A mineralized cuticular structure with connective tissue characteristics on the crowns of human unerupted teeth in amelogenesis imperfecta

Arch oral Biol. Vol.12, ~33.877-889, 1967. PergamonPressLtd. Printedin Gt. Britain.

A MINERALIZED CUTICULAR STRUCTURE WITH CONNECTIVE TISSUE CHARACTERISTICS ON THE CROWNS OF HUMAN UNERUPTED TEETH IN AMELOGENESIS IMPERFECTA A LIGHT

AND ELECTRON M. A.

MICROSCOPIC

STUDY

LISTGARTEN

University of Toronto, Faculty of Dentistry, Toronto, Canada Summary-Premature degeneration and absence of the reduced enamel epithelium was observed in two unerupted teeth from a patient with amelogenesis imperfecta. This epithelial degeneration was accompanied by the patchy formation of a mineralized cuticular structure resembling cementum on the follicular surface facing the enamel. The demineralizd cuticle showed histochemical evidence of a high content of collagen and acid mucopolysaccharide. Electron microscopic studies failed to reveal the presence. in the cuticle of typically banded collagen fibrils. The cuticle was granular and showed a laminated internal structure. Although the cuticle may represent a form of afibrillar cementum, it should be noted that its formation appeared unrelated to direct cellular synthesis. Rather, it appeared to be associated with the degenerative process affecting the reduced enamel epithelium. The possibility that cuticle formation is dependent on adsorption by the enamel of connective tissue components was suggested. The degeneration of the reduced enamel epithelium would merely allow these connective tissue components to gain access to the enamel surface. The resemblance of the coronal cuticle in amelogenesis imperfecta to that found near the cemento-enamel junction of normal human teeth was pointed out, both with respect to ultrastructure and possible origin. It may be that these cuticular structures should be considered under the category of acquired pellicles with the understanding that their formation occurs in unerupted teeth, in areas of the crown not covered by an intact layer of reduced enamel epithelium. INTRODUCTION

recent investigations of the dento-epithelial junction of erupted and unerupted human teeth, a cuticular structure referred to as a type A cuticle was described (LISTGARTEN, 1966a,b). In demineralized sections it appeared as a granular layer up to 10~ thick, extending coronally from the most apical border of the enamel for varying distances of up to 150~. Appositional lines in the structure suggested a succession of deposits progressing from the cemento-enamel junction coronally. The type A cuticle was found over enamel only. It generally appeared as a continuation of the acellular cementurn layer over the enamel, or as a distinct layer internal to the cementurn, separating the latter from the enamel surface. Because of its location and appearance it was postulated that the type A cuticle represented an end product of amelogenesis or perhaps an afibrillar type of cementum never described previously. The possibility that this cuticle arises from an interaction of enamel and connective tissue was suggested by the observation that in unerupted teeth the type A cuticle DURING

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was frequently found in an area, near the apical enamel, devoid of reduced enamel epithelium. In erupted teeth where the epithelial attachment frequently covered this structure, it was assumed that the mature attachment had migrated apically to cover the type A cuticle after it had formed. It is important to note that with light microscopy this layer was not readily distinguishable from cementum. Only one reference was found (GLICKMANand BIBBY, 1943) in which the authors noted an “enamel cuticle” which appeared to correspond to the type A cuticle described above. GLICKMANand BIBBYpostulated that the “enamel cuticle” was an end product of amelogenesis and suggested that it could have a protective influence on the development of caries. The authors also noted its presence on the crown of impacted teeth, but failed to note whether its location was related to degeneration or absence of the reduced enamel epithelium. In 1945, WEINMANNet al. observed that in amelogenesis imperfecta there was a premature degeneration of the cells of the reduced enamel epithelium following the formative stage, thereby rapidly curtailing the maturation stage. In their paper, the authors stated : “The loss of the protective epithelial covering brings the connective tissue in contact with the enamel surface. Beginning at the cemento-enamel junction, a layer of cementum progresses occlusally, but generally does not reach the occlusal surface itself or the incisal edges. The cementum can be considered as a means of protection of the crown against resorption because of the presence of a thin layer of uncalcified cementoid on its surface . . . ” The present study was prompted by the similarity of the type A cuticle to cementurn and the above report of a cementum layer on the unerupted crowns of teeth, the enamel of which is known to lose its protective epithelial covering shortly after completion of the formative stage. It was felt that if the type A cuticle results from the degeneration or absence of an epithelial covering over unerupted crown enamel, unerupted teeth in amelogenesis imperfecta should provide an excellent experimental model to determine if the type A cuticle will form in areas of degenerating epithelium. This study could also be expected to reveal whether the cementum layer described by WEINMANNef al. on the enamel of such teeth is similar to root cementum (HERTING, 1963; SELVIG, 1965; LISTGARTEN,1966a) or whether it may represent a structure similar to the type A cuticle.

METHODS AND MATERIALS Two unerupted teeth enclosed in their follicles were obtained at the time of surgery from a 16-year-old patient whose teeth were all affected by the disease. The specimens consisted of the right mandibular third molar and a supplemental tooth between the right mandibular second premolar and first molar (Figs. 1 and 2). The specimens were collected in an ice cold solution of 5 % glutaraldehyde in 0*17M cacodylate buffer at a pH of 7.4. After 30 min of fixation, the specimens were transferred to a 5 % sucrose-cacodylate buffer wash in which they were immersed for 2 hr.

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Undemineralized portions of the third molar were obtained after subdividing the teeth with an “air-rotor”. The specimens included tissue blocks from the cementoenamel junction. Blocks containing soft tissue were post fixed for 30 min in 2% osmic acid in a veronal-acetate buffer, at pH 7.4. Parts of the third molar and the follicle containing the developing supplemental tooth were demineralized from 1 to 2 weeks in cacodylate buffer containing 0*25M Versene, the pH of the solution having been adjusted to neutrality with 5N sodium hydroxide. After demineralization, the specimens were rinsed in a 5% sucrose buffer wash. Some demineralized blocks were obtained from each tooth, and post-fixed in osmic acid fixative as described above. All specimens for electron microscopy were dehydrated in graded ethanol solutions and embedded in Epon according to the method of LUFT (1961). Sections were cut at 0.1~ on an MT-2 model Porter-Blum microtome and collected on bare or carbonreinforced formvar-coated grids. Sections of soft tissue or demineralized materials were double stained with a saturated solution of uranyl acetate and lead citrate (VENABLE and COGGESHALL, 1965). Undemineralized sections were usually examined unstained or after a brief exposure to uranyl acetate. Specimens were studied in a Philips EM-200 electron microscope. Some demineralized specimens were also embedded in paraffin for light microscopy. These specimens were not fixed in osmic acid, and were subjected to glutaraldehyde fixation only. Paraffin sections were stained using the following procedures (Armed Forces Institute of Pathology, 1960) : Haematoxylin and eosin stain Masson’s trichrome stain Van Gieson’s stain Alcian blue stain Periodic acid-Schiff stain as well as Mtiller’s colloidal iron stain, as described by MOWRY (1958). Some Epon embedded specimens were sectioned for study with phase-contrast microscopy. Sections of undemineralized blocks containing structures of interest were also studied by electron diffraction of selected areas. Areas were selected by the use of a rectangular diaphragm the sides of which are formed by four adjustable platinum blades built into the diffraction lens of the electron microscope.

RESULTS

Pre-operative radiographs of the patient revealed that the right mandibular third molar had a completed crown, whereas the crown of the supplemental tooth was not completed and was still in the formative stage of amelogenesis (Figs. 1 and 2). Examination with the light microscope of sections taken from the third molar indicated that the reduced enamel epithelium had undergone extensive degeneration,

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with large areas showing no epithelium between the enamel matrix and the dental follicle (Fig. 3). In cases where the enamel matrix had been displaced during the preparation of the specimen, tissue blocks of both the superficial enamel and the follicle were examined. In neither case was the reduced enamel epithelium found in a well preserved state, and in most cases it was altogether absent. Since the third molar had been used to obtain undemineralized specimens, the follicle had been opened in several places, and portions of it were absent. It was, therefore, felt that examination of the supplemental follicle, which had not been opened during all of the preparative procedures up to the end of demineralization, might reveal an epithelial layer which conceivably might have been lost from the other tooth. The demineralized follicle was, therefore, carefully cut in half in a longitudinal plane. After division of the follicle and its contents, the dentine core which was loosely connected to the enamel cap was gently separated and discarded. Figure 4 shows a low power view of the specimen embedded for light microscopic study. Note that, although ameloblasts are still actively engaged in amelogenesis near the apical portion of the crown (Fig. 5), there is a rapid degeneration of all the cells of the reduced enamel epithelium coronal to the active ameloblasts. The only site where close contact appears to be maintained between the follicle and the tooth surface is in the region of active amelogenesis. After completion of the formative phase of enamel, the rapid degeneration of the reduced enamel epithelium (Figs. 6 and 7) results in a weakening of the bond between follicle and tooth surface, with the resulting separation, no doubt aggravated by the preparative procedure. Another finding of interest was the presence in patches of a cuticular structure of varying size on the follicular surface facing the enamel. This cuticle varied in width from a barely detectable structure (Fig. 8) to a layer approximately 6~ wide (Fig. 11). This structure was observed in both teeth examined. It appeared preferentially in areas where the reduced enamel epithelium was missing (Fig. 9), although it was also observed on the enamel side of degenerating groups of epithelial cells (Fig. 10). In favourable sections studied by phase-contrast microscopy, the cuticular structure demonstrated appositional lines (Fig. 11). It was felt that the use of the stains listed under Methods and Materials might give us some insight into the possible nature of this cuticular structure. It appeared particularly important to establish whether the staining characteristics of the cuticle would resemble those of enamel matrix or those of certain connective tissue components. The histochemical data are summarized in Table 1. It is interesting to note that the cuticle stained very much like collagen from adjacent connective tissue, and was markedly different from enamel matrix in its staining qualities. Slides treated with Masson’s trichrome and van Gieson’s stains for collagen suggested that the cuticle contained collagen. The periodic acid-Schiff technique stained the cuticle markedly more intensely than the adjacent connective tissue. The intensity of the stain was not affected by prior digestion with diastase. The reaction of the cuticle to staining with colloidal iron and Alcian blue was also markedly stronger than that of adjacent connective tissue.

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TABLE 1

Stain

Cuticle A

Enamel Matrix

Collagen

Basophilic Brownish red to red Very pale pink to colourless

Eosinophilic

H&E

Basophilic

Masson

Green

PAS with diastase

Deep magenta

Alcian blue

Deep blue

Patchy areas light blue to blue

Blue

Colloidal iron

Blue

Colourless

Colourless

Van Gieson

Red

Reddish green

Red

Green Pink

Electron microscopy of the cuticle revealed a laminated structure consisting primarily of a finely granular matrix (Figs. 12-14). Although, successive layers of the cuticle showed some variation in density, the granular character of the structure was fairly uniform throughout, The cuticle was found in most instances in close contact with the adjacent connective tissue (Fig. 9), but could also be detected under groups of degenerating epithelial cells (Figs. 10 and 13). In no instance was there any evidence of typically banded collagen fibrils within the cuticle. The basement lamina separating the follicular connective tissue from the degenerating epithelial cells was also subject to degenerative changes. (Fig. 12) and was frequently missing. Electron diffraction of selected areas of undemineralized unstained sections of the cuticle (Fig. 14) revealed the presence of an apatite pattern with little or no arcing of any of the several reflections from the crystal lattice. This was in marked contrast to the short arcs observable in the diffraction pattern of underlying enamel (Fig. 15). DISCUSSION

Although WEINMANN et al. (1945) were the first to described the premature degeneration of the reduced enamel epithelium in amelogenesis imperfecta, their observations were based on teeth in which amelogenesis had already reached completion. This is the first study, to the author’s knowledge, where various stages in this degenerative process have been followed on the same tooth from the stage of active amelogenesis to complete degeneration of the reduced enamel epithelium. Although the occurrence of all these phases on a single tooth suggests that the rate of degeneration is rapid, it is necessary to be cautious in the interpretation of this observation, since the tooth is a supplemental premolar and it has not been established that supplemental teeth develop at the same rate as normal ones. Of particular interest was the observation that a cuticular structure morphologically similar to the type A cuticle described previously near the cemento-enamel junction of normal teeth (LISTGARTEN, 1966a,b) could also be found in amelogenesis imperfecta. The fact that this cuticle occurred over parts of the crown removed from the cementoenamel junction was of particular interest, since this had not been the case in normal

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teeth. The association of the cuticle with areas of the enamel surface denuded of reduced enamel epithelium or covered with degenerating epithelial cells confirmed a previous suspicion that the formation of this cuticle may depend on prior degeneration of the epithelial barrier between enamel and connective tissue. The histochemical data provided strong support that the cuticle is of connective tissue origin. The strong reactions with the periodic acid-Schiff stain after diastase digestion, and with Alcian blue and colloidal iron stains indicated the presence of an acid mucopolysaccharide component. The van Gieson and Masson stains suggested the presence of a collagenous fraction. In view of these reactions and the results from the haematoxylin and eosin staining, it is not surprising that WEINMANN et al. believed that they were dealing with a cementum layer covering the crown of unerupted teeth in amelogenesis imperfecta. However, examination with the electron microscope of this cuticle clearly indicated that it is morphologically different from root cementum and that it closely resembles the type A cuticle seen near the cemento-enamel junction of normal teeth. The presence in the cuticle of a collagenous component as indicated by histochemical stains, but the absence of the typically banded collagen fibrils with electron microscopy suggested that if collagen is present in this structure, it probably exists in a nonpolymerized form. Although no attempt was made in this study to compare the histochemical properties of the coronal cuticle in amelogenesis imperfecta to those of other types of dental cuticle, it should be noted that some of the staining properties appeared to differ markedly from those described by WERTHEIMER and FULLMER(1962) for the classical dental cuticle, at least for the stains common to both studies such as the periodic acidSchilI, the Alcian blue, and the haematoxylin and eosin stains. Similar differences were noted between this study and that of HOD~ON(1966) for an artificially produced cuticle derived from conglutinated erythrocytes which, the author claims, is similar to the dental cuticle. The results of selected area electron diffraction indicated that the cuticle became mineralized, and that the mineral component was an apatite. However, in contrast to the underlying enamel, the cuticle contained very small crystals (Fig. 17) which, as indicated by the even reflections in the diffraction pattern, show very little, if any, preferential orientation within the organic matrix of the cuticle. In view of the above observations it would appear that the cuticular structure found on the coronal surface of unerupted teeth in amelogenesis imperfecta may represent a form of afibrillar cementum, which appears morphologically similar to the type A cuticle observed in a limited area over the enamel, near the cemento-enamel junction of normal human teeth (LISTGARTEN,1966a). In normal teeth, the origin of the type A cuticle also seemed to be associated with degenerative changes in the reduced enamel epithelium, near the cemento-enamel junction (LISTGARTEN,1966b). The type A cuticle, which may represent an afibrillar type of cementum, is distinctly different from the so-called coronal cementum on human teeth described by LEVINE, GLIMCHERand BONAR(1964), since these authors were referring to tissue corresponding in location to the follicular connective tissue,

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If we assume that a collagen and mucopolysaccharide rich layer forms on enamel in the presence of degenerating reduced enamel epithelium, the question still remains as to how and why such a layer should appear where it does. There is no indication that this cuticle is the product of a cellular process, since the ultrastructural features of adjacent cells do not suggest their active participation in collagen (Ross and BENDITT, 1961: PORTER,1964) or mucopolysaccharide synthesis (NEUTRAand LEBLOND,1966). In view of the degenerative changes in the reduced enamel epithelium and the histochemical findings it is unlikely that epithelial cells are the source of the cuticle. It seems most logical, therefore, to assume that the building blocks for the cuticle are attracted in some fashion to the enamel surface from their normal location in the follicular connective tissue. It is known that hydroxyapatite has excellent adsorbing qualities and is, in fact, used for this reason in the fractionation of proteins (KELLER and BLOCK, 1960). It has been shown that powdered hydroxyapatite is capable of adsorbing various salivary proteins (HAY, 1966; MCGAUGHEYand STOWELL,1966) and preliminary experiments by HAY (1966) have indicated that dental enamel surfaces have similar properties. It is, therefore, reasonable to postulate that under conditions which favour the passage of connective tissue components toward the enamel, that some of these materials will become adsorbed to the enamel surface. Under appropriate conditions this surface layer could mineralize in a manner similar to dental calculus matrix, hence giving rise to a mineralized cuticle. However, if the formation of this cuticle is not due to a cellular process, it would differ radically from the formation of root cementum which is said to be dependent on the cellular activity of cementoblasts. Perhaps, the type A cuticle should then be considered an ‘acquired’ cuticle rather that afibrillar cementum, with the understanding that the acquisition takes place prior to eruption, from material derived from the connective tissue. In that connection it is interesting to compare the above findings to those of LEACHand SAXON(1966) who described the ultrastructure of acquired pellicles taken from the labial surface of erupted human incisors. These authors noted that the acquired pellicle on erupted teeth also showed a laminated appearance when studied with electron microscopy. They noted that the pellicle became mineralized, electron diffraction studies revealing They also determined that the pellicle contained the presence of hydroxyapatite. carbohydrate and protein components as well as glycoproteins, which they assumed to originate from saliva. It appears that adsorption by hydroxyapatite surfaces may play an important role in the formation of surface pellicles on enamel both prior to and following eruption of teeth. It is also possible that adsorption plays a more important role in root cementum formation than has been suspected up to now. Although cementoblasts may produce the raw material, we know that assembly of collagen into fibrils is a process which is not cell dependent. Perhaps, the assembly of these fibrils and the interfibrillar material into cementum is primarily the result of local physico-chemical factors which are only indirectly related to cellular activity. The lack of banded collagen in the mineralized coronal pellicle of teeth affected with amelogenesis imperfecta, as well as in the type A

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cuticle of normal teeth (LISTGARTEN, 1966a,b) may be due to the source of the collagenous material. Collagenolysis in the pericoronal connective tissue may result in the presence of end products of enzymatic digestion which, although chemically similar to collagen, may have lost their ability to reaggregate into characteristically banded collagen fibrils. It is a matter of semantics whether this cementum-like material should be referred to as an afibrillar type of cementum or as a mineralized pellicle acquired prior to eruption. Acknowledgements-The author is grateful to Dr. J. C. DUNCAN and to the Department of Oral Surgery for their assistance in obtaining the specimens used in this study. This work was supported by grant DA-126 from the National Research Council of Canada, Associate Committee on Dental Research.

R&rum&-Une degenerescence prematur&e et une absence d’epithelium de P&mail reduit sont observ6es au niveau de deux dents incluses, chez un patient atteint d’amelogenbse imparfaite. Cette degenerescence epitheliale eat accompagnee de la formation, par places, dune structure cuticulaire minbralis&, analogue au cement, au niveau de la surface folliculaire, faisant face a l’email. La cuticule deminQalis6e montre histochimiquement un contenu eleve en collagene et en mucopolysaccharide acide. La microscopic electronique n’y montre cependant pas la presence de collagene. La cuticule apparait granulaire avec une structure inteme en lamelles. Bien que la cuticule puisse constituer une sorte de cement afibrillaire, sondeveloppement neparaitpas lieaune synthese cellulaire dire&e. 11 semble s’agir plut& dune deg&n&scence affectant l’epithtlium reduit de l’email. 11 se peut que la cuticule se forme par absorption de composants conjonctifs au niveau de l’email. La degenerescence de l’epith6lium adamantin r&iuit permettrait a ces constituants conjonctifs d’arriver a la surface de l’email. La cuticule coronaire, en cas d’am6logen&e imparfaite, semble similaire a celle de la jonction tmail-cement de dents humaines normales. Ces structures cuticulaires devraient &tre class6es dans les pellicules acquises, tout en sachant qu’elles se forment au niveau de dents incluses, dans des r&ions coronaires non recouvertes par une couche intacte d’epithelium adamantin reduit. Zusammenfassung-Die vorzeitige Degeneration sowie das Fehlen des reduzierten Schmelzepithels wurde an zwei nicht durchgebrochenen Zahnen eines Patienten mit erblicher Schmelzhypoplasie beobachtet. Diese epitheliale Degeneration war von einer ungleichmal3igen Ausbildung einer mineralisierten hlutchenartigen, dem Zement lhnlichen Struktur an der follikularen Oberflache des Schmelzes begleitet. Bei der histochemischen Untersuchung wies dieses demineralisierte Hlutchen einen hohen Gehalt an Kollagen sowie an sauren Mucopolysacchariden auf. Durch elektronenmikroskopische Untersuchungen konnte das Vorhandensein von typisch gebtindelten Kollagenlibrillen im HPutchen nicht nachgewiesen werden. Es war eher von kiirniger BeschatIenheit und wies eine blattrige innere Struktur auf. Obwchl das Hautchen eine Art von fibrillenlosem Zement darstellen konnte, sol1 festgestellt werden, da8 seine Bildung nicht mit der zellul&ren Synthese zusammenzuhlngen scheint. Es scheint eher mit dem degenerativen Prozess verbunden zu sein, der auf das reduzierte Schmelzepithel einwirkt. Es wurde die Mbglichkeit erwogen, daD die Formation des Hautchens von der Adsorption von verbindenden Gewebekomponenten durch den Schmelz abhlngt. Die Degeneration von reduziertem Schmelzepithel wtirde diesen verbindenden Gewebekomponenten lediglich Zugang zur Schmelzoberfiache ermoglichen. Es wurde die Ahnlichkeit des koronalen Hautchens bei einer erblichen Schmelzhypoplasie mit dem an der Zement-Dentin-Grenze normaler menschlicher Ziihne vorhandenen diskutiert,

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und zwar im Hinblick auf ihre Ultrastruktur wie such ihrer moglichen Entstehung. Es ist miiglich, dal3 diese kutikuliiren Strukturen in die Kategorie der erworbenen Hlutchen eingereiht werden sollten unter dem Gesichtspunkt, da8 ihre Formation bei nicht durchgebrochenen Zshnen in Bereichen der Krone erfolgt, die nicht durch eine intakte Schicht von reduziertem Schmelzepithel bedeckt sind.

REFERENCES Armed Forces Institute of Pathology. 1960. Manual of Histologic and Special Staining Techniques (2nd ed.). McGraw-Hill, New York. GLICKMAN,I. and BIBBY,B. G. 1943. The existence of cuticular structures on human teeth. J. dent. Res. 22, 91-96. HAY, D. I. 1966. The adsorption of saliva proteins onto apatite and enamel powder. International Association for Dental Research, 44th General Meeting. Abstract 457. HERTING,H. C. 1963. Elektronemnikroskopische Untersuchungen iiber das Zahnwurzelzement des Menschen. Adv. Fluor. Res. J. dent. Caries Prev. 1, 303-312. HODSON,J. J. 1966. A critical review of the dental cuticle with special reference to recent investigations. ht. dent. J. 16, 350-384. KELLER,S. and BLCICK,R. J. 1960. Fractionation of proteins by adsorption and ion exchange. In: A Laboratory Manual of Analytical Methodr of Protein Chemistry (Vol. 1). (Edited by ALEXANDER, P. and BU)CK, R. J.), pp. 70-71. Pergamon Press, New York. LEACHS. A. and SAXTON,C. A. 1966. An electron microscopic study of the acquired pellicle and plaque formed on the enamel of human incisors. Archs oral Biol. 11, 1081-1094. LEVINE,P. T., GLIE~CHER, M. J. and BONAR,L. C. 1964. Collagenous layer covering the crown enamel of unerupted permanent human teeth. Science, N. Y. 46, 16761678. LISTGARTEN,M. A. 1966a. Electron microscopic study of the gingivo-dental junction of man. Am. J. Anat. 119,147-178. LISTGARTEN,M. A. 1966b. Phase-contrast and electron microscopic study of the junction between reduced enamel epithelium and enamel in unerupted human teeth. Archs oral Biol. 11,999-1016. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409-414. __ _ . MCGAUGHEY, C. and STOWELL,E. C. 1966. Adsorption of salivary proteins by hydroxyapatite. International Association for Dental Research. 44th General Meeting. Abstract 456. MOWRY, R. W. 1958. Improved procedure for the staining of acidic pobsaccharides by Miiller’s colloidal (hydrous) ferric oxide and its combination with the Feulgen and the periodic acidSchitf reactions. Lab. Invest. 7, 566-576. NEU~RA, M. and LEBLOND,C. P. 1966. Radioautographic comparison of the uptake of galactose-Ha and glucose-Ha in the golgi region of various cells secreting glycoproteins of mucopolysaccharides. J. Cell Biol. 30, 137-150. PORTER,K. R. 1964. Cell tie structure and biosynthesis of intercellular macromolecules. In: Connective Tissue: Znterceflular Macromolecules, Proc. Symp. New York Heart Association. Little, Brown, Boston. Ross, R. and BEND~IT,E. P. 1961. Wound healing and collagen formation. I. Sequential changes in components of guinea pig skin wounds observed in the electron microscope. J. biophys. biochem. Cytol. 11,677-700. SELVIG,K. A. 1965. The fine structure of human cementum. Acta odont. stand. 23,423-441. VENABLE,J. H. and ~OGOESHALL,R. 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25,407-408. WEINMANN,J. P., SVOBODA,J. F. and WOODS,R. W. 1945. Hereditary disturbances of enamel formation and calcification. J. Am. dent. Ass. 32,397-418. WERTHEIMER, F. W. and Fuw, H. M. 1962. Morphologic and histochemical observations on the human dental cuticle. J. Periodont. 33, 29-39.

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PLATE 1 FIG. 1. Radiograph of unerupted right mandibular third molar. Note that the formative phase of the crown is complete. Although enamel is quantitatively normal, it is abnormally radiolucent, a reflection of incomplete mineralization. FIG. 2. Radiograph of right mandibular amelogenesis is still incomplete.

supplemental

premolar.

Note

that

FIG. 3. Area near the cemento+namel junction (CEJ) of the third molar. The connective tissue (CT) of the dental follicle is devoid of reduced enamel epithelium on the surface facing the enamel space (ES). A cuticle (C) is present, however, near the cemento+namel junction. D, dentine. Demineralized. Phase-contrast. x 450. FIG. 4. Low-power photomicrograph of premolar. The dental follicle comprising mostly connective tissue (CT) surrounds the enamel (E), but is only connected to it in the apical region outlined by a rectangle. This area is shown at higher magnification in Fig. 5. Numbered arrows refer to the regions of the follicle from which x 14. Fig. 6-10 were made. Demineralized. Haematoxylin and eosin. FIG. 5. Magnified view of area outlined in Fig. 4. Note rapid degeneration of ameloblasts (A) and stratum intermedium cells following completion of amelogenesis. This rapid degeneration is accompanied by separation of the reduced enamel epithelium (RF) from enamel (E). CT, connective tissue of dental follicle. Demineralized. Haematoxylin and eosin. x 125.

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PLATE 2

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PLATE 2 FIG. 6. Magnified view of reduced enamel epithelium

(RE) near upper left corner of Fig. 5, where the epithelial lining becomes interrupted (large arrow) so that the connective tissue of the follicle faces directly against enamel (E). Demineralized. Haematoxylin and eosin. x 500. FIG. 7. Only isolated epithelial cells (EP) remain on the connective tissue surface. x 500. E, enamel. Demineralized. Haematoxylin and eosin. FIG. 8. Appearance

enamel.

Demineralized.

of small patches of cuticle (C) on the follicular surface facing Haematoxylin and eosin. x 500.

FIG. 9. Cuticle (C) at the junction of follicular connective Demineralized. Note absence of reduced enamel epithelium. eosin. x 500.

tissue and enamel. Haematoxylin and

FIG. 10. Cuticle (C) may become interrupted by a remaining island of reduced enamel epithelium, although it may also be found under some remaining epithelial cells. EP, epithelial cells. Demineralized. Haematoxylin and eosin. x 500. FIG. 11. Cuticle (C) demonstrating Phase-contrast. x 640.

a laminated internal structure.

Demineralized.

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PLATE3 FIG 12. Electronmicrograph

of laminated cuticle (C) Note that a section (between large arrows) of the basement lamina (BL) is almost completely lysed, thereby permitting connective tissue components (CT) to come in contact with the cuticle. ES, enamel space. Demineralized. x 12,OGO.

FIG. 13. Electronmicrograph of cuticle (C) under degenerating epithelial cells (EP). ES, enamel space. Demineralized. x 12,000.

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PLATE 4 FIG. 14. Section near the cemento+namel junction of the third molar from which the diffraction patterns below were obtained. D, dentine; E, enamel; C, cuticle. Undemineralized and unstained. x 25,000. FIG. 15. Electron diffraction pattern of enamel shown in Fig. 14. Arcing of the 002 reflection indicates a preferred orientation of the crystallites within the enamel matrix. This may be confirmed by examination of Fig. 14. FIG. 16. Electron diffraction pattern obtained from a portion of the cuticle shown in Fig. 14. Note the lack of arcing of any of the reflections indicating a lack of preferred orientation of the crystallites in the cuticle. FIG. 17. A portion of cuticle illustrating the small size, as well as the complete lack of preferred orientation of the crystallites in the structure. The surface facing the connective tissue is visible in the upper right corner. Undemineralized and unstained. x 2M@oo.

H