Cell Tissue Res (1991) 263 : 311-324
Cell and Tissue Research 9 Springer-Verlag 1991
Initiation of acellular extrinsic fiber cementum on human teeth A light- and e l e c t r o n - m i c r o s c o p i c study Dieter D. Bosshardt and Hubert E. Schroeder Department of Oral Structural Biology, Dental Institute, University of Zurich, Plattenstrasse 11, CH-8028 Zurich, Switzerland Accepted September 21, 1990
Summary. The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of A E F C in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 5 0 % - 6 0 % of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in E D T A and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about i mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 gm from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 ~tm from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process o f dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization. D. Bosshardt Abbreviations: A E F C acellular extrinsic fiber cementum; A I F C acellular intrinsic fiber cementum ; CIFC cellular intrinsic fiber cementum; C M S C cellular mixed stratified cementum; A R E advancing root edge; CP cytoplasmic process; D dentin; D C J dentinocemental junction; E enamel; E B L external basal lamina; EC epithelial cell; ED TA ethylene diaminetetraacetic acid; E R M epithelial rests of Malassez; F F fiber fringe; H R S Hertwig's epithelial root sheath; IBL internal basal lamina; M D mineralized dentin; N M D non-mineralized dentin; OB odontoblast; PD predentin; P L periodontal ligament
Key words: Dental root surface - Periodontal fiber fringe - Dentino-cemental junction - Electron microscopy Human
In human teeth, cementum covers the entire surface of the roots, increases in thickness with age, and serves in fully erupted and functioning teeth for the attachment of the principal periodontal ligament fiber bundles, which run from alveolar bone to cementum. The two major types of human root cementum are acellular extrinsic fiber cementum (AEFC) and cellular mixed stratified cementum (CMSC). A E F C is found primarily on the cervical and middle third o f the root, whereas CMSC occurs primarily in the apical third of the root and the furcations (Schroeder 1986, 1988). The genesis of both types of cementum on growing roots of human teeth is unknown: only two investigations, one microradiographic (Owens 1976) and one light- and electron-microscopic study (Bosshardt and Schroeder 1990) have dealt with particular aspects of human cementogenesis. However, to our knowledge, there is not a single study describing the genesis of A E F C in man. On the other hand, there are numerous
Offprint requests to." D.
Fig. 1a--c. Examples of X-ray photographs of a maxillary first (a), a mandibular first (b), and a mandibular second (e) premolar showing roots developed to 50% (a, e) and 60% (b) of their final length. a--c x2.5
b.~
313 studies d e a l i n g with established c e m e n t u m o n fully f o r m e d roots. F o r example, the structure o f A E F C has been e x a m i n e d light- a n d e l e c t r o n - m i c r o s c o p i c a l l y in hum a n d e c i d u o u s (Keller 1964; F u r s e t h 1967) a n d p e r m a n e n t (Dewey 1926; K r o n f e l d 1928; Schmid 1951; Herting 1962, 1964; Selvig 1965, 1967; F u r s e t h 1974) teeth. I n a d d i t i o n , m i c r o r a d i o g r a p h i c i n v e s t i g a t i o n s (Soni et al. 1962; Dreyfuss a n d F r a n k 1964; Selvig 1965; F u r s e t h 1967; O w e n s 1976) o n fully developed h u m a n teeth have revealed h e t e r o g e n e o u s calcification o f the d e n t i n o - c e m e n t a l j u n c t i o n (DCJ). O n a c c o u n t o f the absence of i n f o r m a t i o n o n the genesis o f A E F C o n h u m a n teeth, studies were initiated to describe its initial f o r m a t i o n in the p r e f u n c t i o n a l phase. I n p a r t i c u l a r , it was necessary to clarify the p r o b lems o f the initial genesis o f p e r i o d o n t a l fibers a n d their a t t a c h m e n t to the d e n t i n a l surface, i.e,, the d e v e l o p m e n t of the DCJ.
Materials and methods Ten premolars (6 maxillary and 4 mandibular, 7 first and 3 second), extracted for orthodontic reasons from 7 girls and 3 boys, 9-111/2 years of age, were selected from a larger collection of freshly extracted human teeth and processed for light- and electronmicroscopic study. The selected teeth presented incomplete roots developed to 50%-60% of their final length. These premolars were processed with the intention of studying the initiation, establishment and further growth of AEFC. Its structural aspects, attachment to dentin and association with the fibers of Sharpey were also examined. Immediately after extraction, the teeth were fixed in halfstrength Karnovsky's fixative (Karnovsky 1965), buffered (pH 7.4) with 0.02 M sodium cacodylate for 24-48 h. Thereafter, the teeth were briefly washed in 0.185 M sodium cacodylate buffer (pH 7.4; 350 mOsm) and exposed to a decalcifying solution containing 0.15 M EDTA, 2.5% glutaraldehyde with or without supplementation with 0.2 M sucrose. Decalcification was achieved at room temperature over 4-8 weeks, under constant stirring. The solution was changed twice weekly. Following decalcification, slices cut vertical to the root surface in a corono-apical direction, were severed with the use of razor blades from the teeth at mesial and distal sites. The slices were about 4 mm in length and included either the apical or the cervical portion of the teeth. This resulted in about 10 slices per tooth. These slices were briefly washed in 0.185 M sodium
cacodylate buffer and postfixed in 1.33% OsO4, buffered in 0.067 M S-collidine, for 2 h. Thereafter, the slices were washed again in buffer, dehydrated in ethanol and embedded in Epon (Luft 1961). From each block, 1 to 2-pro-thick sections were cut using glass knives and a Reichert OMU-2 ultramicrotome. These sections were stained with a combination of PAS and a mixture of methyleneblue/Azure II (Schroeder et al. 1980) and served for light-microscopic evaluation, photography and sample selection. Light micrographs were obtained using a Leitz Orthoplan/Orthomat equipment. Sample sites for electron-microscopic study were (1) root surface areas either close to or remote (up to 4-5 mm) from the advancing edge of the root, and (2) root surface areas close to the cemento-enameljuntion. From the selected areas, ultrathin sections (60-80 nm) were cut, using a diamond knife and an LKB-Ultrotome III, and contrasted with uranyI acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Electron-microscopic examination and recording were performed with a Philips 201 transmission electron microscope. The investigated teeth were extracted prior to or during their emergence into the oral cavity. In particular, three premolars (Fig. 1) with the best possible degree of tissue preservation were selected for an in-depth study with light- and electron-microsocpic techniques. This selection was made for the following reasons: (1) Fully developed roots of human premolars are covered by pure AEFC for 60%-80% of their length, as measured from the cemento-enamel junction (Schroeder 1988). This applies to all aspects of their roots, i.e., buccal, mesial, oral and distal sites. (2) The initiation and early establishment of pure AEFC follow an apicocoronal gradient, with matrix production starting near the advancing root edge. Because of these features, it was reasonable to assume that no other type of cementum but AEFC would develop along the entire length of the selected root surfaces. The total number of such blocks investigated was 120. All abreviations used in this paper are explained on the title-page.
Results The degree o f tissue p r e s e r v a t i o n varied c o n s i d e r a b l y a n d u n p r e d i c t a b l y , because of e x t r a c t i o n t r a u m a . U n suitable blocks, as j u d g e d f r o m light-microscopic sections, were discarded. I n general, all l i g h t - m i c r o s c o p i c sections f r o m sufficiently well preserved blocks revealed a basic p a t t e r n of d e v e l o p m e n t a l features: Hertwig's epithelial r o o t sheath, a l t h o u g h associated with the edge of the a d v a n c i n g root, was never seen to follow the external surface o f the r o o t d e n t i n for m o r e t h a n a few gm. Instead, the m o s t apical p a r t (i.e., a l o n g 100 g m c o r o n a l
Fig. 2 a-c. Light-(a) and electron-(b,c) microscopic views of the mesial, apical portion of a mandibular second premolar (with a root developed to 50% of its final length), extracted from an 11-year-old girl. The area outlined in a corresponds to b, and that in b corresponds to c. The region encircled in c is shown in the inset. At the light-microscopic level, intensely basophilic cells are seen closely abutting the external dentinal surface (a) immediately coronally to the advancing root edge (ARE). At a distance of about 200 pm coronal to the ARE, a fiber fringe (FF, arrowheads in a) oriented perpendicular to the root surface is apparent. Electron-microscopically, the cells lining the external and apical dentin surface reveal an elongated fibroblast-like appearance (b, c). Between these cells and the dentin matrix, collagen fibrils are demarcated from the randomly oriented dentinal fibrils by their parallel arrangement and appearance in either cross- (c, arrowheads and inset) or longitudinal (c, arrows) planes of sectioning. The latter are oriented approximately perpendicular to the dentinal surface and extend toward the adjacent cells (c, arrows). D dentin; PD not yet calcified predentin; PL periodontal ligament, a • 580, b x 3015, c x 6700; inset: • 14400
Fig. 3. Light-(a,b) and electron-microscopic (c) views of the advancing root edge (ARE) at the distal site of a mandibular first premolar (with a root developed to 60% of its final length), extracted from an 11 1/2-year-old girl. The area outlined in a corresponds to b, the lower area outlined in h corresponds to c. Note the presence of a fiber fringe (FF, arrowheads in a, b), oriented perpendicular to the root surface and beginning about 100 gm coronal to the ARE. Within the apical 100 pm, the space between the remnants of Hertwig's root sheath (*) and the dentinal root surface is populated with intensely stained, basophilic cells (a, b). These elongated and fibroblast-like cells are in close contact with the external dentin matrix (b, c). Note that all these cells are similar morphologically, with a typical nuclear fibrous lamina (inset), and are in contact with one another. The external predentin matrix is still non-mineralized (c). D dentin; PD predentin; EC epithelial cell; IBL/EBL internal and external basal lamina; OB odontoblasts ; PL periodontal ligament, a x580, b x900, c x3015; inset: x62500
314
F~.3.
315
Fig. 4.
316 to the advancing root edge) of this surface was covered by intensely basophilic and heavily contrasted cells, populating a space between the external dentinal surface and Hertwig's root sheath, and closely abutting the notyet mineralized dentinal matrix (Fig. 9). Further coronally, within another 100 gm from the root edge, a fiber fringe was seen light-microscopically; this covered the external root surface and became progressively more distinct. It eventually extended to the cemento-enamel junct i o n and, in more cervical root regions, became part of the first, very thin layer of AEFC. The following observations are devoted to the events occurring within a distance of about I m m coronal to the advancing root edge (ARE). Along the first 100 gm coronal to the A R E (Fig. 9), the external surface o f root dentin, still non-mineralized, was covered by a dense population of intensely stained, basophilic cells (Figs. 2 a, 3 a, b). These cells populated the space between the external dentinal surface and Hertwig's root sheath (Fig. 3c). Ultrastructurally, they were similar morphologically. They had an elongated, sometimes slender shape (Figs. 2b, 3c) and a fine-structural appearance typical o f fibroblast-like cells, with an abundant basophilic cytoplasm rich in organelles, particularly rough endoplasmic reticulum (Figs. 2c, 4a). In addition, these cells contained small amounts of glycogen storage granules. The nuclei were elaborately convoluted and rather rich in euchromatin, displaying a typical, about 50-nm-thick, fibrous lamina (Fig. 3, inset). These fibroblast-like cells never revealed closely abutting plasma membranes, but were regularly connected to one another by desmosome-like junctions (Fig. 4, insets). The intercellular spaces were sparsely filled with cross-cut collagen fibrils (Figs. 2c, 4a, arrowheads). Cells located immediately coronal to the A R E projected numerous cytoplasmic processes towards the external dentin matrix (Fig. 4a). Occasionally, such processes were found to insert between and to contact the dentinal fibrils (Fig. 4c). The external dentinal matrix was still nonmineralized and revealed an uniform appearance of randomly oriented collagen fibrils (Figs. 2c, 3c, 4a, c). Between the latter and the closely abutting fibroblast-like cells, a moderate developmental gradient o f tiny bundles of collagen fibrils was apparent. This gradient increased in the apico-coronal direction. The initially bundled fibrils were seen adjacent to the fibrils of the external
Fig. 5a, b. Electron-microscopic views of the upper (a) and the middle (b) area outlined in Fig. 3b. The middle area (b), 100 gm coronal to the advancing root edge, shows bundles of collagen fibrils that are densely packed in parallel arrays, and oriented perpendicular to the root surface. These fibers are separated from each other by the cytoplasm of adjacent connective tissue cells and appear in cross-, oblique- and longitudinal-planes of sectioning. They terminate at the external dentin surface and intermingle with the randomly oriented fibrils of the still non-calcified predentin. This transitional zone of fibril-interdigitation determines the future dentino-cemental junction (DC3). A few lam pulpwards from this zone, the dentinal fibrils become obscured by ground substance, indicating the external mineralization front of dentin (D). Bundled and longitudinally cut fibrils of an advanced fiber fringe, located 200 gm coronal to the advancing root edge (a), are mainly oriented perpendicular to the dentin surface and terminate in the transitional zone. Other fibrils of the inner fringe portion appear either bundled and cross-cut or are arranged irregularly (a). The dentin matrix is obscured at a front almost reaching the DCJ, indicating the advancement of mineralization. D dentin, a x 6700; b x 14400
317
318
319 ented dentinal fibrils. In this transitional zone, the future DCJ developed in an apico-coronal direction. The external dentinal fibrils became increasingly obscured by ground substance, indicating that the external mineralization front progressively approached the DCJ. These developmental features are illustrated in Figs. 3 b and 5 b. In the middle area outlined in Fig. 3 b, located about 100 pm coronal to the ARE, fringe fibers at the root surface were rarely seen at the light-microscopic level. Adjacent cells were indistinct. Ultrastructurally (Fig. 5 b), the same area revealed an early developmental stage o f fiber formation: bundles o f collagen fibrils were densely packed in parallel arrays but these fibers were separated from each other by the cytoplasm of adjacent connective tissue cells and appeared in cross-, oblique- and longitudinal-planes of sectioning. Near the dentinal matrix, longitudinally cut collagen fibrils of the fiber fringe were oriented mainly perpendicular to and terminated at the external dentin surface. About 200 gm coronal to the ARE, the upper outlined area in Fig. 3b shows an advanced fiber fringe. Bundled and longitudinally cut fibrils of this fringe were oriented mainly perpendicular to the dentin surface and terminated in the transitional zone (DCJ). Other fibrils of the inner fringe portion appeared either bundled and cross-cut or were arranged irregularly (Fig. 5 a). The front of the obscured dentinal matrix reached almost to the DCJ. Coronal to the above described region, a well-developed and established cell-fiber fringe meshwork was encountered (Figs. 6a, 7a). The overall character of this network did not differ significantly along the root surface: the enmeshed cells varied in shape, ranging from elongated to ovoid or round (Figs. 6a, b and 7a, b). They had a voluminous, organelle-rich cytoplasm, although their rough endoplasmic reticulum was less dense than that in more apically located cells (Figs. 6b, c and 7b, c). Glycogen storage granules were regularly seen. All enmeshed connective tissue cells contained an euchromatin-rich nucleus with a prominent, about 50-nmthick, fibrous lamina (Fig. 6, inset). The fiber fringe, enmeshing these cells, appeared variably dense in the light microscope (Figs. 6a, 7a). Some
of its fibers continued for a short distance towards the periodontal ligament tissue. A series of light-microscopic sections (Fig. 8 a-j) revealed, at least in part, the threedimensional arrangement o f the cell-fiber fringe meshwork. This particular region, illustrated in Fig. 8, was located about i mm coronal to the ARE. The fiber fringe consisted of 10 to 15-gm-long and variably thick fibers oriented vertical to the dentinal surface, and of short fiber stubs, with fibers and fiber stubs occurring side by side or in an irregular sequence. The latter was associated with the occurrence and shape of the enmeshed cells (Fig. 8a, d, f). Following a particular, well demarcated fiber (Fig. 8a, arrowhead) through a series of 10 sections (Fig. 8a-j), it became clear that fiber stubs were the consequence of a particular cell, or a group of cells, being located close to the base of the fiber fringe (Fig. 8 d h). In front (Fig. 8 ~ c , arrowhead) and behind such a cell (Fig. 8 h @ double arrowhead), one or two different fibers could be detected. This pattern was seen more clearly in the electron microscope (Figs. 6b, c and 7b, c). Fiber stubs appeared most clearly in the more coronal regions. Their density was at its maximum, i.e., at the base of the fiber fringe, fiber stubs were packed one against the next (Fig. 7 b). Whereas the collagen fibrils of these stubs began their course mostly vertical to the root surface, they soon deviated from this course: the collagen fibrils thus appeared in a longitudinal, oblique or cross plane of sectioning (Figs. 6c, 7b, c). Immediately on top of these fiber stubs, large cells were located (Figs. 6 b, c and 7 b, c). At their root-related termination, longitudinally cut fibers and fiber stubs fanned out into single collagen fibrils or groups of fibrils, which were oriented vertical to the dentinal surface (Figs. 6c, 7c, insets). These fibrils intermingled with the collagen fibrils of the dentinal matrix, as seen prior to mineralization of this region. The front o f mineralization appeared irregular in outline, and formed the border of a heavily contrasted and obscured dentinal matrix (Figs. 6c, 7b, c). In an apico-coronal direction, this front was seen first near to (Fig. 6) and later, i.e. more coronally, at (Fig. 7) the base of the fiber fringe.
Cell and Tissue Research 9 Springer-Verlag 1991
Initiation of acellular extrinsic fiber cementum on human teeth A light- and e l e c t r o n - m i c r o s c o p i c study Dieter D. Bosshardt and Hubert E. Schroeder Department of Oral Structural Biology, Dental Institute, University of Zurich, Plattenstrasse 11, CH-8028 Zurich, Switzerland Accepted September 21, 1990
Summary. The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of A E F C in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 5 0 % - 6 0 % of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in E D T A and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about i mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 gm from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 ~tm from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process o f dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization. D. Bosshardt Abbreviations: A E F C acellular extrinsic fiber cementum; A I F C acellular intrinsic fiber cementum ; CIFC cellular intrinsic fiber cementum; C M S C cellular mixed stratified cementum; A R E advancing root edge; CP cytoplasmic process; D dentin; D C J dentinocemental junction; E enamel; E B L external basal lamina; EC epithelial cell; ED TA ethylene diaminetetraacetic acid; E R M epithelial rests of Malassez; F F fiber fringe; H R S Hertwig's epithelial root sheath; IBL internal basal lamina; M D mineralized dentin; N M D non-mineralized dentin; OB odontoblast; PD predentin; P L periodontal ligament
Key words: Dental root surface - Periodontal fiber fringe - Dentino-cemental junction - Electron microscopy Human
In human teeth, cementum covers the entire surface of the roots, increases in thickness with age, and serves in fully erupted and functioning teeth for the attachment of the principal periodontal ligament fiber bundles, which run from alveolar bone to cementum. The two major types of human root cementum are acellular extrinsic fiber cementum (AEFC) and cellular mixed stratified cementum (CMSC). A E F C is found primarily on the cervical and middle third o f the root, whereas CMSC occurs primarily in the apical third of the root and the furcations (Schroeder 1986, 1988). The genesis of both types of cementum on growing roots of human teeth is unknown: only two investigations, one microradiographic (Owens 1976) and one light- and electron-microscopic study (Bosshardt and Schroeder 1990) have dealt with particular aspects of human cementogenesis. However, to our knowledge, there is not a single study describing the genesis of A E F C in man. On the other hand, there are numerous
Offprint requests to." D.
Fig. 1a--c. Examples of X-ray photographs of a maxillary first (a), a mandibular first (b), and a mandibular second (e) premolar showing roots developed to 50% (a, e) and 60% (b) of their final length. a--c x2.5
b.~
313 studies d e a l i n g with established c e m e n t u m o n fully f o r m e d roots. F o r example, the structure o f A E F C has been e x a m i n e d light- a n d e l e c t r o n - m i c r o s c o p i c a l l y in hum a n d e c i d u o u s (Keller 1964; F u r s e t h 1967) a n d p e r m a n e n t (Dewey 1926; K r o n f e l d 1928; Schmid 1951; Herting 1962, 1964; Selvig 1965, 1967; F u r s e t h 1974) teeth. I n a d d i t i o n , m i c r o r a d i o g r a p h i c i n v e s t i g a t i o n s (Soni et al. 1962; Dreyfuss a n d F r a n k 1964; Selvig 1965; F u r s e t h 1967; O w e n s 1976) o n fully developed h u m a n teeth have revealed h e t e r o g e n e o u s calcification o f the d e n t i n o - c e m e n t a l j u n c t i o n (DCJ). O n a c c o u n t o f the absence of i n f o r m a t i o n o n the genesis o f A E F C o n h u m a n teeth, studies were initiated to describe its initial f o r m a t i o n in the p r e f u n c t i o n a l phase. I n p a r t i c u l a r , it was necessary to clarify the p r o b lems o f the initial genesis o f p e r i o d o n t a l fibers a n d their a t t a c h m e n t to the d e n t i n a l surface, i.e,, the d e v e l o p m e n t of the DCJ.
Materials and methods Ten premolars (6 maxillary and 4 mandibular, 7 first and 3 second), extracted for orthodontic reasons from 7 girls and 3 boys, 9-111/2 years of age, were selected from a larger collection of freshly extracted human teeth and processed for light- and electronmicroscopic study. The selected teeth presented incomplete roots developed to 50%-60% of their final length. These premolars were processed with the intention of studying the initiation, establishment and further growth of AEFC. Its structural aspects, attachment to dentin and association with the fibers of Sharpey were also examined. Immediately after extraction, the teeth were fixed in halfstrength Karnovsky's fixative (Karnovsky 1965), buffered (pH 7.4) with 0.02 M sodium cacodylate for 24-48 h. Thereafter, the teeth were briefly washed in 0.185 M sodium cacodylate buffer (pH 7.4; 350 mOsm) and exposed to a decalcifying solution containing 0.15 M EDTA, 2.5% glutaraldehyde with or without supplementation with 0.2 M sucrose. Decalcification was achieved at room temperature over 4-8 weeks, under constant stirring. The solution was changed twice weekly. Following decalcification, slices cut vertical to the root surface in a corono-apical direction, were severed with the use of razor blades from the teeth at mesial and distal sites. The slices were about 4 mm in length and included either the apical or the cervical portion of the teeth. This resulted in about 10 slices per tooth. These slices were briefly washed in 0.185 M sodium
cacodylate buffer and postfixed in 1.33% OsO4, buffered in 0.067 M S-collidine, for 2 h. Thereafter, the slices were washed again in buffer, dehydrated in ethanol and embedded in Epon (Luft 1961). From each block, 1 to 2-pro-thick sections were cut using glass knives and a Reichert OMU-2 ultramicrotome. These sections were stained with a combination of PAS and a mixture of methyleneblue/Azure II (Schroeder et al. 1980) and served for light-microscopic evaluation, photography and sample selection. Light micrographs were obtained using a Leitz Orthoplan/Orthomat equipment. Sample sites for electron-microscopic study were (1) root surface areas either close to or remote (up to 4-5 mm) from the advancing edge of the root, and (2) root surface areas close to the cemento-enameljuntion. From the selected areas, ultrathin sections (60-80 nm) were cut, using a diamond knife and an LKB-Ultrotome III, and contrasted with uranyI acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Electron-microscopic examination and recording were performed with a Philips 201 transmission electron microscope. The investigated teeth were extracted prior to or during their emergence into the oral cavity. In particular, three premolars (Fig. 1) with the best possible degree of tissue preservation were selected for an in-depth study with light- and electron-microsocpic techniques. This selection was made for the following reasons: (1) Fully developed roots of human premolars are covered by pure AEFC for 60%-80% of their length, as measured from the cemento-enamel junction (Schroeder 1988). This applies to all aspects of their roots, i.e., buccal, mesial, oral and distal sites. (2) The initiation and early establishment of pure AEFC follow an apicocoronal gradient, with matrix production starting near the advancing root edge. Because of these features, it was reasonable to assume that no other type of cementum but AEFC would develop along the entire length of the selected root surfaces. The total number of such blocks investigated was 120. All abreviations used in this paper are explained on the title-page.
Results The degree o f tissue p r e s e r v a t i o n varied c o n s i d e r a b l y a n d u n p r e d i c t a b l y , because of e x t r a c t i o n t r a u m a . U n suitable blocks, as j u d g e d f r o m light-microscopic sections, were discarded. I n general, all l i g h t - m i c r o s c o p i c sections f r o m sufficiently well preserved blocks revealed a basic p a t t e r n of d e v e l o p m e n t a l features: Hertwig's epithelial r o o t sheath, a l t h o u g h associated with the edge of the a d v a n c i n g root, was never seen to follow the external surface o f the r o o t d e n t i n for m o r e t h a n a few gm. Instead, the m o s t apical p a r t (i.e., a l o n g 100 g m c o r o n a l
Fig. 2 a-c. Light-(a) and electron-(b,c) microscopic views of the mesial, apical portion of a mandibular second premolar (with a root developed to 50% of its final length), extracted from an 11-year-old girl. The area outlined in a corresponds to b, and that in b corresponds to c. The region encircled in c is shown in the inset. At the light-microscopic level, intensely basophilic cells are seen closely abutting the external dentinal surface (a) immediately coronally to the advancing root edge (ARE). At a distance of about 200 pm coronal to the ARE, a fiber fringe (FF, arrowheads in a) oriented perpendicular to the root surface is apparent. Electron-microscopically, the cells lining the external and apical dentin surface reveal an elongated fibroblast-like appearance (b, c). Between these cells and the dentin matrix, collagen fibrils are demarcated from the randomly oriented dentinal fibrils by their parallel arrangement and appearance in either cross- (c, arrowheads and inset) or longitudinal (c, arrows) planes of sectioning. The latter are oriented approximately perpendicular to the dentinal surface and extend toward the adjacent cells (c, arrows). D dentin; PD not yet calcified predentin; PL periodontal ligament, a • 580, b x 3015, c x 6700; inset: • 14400
Fig. 3. Light-(a,b) and electron-microscopic (c) views of the advancing root edge (ARE) at the distal site of a mandibular first premolar (with a root developed to 60% of its final length), extracted from an 11 1/2-year-old girl. The area outlined in a corresponds to b, the lower area outlined in h corresponds to c. Note the presence of a fiber fringe (FF, arrowheads in a, b), oriented perpendicular to the root surface and beginning about 100 gm coronal to the ARE. Within the apical 100 pm, the space between the remnants of Hertwig's root sheath (*) and the dentinal root surface is populated with intensely stained, basophilic cells (a, b). These elongated and fibroblast-like cells are in close contact with the external dentin matrix (b, c). Note that all these cells are similar morphologically, with a typical nuclear fibrous lamina (inset), and are in contact with one another. The external predentin matrix is still non-mineralized (c). D dentin; PD predentin; EC epithelial cell; IBL/EBL internal and external basal lamina; OB odontoblasts ; PL periodontal ligament, a x580, b x900, c x3015; inset: x62500
314
F~.3.
315
Fig. 4.
316 to the advancing root edge) of this surface was covered by intensely basophilic and heavily contrasted cells, populating a space between the external dentinal surface and Hertwig's root sheath, and closely abutting the notyet mineralized dentinal matrix (Fig. 9). Further coronally, within another 100 gm from the root edge, a fiber fringe was seen light-microscopically; this covered the external root surface and became progressively more distinct. It eventually extended to the cemento-enamel junct i o n and, in more cervical root regions, became part of the first, very thin layer of AEFC. The following observations are devoted to the events occurring within a distance of about I m m coronal to the advancing root edge (ARE). Along the first 100 gm coronal to the A R E (Fig. 9), the external surface o f root dentin, still non-mineralized, was covered by a dense population of intensely stained, basophilic cells (Figs. 2 a, 3 a, b). These cells populated the space between the external dentinal surface and Hertwig's root sheath (Fig. 3c). Ultrastructurally, they were similar morphologically. They had an elongated, sometimes slender shape (Figs. 2b, 3c) and a fine-structural appearance typical o f fibroblast-like cells, with an abundant basophilic cytoplasm rich in organelles, particularly rough endoplasmic reticulum (Figs. 2c, 4a). In addition, these cells contained small amounts of glycogen storage granules. The nuclei were elaborately convoluted and rather rich in euchromatin, displaying a typical, about 50-nm-thick, fibrous lamina (Fig. 3, inset). These fibroblast-like cells never revealed closely abutting plasma membranes, but were regularly connected to one another by desmosome-like junctions (Fig. 4, insets). The intercellular spaces were sparsely filled with cross-cut collagen fibrils (Figs. 2c, 4a, arrowheads). Cells located immediately coronal to the A R E projected numerous cytoplasmic processes towards the external dentin matrix (Fig. 4a). Occasionally, such processes were found to insert between and to contact the dentinal fibrils (Fig. 4c). The external dentinal matrix was still nonmineralized and revealed an uniform appearance of randomly oriented collagen fibrils (Figs. 2c, 3c, 4a, c). Between the latter and the closely abutting fibroblast-like cells, a moderate developmental gradient o f tiny bundles of collagen fibrils was apparent. This gradient increased in the apico-coronal direction. The initially bundled fibrils were seen adjacent to the fibrils of the external
Fig. 5a, b. Electron-microscopic views of the upper (a) and the middle (b) area outlined in Fig. 3b. The middle area (b), 100 gm coronal to the advancing root edge, shows bundles of collagen fibrils that are densely packed in parallel arrays, and oriented perpendicular to the root surface. These fibers are separated from each other by the cytoplasm of adjacent connective tissue cells and appear in cross-, oblique- and longitudinal-planes of sectioning. They terminate at the external dentin surface and intermingle with the randomly oriented fibrils of the still non-calcified predentin. This transitional zone of fibril-interdigitation determines the future dentino-cemental junction (DC3). A few lam pulpwards from this zone, the dentinal fibrils become obscured by ground substance, indicating the external mineralization front of dentin (D). Bundled and longitudinally cut fibrils of an advanced fiber fringe, located 200 gm coronal to the advancing root edge (a), are mainly oriented perpendicular to the dentin surface and terminate in the transitional zone. Other fibrils of the inner fringe portion appear either bundled and cross-cut or are arranged irregularly (a). The dentin matrix is obscured at a front almost reaching the DCJ, indicating the advancement of mineralization. D dentin, a x 6700; b x 14400
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319 ented dentinal fibrils. In this transitional zone, the future DCJ developed in an apico-coronal direction. The external dentinal fibrils became increasingly obscured by ground substance, indicating that the external mineralization front progressively approached the DCJ. These developmental features are illustrated in Figs. 3 b and 5 b. In the middle area outlined in Fig. 3 b, located about 100 pm coronal to the ARE, fringe fibers at the root surface were rarely seen at the light-microscopic level. Adjacent cells were indistinct. Ultrastructurally (Fig. 5 b), the same area revealed an early developmental stage o f fiber formation: bundles o f collagen fibrils were densely packed in parallel arrays but these fibers were separated from each other by the cytoplasm of adjacent connective tissue cells and appeared in cross-, oblique- and longitudinal-planes of sectioning. Near the dentinal matrix, longitudinally cut collagen fibrils of the fiber fringe were oriented mainly perpendicular to and terminated at the external dentin surface. About 200 gm coronal to the ARE, the upper outlined area in Fig. 3b shows an advanced fiber fringe. Bundled and longitudinally cut fibrils of this fringe were oriented mainly perpendicular to the dentin surface and terminated in the transitional zone (DCJ). Other fibrils of the inner fringe portion appeared either bundled and cross-cut or were arranged irregularly (Fig. 5 a). The front of the obscured dentinal matrix reached almost to the DCJ. Coronal to the above described region, a well-developed and established cell-fiber fringe meshwork was encountered (Figs. 6a, 7a). The overall character of this network did not differ significantly along the root surface: the enmeshed cells varied in shape, ranging from elongated to ovoid or round (Figs. 6a, b and 7a, b). They had a voluminous, organelle-rich cytoplasm, although their rough endoplasmic reticulum was less dense than that in more apically located cells (Figs. 6b, c and 7b, c). Glycogen storage granules were regularly seen. All enmeshed connective tissue cells contained an euchromatin-rich nucleus with a prominent, about 50-nmthick, fibrous lamina (Fig. 6, inset). The fiber fringe, enmeshing these cells, appeared variably dense in the light microscope (Figs. 6a, 7a). Some
of its fibers continued for a short distance towards the periodontal ligament tissue. A series of light-microscopic sections (Fig. 8 a-j) revealed, at least in part, the threedimensional arrangement o f the cell-fiber fringe meshwork. This particular region, illustrated in Fig. 8, was located about i mm coronal to the ARE. The fiber fringe consisted of 10 to 15-gm-long and variably thick fibers oriented vertical to the dentinal surface, and of short fiber stubs, with fibers and fiber stubs occurring side by side or in an irregular sequence. The latter was associated with the occurrence and shape of the enmeshed cells (Fig. 8a, d, f). Following a particular, well demarcated fiber (Fig. 8a, arrowhead) through a series of 10 sections (Fig. 8a-j), it became clear that fiber stubs were the consequence of a particular cell, or a group of cells, being located close to the base of the fiber fringe (Fig. 8 d h). In front (Fig. 8 ~ c , arrowhead) and behind such a cell (Fig. 8 h @ double arrowhead), one or two different fibers could be detected. This pattern was seen more clearly in the electron microscope (Figs. 6b, c and 7b, c). Fiber stubs appeared most clearly in the more coronal regions. Their density was at its maximum, i.e., at the base of the fiber fringe, fiber stubs were packed one against the next (Fig. 7 b). Whereas the collagen fibrils of these stubs began their course mostly vertical to the root surface, they soon deviated from this course: the collagen fibrils thus appeared in a longitudinal, oblique or cross plane of sectioning (Figs. 6c, 7b, c). Immediately on top of these fiber stubs, large cells were located (Figs. 6 b, c and 7 b, c). At their root-related termination, longitudinally cut fibers and fiber stubs fanned out into single collagen fibrils or groups of fibrils, which were oriented vertical to the dentinal surface (Figs. 6c, 7c, insets). These fibrils intermingled with the collagen fibrils of the dentinal matrix, as seen prior to mineralization of this region. The front o f mineralization appeared irregular in outline, and formed the border of a heavily contrasted and obscured dentinal matrix (Figs. 6c, 7b, c). In an apico-coronal direction, this front was seen first near to (Fig. 6) and later, i.e. more coronally, at (Fig. 7) the base of the fiber fringe.
