, H. E. 1969. Ultrastructure of the Frank, R., G. Fiore-Donno, G. Cimason & junctional epithelium of the human gingiva. Ogilvie, A. 1972. Gingival reattachment after Helv. Odont. Acta. 13: 65-83. surgery in man: an electron microscopic Schroeder, H. E. & Listgarten, M. A. 1971. study. J. Periodontol. 43: 597-605. Eine structure of tbe developing epithelial Hirose, S. 1970. Electron microscopic study of attachment of human teetb. "Monographs in tbe epithelial reattachment of the human Developmental Biology", Ed. A. Wolsky, S. teeth. Jap. J. Oral Biol. 12: 327-345. Karger, Basel, Vol. 2. Ijuhin,, N., G. G. Rose & Mahan, C. J. 1976. Taylor, A. C. 1970. Adhesion of cells to surfaces. In "Adhesion of Biological Systems", Subcellular organization of human gingival Ed. R. S. Manly, pp. 51-71, New York, epithelium cultivated in circumfasion sysAcademic Press. tems. Arch. Oral Biol, in press. Ito, H., S. Enomoto & Kobayashi, K. 1967. Taylor, A. C & M. M. Campbell. 1972. Reattachment of gingival epithelium to the Electron microscopic study of the human teeth. /. Periodontol. 43: 281-293. epithelial attachment. Bull. Tokyo med. Thilander, H. & A. Hugoson. 1970. The border dent. Univ. 14: 267-277. zone too'b-enatnel and epithelium after peKarnovsky, M. J. 1965. A formaldehyde-gluriodontal treatment. An experimental electaraidehyde fixative of high osmolality for tron microscopic study in the cat. Acta use in electron microscopy. J. Cell Biol. 27: Odont. Scatid. 28: 147-155. 137A.
between negatively charged substances. The true significance of the cuticle then remains a mystery which begs for a more concerted effort of study than possible with conventional observations. Whatever the origin and role the dental cuticle might have, we found the complete unit of adhesion, i.e., the attachment apparatus plus the sub-lamina lucida, was a constant structure which united the junctional epithelium with either the dental cuticle, the enannel, the afibrillar cementum or fibrillar cementum.
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Warshawsky, H. & G. Moore. 1967. A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytoehem. IS: 542-549. Weiss, L. & M. E. Neiders. 1971. A biophysical approach to the adhesion of human
Address: The University of Te.xas, Dental Branch P. O. Box 20068 Houston, Texas 77025
gingiva! epithelial cells to tooth and glass surfaces. J. Periodont. Res. 6: 28-37. Yaoi, Y. & T. Kanaseki. 1972. Role of microexudate carpet in cell division. Nature (Land.) 237: 283, 285.
