Cell Tissue Res (1992) 267:321 335
Cell kTissue Research 9 Springer-Verlag 1992
Initial formation of cellular intrinsic fiber cementum in developing human teeth A light- and electron-microscopic study Dieter D. Bosshardt and Hubert E. Schroeder Department of Oral Structural Biology,Dental Institute, Universityof Zfirich, Plattenstrasse 11, CH-8028 Zfirich,Switzerland Received April I5, 1991 / Accepted September 2, 1991
Summary. The present study describes the formative process of the initiation of cellular intrinsic fiber cementum (CIFC) in still growing human teeth. From 29 premolars and molars with incomplete roots developed to 60-90% of their final length, 8 premolars (with roots formed to three quarters of their final length) were selected for electron-microscopic investigation. All teeth were clinically intact and prefixed in Karnovsky's fixative immediately after extraction. Most of them were decalcified in ethylene diaminetetraacetic acid (EDTA), and the apical part of the roots was divided axially into mesial and distal portions that were subdivided in about 5 slices each. Following osmication and embedding in Epon, these blocks were cut for light- and electron-microscopic examination. In addition, 5 teeth with incomplete roots were freed from organic material and processed for scanning electron microscopy. It was found that CIFC-initiation commenced very close to the advancing root edge and resulted in a rapid cementum thickening. Thereafter, appositional growth continued on the already established cementum surface. Large, basophilic and rough endoplasmic reticulum-rich cementoblasts, some of which became cementocytes, were responsible for both fast and slow CIFC-formation. The CIFC-matrix was free of Sharpey's fibers and composed of more or less organized intrinsic collagen fibrils, in part fibril bundles, that ran roughly parallel to the root surface. Initially, the cementum fibrils intermingled with those of the dentinal collagen fibrils, which were not yet mineralized. This boundary subsequently underwent calcification. The development of collagen fibril bundles and their extracellular arrangement were associated with cytoplasmic processes probably involved in fibril formation and fibril assembly. Many cementoblasts contained intracytoplasmic, membrane-bounded collagen fibrils, which probably were related to fibril formation rather than degradation. Key words: Teeth Cementum-Cementoblasts Matrix production - Electron microscopy - Human Offprint requests to: D.D. Bosshardt
In fully erupted and functioning human teeth, the roots are entirely covered by cementum, which among other functions serves for the attachment of principal periodontal ligament fibers that combine root with bone. The two major types of human root cementum are acellular extrinsic fiber cementum (AEFC) and cellular mixed stratified cementum (CMSC). AEFC is found primarily on the cervical and middle third of the roots. Initiation and early establishment of AEFC occur mainly during tooth eruption, i.e., prior to the organization of the tooth-supporting fiber apparatus and before any other type of cementum is being formed (Bosshardt and Schroeder 1991 a, b). The apical third of human roots and the furcations are covered by CMSC. This is a heterogeneous tissue composed of up to 3 different types of root cementum, i.e., alternating or merging layers of cellular intrinsic fiber cementum (CIFC), acellular intrinsic fiber cementum (AIFC) and AEFC. CIFC (containing cementocytes) and AIFC, both being composed entirely of intrinsic fibers, result from two fundamentally different modes of cementum matrix production (Bosshardt and Schroeder 1990): CIFC is formed by a multipolar, AIFC by a unipolar mode. It is safe to say that the multipolar mode is a rapid whereas the unipolar mode is a slow-rate procedure. Previous light- and electron-microscopic studies dealing with CIFC on human teeth described either the arrangement and structure of matrix fibers (Herring 1962; Dreyfuss and Frank 1964; Selvig 1965) or the fine structure of its cellular elements, i.e., cementoblasts and cementocytes (Furseth 1967, 1969). Apart from the preliminary study mentioned above (Bosshardt and Schroeder 1990), no studies exist on the formal genesis of CIFC on human teeth. In this report, observations on CIFC matrix production were restricted to the apical regions of still-growing human teeth, where CIFC-formation was seen exclusively at or near the advancing root edge. CIFC matrix production comprised the initial collagen fibril formation on the newly formed dentinal matrix, i.e., the development of the dentino-cemental junction, and the subse-
322 treated for 8 10 h with 5% sodium hypochlorite at room temperature, thus removing the organic material adherent to their surfaces. Following a rinse in tap water, teeth were dehydrated in ethanol and, for 3 times 1 h, incubated in Freon 113 (Du Pont, Dordrecht, The Netherlands). The specimens were air-dried in a dessicator containing silica-gel. The dried teeth were mounted on metal stubs and sputter-coated with 20-30 nm of gold (Edwards, S-150B). SEM-examination was carried out with a Cambridge Stereoscan S-180 at 12 kV.
Results
Fig. 1 a-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 75% of their final length, a-e x 2.5
q u e n t a d d i t i o n o f c e m e n t u m m a t r i x to an a l r e a d y established layer o f C I F C .
Materials and methods From a large collection of freshly extracted and healthy human teeth removed for orthodontic and surgical reasons, 21 premolars (12 maxillary and 9 mandibular, 13 first and 8 second, from 7 girls and 14 boys, 9 to 14 years of age) and 8 third molars (4 maxillary and 4 mandibular, from 4 females and 4 males, about 20 years of age) were selected as being suitable for studying the initiation and early establishment of CIFC. These 29 teeth presented with incomplete roots developed to 60-90% of their final length and were processed for light- and electron-microscopic investigations as described previously (Bosshardt and Schroeder 1991a). Following decalcification in ethylene diaminetetraacetic acid (EDTA), the apical part, i.e., 4-5 mm of the incomplete root, was severed from each tooth with the use of razor blades. Such parts of premolars were separated into mesial and distal portions, and these were subdivided in 5 slices each, cut vertical to the root surface in a corono-apical direction. Slices from third molars, cut in the same direction as in premolars, originated from various aspects around the incomplete roots. From the resulting 157 demineralized Epon blocks, 1-2 gm thick sections were cut using a diamond knife (Diatome, Biel, Switzerland) and a Reichert OMU-2 ultramicrotome. Sections were stained with a combination of PAS and a mixture of methylene-blue/Azure II (Schroeder et al. 1980) and served for light-microscopic evaluation and sample selection. Light micrographs were obtained using a Leitz Orthoplan/Orthomat microscope. Unsuitable blocks showing less than optimal tissue preservation were discarded. From the remaining material, 22 blocks originating from the mesial and distal aspects of 8 premolars [with roots (Fig. 1) formed to three quarters of their final length] were selected and prepared for electron microscopy. Ultrathin sections (about 80 nm thick) cut with a diamond knife (Diatome, Biel, Switzerland) and an LKB-Ultrotome III, were contrasted with uranyl acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Examination and recording was carried out with a Philips 201 transmission electron microscope. In addition, 5 premolars and molars demonstrating incomplete root development were processed for scanning electron-microscopic examination. Immediately after extraction, such teeth were fixed in half-strength Karnovsky's (Karnovsky 1965) fixative for 24 h. Thereafter, the teeth were washed in 0.185 M sodium cacodylate buffer and stored in the same solution at 4~ C. Teeth were then
In the selected g r o u p o f h u m a n p r e m o l a r s (with r o o t s d e v e l o p e d to 7 5 % o f their final length) active m a t r i x f o r m a t i o n o f cellular intrinsic fiber c e m e n t u m ( C I F C ) was seen exclusively at the a d v a n c i n g r o o t edge ( A R E ) , p a r t i c u l a r l y in the slight concavities o f the mesial a n d distal r o o t aspects. A t these sites, newly p r o d u c e d collagen fibrils were o b s e r v e d directly o n recently f o r m e d dentin, i.e., at o r n e a r the A R E . F o l l o w i n g this initiation, m a t r i x p r o d u c t i o n c o n t i n u e d on the surface o f the already formed and more coronally established cementum layer. In b o t h these regions, fibers o f S h a r p e y were absent, i.e., the c e m e n t u m was c o m p o s e d entirely o f intrinsic fibers t h a t r e m a i n e d w i t h i n the c e m e n t u m m a t r i x . I n i t i a t i o n o f C I F C was o b s e r v e d a l o n g the first 1 0 0 200 ~tm c o r o n a l to the A R E . C o l l a g e n fibril a s s e m b l y was seen a g a i n s t the surface o f recently f o r m e d d e n t i n as well as b e t w e e n c e m e n t o b l a s t s . T h e C I F C - m a t r i x increased r a p i d l y in thickness in the a p i c o - c o r o n a l direction. T h e e n d - p o i n t level o f this r a p i d t h i c k e n i n g corres p o n d e d a p p r o x i m a t e l y to t h a t o f the m i n e r a l i z e d edge o f the g r o w i n g r o o t (Fig. 2c). A l o n g this edge, the inorganic C I F C , w h e n seen f r o m outside, s h o w e d n u m e r o u s l a c u n a e o f c e m e n t o c y t e s (Fig. 2a). C I F C - i n i t i a t i o n c o m m e n c e d a l m o s t d i r e c t l y at the A R E : H e r t w i g ' s r o o t s h e a t h ( H R S ) was a s s o c i a t e d with the A R E for o n l y a few g m a n d , thereafter, c o n t i n u e d c o r o n a l l y in increasing d i s t a n c e f r o m the d e n t i n a l r o o t surface (Fig. 2 b , c). T h e resulting space b e t w e e n the d e v i a t i n g H R S a n d the e x t e r n a l d e n t i n a l m a t r i x was densely p o p u l a t e d b y cem e n t o b l a s t s (Figs. 2 b , c, 3a, 4a). These b a s o p h i l i c cem e n t o b l a s t s (Fig. 2c) were large a n d v a r i a b l e in shape.
Fig. 2. Light- (c) and electron-microscopic (b) views of the advancing root edge (ARE) at the distal aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. The area outlined in e corresponds to b. Note the accumulation of cementoblasts (CB) in the space between Hertwig's root sheath (HRS) and the not yet mineralized external dentinal surface (e) and their close contact with the dentinal matrix fibrils, immediately at the ARE (b). The cementummatrix, i.e., cellular intrinsic fiber cementum (CIFC) increases rapidly in thickness and CBs become cementocytes (CC in c). The dentinal mineralization front is continuous with that of cementum (arrowheads in e). In the scanning electron microscope (a), the external aspect of the inorganic root surface along the mineralized edge of a growing premolar is completely covered by CIFC, showing numerous CC-lacunae. The interrupted line in c corresponds to the dentino-cemental junction. D Dentin; OB odontoblasts; PD predentin; PL periodontal ligament, a x 350, b • 2100, c x 570
323
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325 Their most characteristic feature was the abundant cytoplasm rich in cytoplasmic organelles, particularly rough endoplasmic reticulum (Figs. 3a, b, 4a). Their nuclei were euchromatin-rich (Figs. 2b, 3a, 4a). Cells located immediately coronal to the A R E were occasionally seen to possess numerous long cytoplasmic extensions pointing towards the external dentinal matrix (Fig. 3 a). Directly coronal to the interdigitation of these extensions with the newly formed dentinal collagen fibrils, cementoblasts formed an almost continuous matrix layer covering the external root surface (Figs. 2a, 3a). At the root-related cementoblast-matrix interface, these cementoblasts formed numerous, slender and fingerlike cytoplasmic processes, which interdigitated with collagen fibrils of the initially produced C I F C - m a t r i x (Fig. 3c, CP1, inset in Fig. 4a). Neither these cytoplasmic processes nor the collagen fibrils reflected a favored orientation. The initially formed CIFC-fibrils interdigitated with the collagen fibrils of the still non-mineralized dentinal matrix. In this transitional zone, the future dentinocemental junction (DCJ) developed. With increasing distance from the A R E , the C I F C - m a t r i x increased in thickness and, as a result, cementoblasts became separated from the D C J (see interrupted line in Fig. 5a-c). Occasionally, discrete bundles of cemental collagen fibrils were clearly demarcated f r o m the randomly oriented dentinal collagen fibrils (Fig. 5a). Consequently, the demarcation of C I F C from predentin varied in distinctiveness and was mainly based on structural differences between the more r a n d o m fibril orientation and the homogeneous pattern of the dentinal matrix and the loosely arranged or partly oriented C I F C - m a t r i x elements (Fig. 5 ~ c ) . Concurrently with the appearance of C I F C - m a t r i x fibrils at the root-related cementoblast-surface, a matrix rich in collagen fibrils was observed on the opposite side of the cementoblasts, thus enclosing these cells (Fig. 3 b). Neighbouring cementoblasts were therefore either separated from each other by a narrow intercellular space or by variable amounts of C I F C - m a t r i x (Figs. 3 a, 4a). Collagen fibrils f r o m the central regions of such accumulated masses of C I F C - m a t r i x appeared to be arranged randomly (Fig. 4c), whereas collagen fibrils of
peripheral regions were loosely bundled (Fig. 4b). These fibril aggregates were located in indentations of the cementoblast-surface, which occurred at various sites around the cementoblasts (arrowheads in Fig. 4a). The aggregated fibrils demonstrated great variation in diameter. At the aggregate periphery, fibrils were intimately associated with narrow cytoplasmic processes cut in the same cross-sectional plane as the surrounding extracellular collagen fibrils (Fig. 4b, CP1). Following this initial and rapid CIFC-production, the cementum layer began to establish itself (Fig. 2c). Cementocytes became embedded in the C I F C - m a t r i x in varying numbers. They revealed numerous, broadbased, triangular cytoplasmic processes projecting into the surrounding matrix (CP2, insets in Fig. 6, Fig. 7). This C I F C - m a t r i x c o m m o n l y exhibited a heterogeneous pattern composed of randomly oriented collagen fibrils and small bundles of such fibrils cut in cross (stars in Fig. 6), oblique or tangential planes of sectioning (Figs. 6, 8a, b, 9a). In comparison to more apical regions, the cementum matrix was less-well demarcated from the dentinal matrix (Fig. 6). This was particularly true after the D C J had been obscured by the mineralization process. On the established cementum surface, cementoblasts often formed a continuous covering. They were variable in shape (Figs. 6, 8a, b, 9a) and had a fine-structural appearance similar to those cells located more apically, although their rough endoplasmic reticulum appeared
Fig. 3 a-e. Electron-microscopic view (a) of the advancing root edge (ARE) at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. The area outlined in a corresponds to h, that in b to e. Cementoblasts (CB) form a continuous covering on the external root surface, i.e., the predentin (PD in a). Immediately coronal to the ARE, numerous cytoplasmic extensions (CE) project towards the dentinal matrix and penetrate between and contact the dentinaI collagen fibrils (inset in a). About 50 gm from the ARE, a particular CB is surrounded by CIFC (b). The root-related CB-matrix interface shows numerous slender and fingerlike cytoplasmic processes (CP~) originating from the CB surface and interdigitating with collagen fibrils of the cementum matrix (e). Neither these CPs nor collagen fibrils are preferably oriented. The interrupted line in b corresponds to the dentino-cemental junction (DCJ). a x 3000, b • e • 6700
Fig. 5. The photomicrographs in a-c were aligned to allow the dentino-cemental junction (interrupted line) to form a straight Dine. With increasing distance from the advancing root edge (a=40 pm, b=60 pm, e=100 gm), the initially formed matrix of cellular intrinsic fiber cementum (CIFC) increases rapidly in thickness in the apico-coronal direction. The collagen fibrils of CIFC-matrix show an irregular orientation (a-e) or are occasionally bundled (a). Consequently, these fibrils are more or less clearly demarcated from the randomly oriented collagen fibrils of the not yet mineralized dentinal matrix (PD). In more coronal root regions, i.e., on established CIFC-surfaces, cementoblasts (CB) reveal many surface indentations that contain cross-sectional, slender cytoplasmic processes (CP~) and engulf cross-sectioned collagen fibrils of homogeneous diameter (d, e). Longitudinally cut collagen fibrils appear to radiate perpendicularly from the CBs plasma membrane (f). PD Predentin. a-c x 8000, d-f x 27000
Fig. 4a-e. Electron-microscopic view (a) of the external root surface at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2year-old boy. The left lower area outlined in a corresponds to b, the left upper area in a to c, and the right rectangular area in a to the left inset in a. Cementoblasts (CB) located approximately 50 btm from the advancing root edge (ARE), show well-developed rough endoplasmic reticulum (a). Numerous surface indentations are seen at various sites around the CB's (arrowheads in a). Surface recesses surround bundles of cross-sectioned collagen fibrils in intimate association with slender and fingerlike cytoplasmic processes (CPI in b). These fibrils are very heterogeneous in diameter (b). Collagen fibrils from central regions of CIFC-matrix show no preferable orientation (e). Towards the dentino-cemental junction (DCJ, interrupted line), many slender CPs originating from the CB surface interdigitate with the collagen fibrils of CIFC (CP~, inset in a). PD Predentin. a x 5400, b, e x 27000; inset: x 21000
326
Fig. 4 a - c
327
Fig. 5 a - f
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329
Fig. 7. Large cementocyte not distant from the cementum surface and near the advancing root edge, embedded in cementoid. This cell revealing well-developed rough endoplasmic reticulum, shows
two broad-based cytoplasmic processes (CP2) extending into the mineralized matrix of cellular intrinsic fiber cementum (CIFC). • 9000
less dense. Their nuclei were euchromatin-rich. These cementoblasts showed irregular profiles along the cementum surface, caused by numerous surface indentations (arrowheads in Figs. 6 and 8 a, b). The latter were filled with cross-cut and bundled collagen fibrils that were surrounded, in part, by variably long, broad-based cytoplasmic processes (CP2, Fig. 5d, e). These surface indentations intimately engulfed cross-sectioned collagen fibrils of similar diameter as well as cross-sectioned, narrow and fingerlike cytoplasmic processes distributed among the collagen fibrils (CP1, Fig. 5d, e; inset in Fig. 8). In tangential planes of sectioning, collagen fibrils 9 appeared to radiate perpendicularly from the cytoplas-
mic membrane of cementoblasts (Fig. 5f). Frequently, the slender and fingerlike cytoplasmic processes (Fig. 9 e, f and large inset) and their cytoplasmic bases (Fig. 9 b) enclosed one (Fig. 9e, f a n d large inset) or more (Fig. 9b) collagen fibrils. Such collagen fibrils were also seen within the cementoblast cytoplasm in close proximity to the plasma membrane (small inset in Fig. 9). Both collagen fibrils within slender cytoplasmic processes and those located near the cell periphery were enclosed in membrane-bounded compartments and were oriented parallel to the bundles of extracellular collagen fibrils. The cross-sectional diameter of the membrane-bounded collagen fibrils and their electron density were comparable to that of extracellular collagen fibrils. In more central portions of the cementoblast cytoplasm, single or small groups of collagen fibrils were observed in both longitudinal and cross-sectional planes of sectioning (Fig. 9c, d). A limiting membrane was usually observed between these fibrils and the cytoplasm, suggesting that such collagen fibrils were also enclosed within a membranebounded compartment. The latter appeared electron-lucent and, as seen in longitudinal sections, formed large intracytoplasmic channels (Fig. 9d). The fibrillar content of these channels was morphologically similar to the collagen fibrils in the extracellular space, showing a typical 64-nm pattern. Occasionally, intracytoplasmic channels contained moderately dense spherules along the course of the fibrils (Fig. 9d). Varicosities occurring
Fig. 6. Electron-microscopic view of the established, 30-50 gm-
thick, layer of cellular intrinsic fiber cementum (C1FC) that originated from the distal aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-oldboy. Note that 151~200 pm coronal to the advancing root edge, cementoblasts (CB) form a continuous covering over the established cementum surface. They show an irregular profile along the cementum-matrix, caused by numerous cementoblastsurface indentations (arrowheads). Cementocytes (CC and insets) are embedded in the CIFC-matrix. The CCs have numerous broadbased cytoplasmic processes (CP2) and are completely surrounded by collagen fibrils (insets). The matrix of CIFC is not well demarcated from the dentinal matrix (PD). PD Predentin. • 3000; small inset: x 5600, large inset: • 6700
330
331 along the course of the membrane profiles and amorphous, electron-dense material suggestive of collagenolytic activity were not observed within these intracytoplasmic (collagen-containing) compartments. Intracytoplasmic channels containing collagen fibrils were also observed in perinucelar regions of the cementoblasts (not shown). Cementoblasts containing membrane-bounded collagen fibrils were seen in all parts of developing CIFC. In regions of its initiation, cementoblasts included single fibrils, while those cells covering the established CIFCsurface contained both single and groups of membranebounded collagen fibrils, the latter confined to intracytoplasmic channels.
Discussion
In continuation of two preceding papers describing the initiation and establishment of acellular extrinsic fiber cementum (AEFC) on human teeth (Bosshardt and Schroeder 1991 a, b), the present investigation provides, for the first time, detailed information on how cellular intrinsic fiber cementum (CIFC) develops on growing human roots. The findings can be summarized as follows: (1) Hertwig's root sheath did not cover the newly formed root dentin but immediately deviated from the advancing root edge. Consequently, connective tissue cells had access to the external root surface up to the ARE. (2) CIFC-formation commenced almost directly at the advancing root edge and resulted in a rapid cementum thickening along the first 100-200 gm coronal to this edge. (3) This rapid thickening terminated approximately at level with the mineralized edge of the growing root. However, CIFC continued to grow, although more slowly. (4) Large, basophilic and RER-rich cementoblasts, which rapidly became cementocytes, were responsible for fast CIFC-formation, while further cementum
Fig. 8 a, b. Electron-microscopicviews of the periodontal ligament-
related root surface at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. These regions are located 0.5 mm (a) and 1.0 mm (b) coronal to the advancing root edge. The boundary between cementoblasts (CB) and the established layer of cellular intrinsic fiber cementum (CIFC) shows irregular profiles caused by numerous cross-sectioned cell surface indentations (arrowheads in a, b). These indentations are filled with bundled collagen fibrils that are surrounded, in part, by broad-based cytoplasmic processes. Other very slender processes (CP1, inset) are seen among the bundled collagen fibrils. The latter are newly added to the CIFC-matrix that commonly exhibits a heterogeneous pattern. The mineralization front is uneven, and numerous calcified globules are present in the cementoid (a, b). PL Periodontal ligament, a, b x 3000; inset: x 27000
growth was due to superficial cementoblasts which covered most of the newly established CIFC-surface. (5) The CIFC-matrix was composed entirely of intrinsic collagen fibrils which, at the dentino-cemental junction, initially intermingled with the non-mineralized dentinal collagen fibrils. (6) Developing bundles of collagen fibrils were intimately associated with slender cytoplasmic processes and cementoblast surface indentations. (7) Intracytoplasmic, membrane-bounded collagen fibrils were a common finding within many cementoblasts. Although the light- and electron-microscopic observations were based on demineralized sections only, the front of mineralization could be detected indirectly by noting the light- and ultrastructural changes accompanying calcification. Furthermore, the results described were derived from static observations. Unlike AEFC, which is formed very regularly and very slowly (Nalbandian 1978; Schroeder 1986; Bosshardt etal. 1989), initial CIFC-formation is a very irregular and a rapid process (Bosshardt et al. 1989; Bosshardt and Schroeder 1990). This difference in cementum apposition amounts to the fact that initial CIFC-formation may be up to 30-fold faster than AEFC-formation, as revealed by fluorescence labelling lines in developing deciduous teeth of a non-human primate (Bosshardt et al. 1989). The high rate of apposition renders a developmental description of CIFC, being based on static observations, much more difficult than that of AEFC, the gradual development of which was possible to be shown step-by-step over a long period of time and distance (Bosshardt and Schroeder 199l a, b). The fact that all 8 premolars included in this study were developed to about 75% of their final root length indicates that initial CIFC-formation occurs and is subject to investigation at this particular stage of premolar development. Our findings confirm the high rate of initial CIFCapposition found in the deciduous teeth of a non-human primate (Bosshardt et al. 1989), and also corroborate the assumption of a multipolar mode of CIFC-matrix production observed in human teeth (Bosshardt and Schroeder 1990). In addition, this rapid and multipolar mode of matrix synthesis appears to be the reason for the incorporation of cementocytes, as already suggested by Paynter and Pudy (1958), Formicola et al. (1971) and Bosshardt and Schroeder (1990). A rather dense cementocyte distribution may, therefore, not only be found in the initially formed CIFC-matrix described in this study, but whenever this rapid form of intrinsic fiber cementum formation occurs, e.g., in resorption lacunae. On the other hand, the more coronally located cementoblasts and their cell-matrix interaction resembled the slow, unipolar, acellular intrinsic fiber cementum (A1FC) production described recently (Bosshardt and Schroeder 1990). However, there is still a significant difference between the slow-rate cementum apposition leading to A I F C and the type of cementum formation at the established CIFC-surface as observed in this study: In the latter case, the cementoblasts formed a
332
333 continuous but not strictly unicellular layer on the cementum-surface and the collagenous matrix fibers produced were less well organized. Thus, AIFC probably shows the highest degree of fibril bundle organization, whereas initially produced CIFC-matrix reflects the least degree. However, all cells engaged in CIFC-formation were remarkably large, had euchromatin-rich, i.e., activated nuclei, and revealed a cytoplasmic composition typical for actively protein synthesizing cells (Furseth 1969). Based on their morphological features and their size, these cells are undoubtedly cementoblasts. They are identical with the cementoblasts observed by Furseth (1969) and by Bosshardt and Schroeder (/990) and strongly resemble osteoblasts (Marks and P o p o f f 1988), but they are unlike connective tissue cells (presumably fibroblasts) responsible for AEFC-formation (Beertsen and Everts 1990; Bosshardt and Schroeder 1991a, b). Nevertheless, the mechanism of the initial attachment of matrix fibrils to dentin is the same for both A E F C and CIFC: collagen fibrils of the two populations interdigitate in a transitional zone defining the future dentino-cemental junction before the mineralization front has reached this boundary. Since CIFC is rather similar to bone morphologically, the present observations offer an opportunity to compare the formal genesis of these two tissues. Marks and P o p o f f (1988) stated: "Protein secretion is generally polarized toward the bone surface, but at regular intervals along the surface of newly forming bone an osteoblast will secrete matrix away from the surface, eventually surrounding itself to become an osteocyte". In an electron-microscopic autoradiographic study, Frank and Frank (1969) showed that the osteoblast is a polarized cell with an alternating protein secretory activity, i.e., directed either toward the osteoid layer or to the opposite side. A similar alternating secretory activity might be true for the cementoblast producing CIFC-matrix in a multipolar fashion. Further investigations such as electron-microscopic autoradiography are needed to deter
Cell kTissue Research 9 Springer-Verlag 1992
Initial formation of cellular intrinsic fiber cementum in developing human teeth A light- and electron-microscopic study Dieter D. Bosshardt and Hubert E. Schroeder Department of Oral Structural Biology,Dental Institute, Universityof Zfirich, Plattenstrasse 11, CH-8028 Zfirich,Switzerland Received April I5, 1991 / Accepted September 2, 1991
Summary. The present study describes the formative process of the initiation of cellular intrinsic fiber cementum (CIFC) in still growing human teeth. From 29 premolars and molars with incomplete roots developed to 60-90% of their final length, 8 premolars (with roots formed to three quarters of their final length) were selected for electron-microscopic investigation. All teeth were clinically intact and prefixed in Karnovsky's fixative immediately after extraction. Most of them were decalcified in ethylene diaminetetraacetic acid (EDTA), and the apical part of the roots was divided axially into mesial and distal portions that were subdivided in about 5 slices each. Following osmication and embedding in Epon, these blocks were cut for light- and electron-microscopic examination. In addition, 5 teeth with incomplete roots were freed from organic material and processed for scanning electron microscopy. It was found that CIFC-initiation commenced very close to the advancing root edge and resulted in a rapid cementum thickening. Thereafter, appositional growth continued on the already established cementum surface. Large, basophilic and rough endoplasmic reticulum-rich cementoblasts, some of which became cementocytes, were responsible for both fast and slow CIFC-formation. The CIFC-matrix was free of Sharpey's fibers and composed of more or less organized intrinsic collagen fibrils, in part fibril bundles, that ran roughly parallel to the root surface. Initially, the cementum fibrils intermingled with those of the dentinal collagen fibrils, which were not yet mineralized. This boundary subsequently underwent calcification. The development of collagen fibril bundles and their extracellular arrangement were associated with cytoplasmic processes probably involved in fibril formation and fibril assembly. Many cementoblasts contained intracytoplasmic, membrane-bounded collagen fibrils, which probably were related to fibril formation rather than degradation. Key words: Teeth Cementum-Cementoblasts Matrix production - Electron microscopy - Human Offprint requests to: D.D. Bosshardt
In fully erupted and functioning human teeth, the roots are entirely covered by cementum, which among other functions serves for the attachment of principal periodontal ligament fibers that combine root with bone. The two major types of human root cementum are acellular extrinsic fiber cementum (AEFC) and cellular mixed stratified cementum (CMSC). AEFC is found primarily on the cervical and middle third of the roots. Initiation and early establishment of AEFC occur mainly during tooth eruption, i.e., prior to the organization of the tooth-supporting fiber apparatus and before any other type of cementum is being formed (Bosshardt and Schroeder 1991 a, b). The apical third of human roots and the furcations are covered by CMSC. This is a heterogeneous tissue composed of up to 3 different types of root cementum, i.e., alternating or merging layers of cellular intrinsic fiber cementum (CIFC), acellular intrinsic fiber cementum (AIFC) and AEFC. CIFC (containing cementocytes) and AIFC, both being composed entirely of intrinsic fibers, result from two fundamentally different modes of cementum matrix production (Bosshardt and Schroeder 1990): CIFC is formed by a multipolar, AIFC by a unipolar mode. It is safe to say that the multipolar mode is a rapid whereas the unipolar mode is a slow-rate procedure. Previous light- and electron-microscopic studies dealing with CIFC on human teeth described either the arrangement and structure of matrix fibers (Herring 1962; Dreyfuss and Frank 1964; Selvig 1965) or the fine structure of its cellular elements, i.e., cementoblasts and cementocytes (Furseth 1967, 1969). Apart from the preliminary study mentioned above (Bosshardt and Schroeder 1990), no studies exist on the formal genesis of CIFC on human teeth. In this report, observations on CIFC matrix production were restricted to the apical regions of still-growing human teeth, where CIFC-formation was seen exclusively at or near the advancing root edge. CIFC matrix production comprised the initial collagen fibril formation on the newly formed dentinal matrix, i.e., the development of the dentino-cemental junction, and the subse-
322 treated for 8 10 h with 5% sodium hypochlorite at room temperature, thus removing the organic material adherent to their surfaces. Following a rinse in tap water, teeth were dehydrated in ethanol and, for 3 times 1 h, incubated in Freon 113 (Du Pont, Dordrecht, The Netherlands). The specimens were air-dried in a dessicator containing silica-gel. The dried teeth were mounted on metal stubs and sputter-coated with 20-30 nm of gold (Edwards, S-150B). SEM-examination was carried out with a Cambridge Stereoscan S-180 at 12 kV.
Results
Fig. 1 a-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 75% of their final length, a-e x 2.5
q u e n t a d d i t i o n o f c e m e n t u m m a t r i x to an a l r e a d y established layer o f C I F C .
Materials and methods From a large collection of freshly extracted and healthy human teeth removed for orthodontic and surgical reasons, 21 premolars (12 maxillary and 9 mandibular, 13 first and 8 second, from 7 girls and 14 boys, 9 to 14 years of age) and 8 third molars (4 maxillary and 4 mandibular, from 4 females and 4 males, about 20 years of age) were selected as being suitable for studying the initiation and early establishment of CIFC. These 29 teeth presented with incomplete roots developed to 60-90% of their final length and were processed for light- and electron-microscopic investigations as described previously (Bosshardt and Schroeder 1991a). Following decalcification in ethylene diaminetetraacetic acid (EDTA), the apical part, i.e., 4-5 mm of the incomplete root, was severed from each tooth with the use of razor blades. Such parts of premolars were separated into mesial and distal portions, and these were subdivided in 5 slices each, cut vertical to the root surface in a corono-apical direction. Slices from third molars, cut in the same direction as in premolars, originated from various aspects around the incomplete roots. From the resulting 157 demineralized Epon blocks, 1-2 gm thick sections were cut using a diamond knife (Diatome, Biel, Switzerland) and a Reichert OMU-2 ultramicrotome. Sections were stained with a combination of PAS and a mixture of methylene-blue/Azure II (Schroeder et al. 1980) and served for light-microscopic evaluation and sample selection. Light micrographs were obtained using a Leitz Orthoplan/Orthomat microscope. Unsuitable blocks showing less than optimal tissue preservation were discarded. From the remaining material, 22 blocks originating from the mesial and distal aspects of 8 premolars [with roots (Fig. 1) formed to three quarters of their final length] were selected and prepared for electron microscopy. Ultrathin sections (about 80 nm thick) cut with a diamond knife (Diatome, Biel, Switzerland) and an LKB-Ultrotome III, were contrasted with uranyl acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Examination and recording was carried out with a Philips 201 transmission electron microscope. In addition, 5 premolars and molars demonstrating incomplete root development were processed for scanning electron-microscopic examination. Immediately after extraction, such teeth were fixed in half-strength Karnovsky's (Karnovsky 1965) fixative for 24 h. Thereafter, the teeth were washed in 0.185 M sodium cacodylate buffer and stored in the same solution at 4~ C. Teeth were then
In the selected g r o u p o f h u m a n p r e m o l a r s (with r o o t s d e v e l o p e d to 7 5 % o f their final length) active m a t r i x f o r m a t i o n o f cellular intrinsic fiber c e m e n t u m ( C I F C ) was seen exclusively at the a d v a n c i n g r o o t edge ( A R E ) , p a r t i c u l a r l y in the slight concavities o f the mesial a n d distal r o o t aspects. A t these sites, newly p r o d u c e d collagen fibrils were o b s e r v e d directly o n recently f o r m e d dentin, i.e., at o r n e a r the A R E . F o l l o w i n g this initiation, m a t r i x p r o d u c t i o n c o n t i n u e d on the surface o f the already formed and more coronally established cementum layer. In b o t h these regions, fibers o f S h a r p e y were absent, i.e., the c e m e n t u m was c o m p o s e d entirely o f intrinsic fibers t h a t r e m a i n e d w i t h i n the c e m e n t u m m a t r i x . I n i t i a t i o n o f C I F C was o b s e r v e d a l o n g the first 1 0 0 200 ~tm c o r o n a l to the A R E . C o l l a g e n fibril a s s e m b l y was seen a g a i n s t the surface o f recently f o r m e d d e n t i n as well as b e t w e e n c e m e n t o b l a s t s . T h e C I F C - m a t r i x increased r a p i d l y in thickness in the a p i c o - c o r o n a l direction. T h e e n d - p o i n t level o f this r a p i d t h i c k e n i n g corres p o n d e d a p p r o x i m a t e l y to t h a t o f the m i n e r a l i z e d edge o f the g r o w i n g r o o t (Fig. 2c). A l o n g this edge, the inorganic C I F C , w h e n seen f r o m outside, s h o w e d n u m e r o u s l a c u n a e o f c e m e n t o c y t e s (Fig. 2a). C I F C - i n i t i a t i o n c o m m e n c e d a l m o s t d i r e c t l y at the A R E : H e r t w i g ' s r o o t s h e a t h ( H R S ) was a s s o c i a t e d with the A R E for o n l y a few g m a n d , thereafter, c o n t i n u e d c o r o n a l l y in increasing d i s t a n c e f r o m the d e n t i n a l r o o t surface (Fig. 2 b , c). T h e resulting space b e t w e e n the d e v i a t i n g H R S a n d the e x t e r n a l d e n t i n a l m a t r i x was densely p o p u l a t e d b y cem e n t o b l a s t s (Figs. 2 b , c, 3a, 4a). These b a s o p h i l i c cem e n t o b l a s t s (Fig. 2c) were large a n d v a r i a b l e in shape.
Fig. 2. Light- (c) and electron-microscopic (b) views of the advancing root edge (ARE) at the distal aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. The area outlined in e corresponds to b. Note the accumulation of cementoblasts (CB) in the space between Hertwig's root sheath (HRS) and the not yet mineralized external dentinal surface (e) and their close contact with the dentinal matrix fibrils, immediately at the ARE (b). The cementummatrix, i.e., cellular intrinsic fiber cementum (CIFC) increases rapidly in thickness and CBs become cementocytes (CC in c). The dentinal mineralization front is continuous with that of cementum (arrowheads in e). In the scanning electron microscope (a), the external aspect of the inorganic root surface along the mineralized edge of a growing premolar is completely covered by CIFC, showing numerous CC-lacunae. The interrupted line in c corresponds to the dentino-cemental junction. D Dentin; OB odontoblasts; PD predentin; PL periodontal ligament, a x 350, b • 2100, c x 570
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325 Their most characteristic feature was the abundant cytoplasm rich in cytoplasmic organelles, particularly rough endoplasmic reticulum (Figs. 3a, b, 4a). Their nuclei were euchromatin-rich (Figs. 2b, 3a, 4a). Cells located immediately coronal to the A R E were occasionally seen to possess numerous long cytoplasmic extensions pointing towards the external dentinal matrix (Fig. 3 a). Directly coronal to the interdigitation of these extensions with the newly formed dentinal collagen fibrils, cementoblasts formed an almost continuous matrix layer covering the external root surface (Figs. 2a, 3a). At the root-related cementoblast-matrix interface, these cementoblasts formed numerous, slender and fingerlike cytoplasmic processes, which interdigitated with collagen fibrils of the initially produced C I F C - m a t r i x (Fig. 3c, CP1, inset in Fig. 4a). Neither these cytoplasmic processes nor the collagen fibrils reflected a favored orientation. The initially formed CIFC-fibrils interdigitated with the collagen fibrils of the still non-mineralized dentinal matrix. In this transitional zone, the future dentinocemental junction (DCJ) developed. With increasing distance from the A R E , the C I F C - m a t r i x increased in thickness and, as a result, cementoblasts became separated from the D C J (see interrupted line in Fig. 5a-c). Occasionally, discrete bundles of cemental collagen fibrils were clearly demarcated f r o m the randomly oriented dentinal collagen fibrils (Fig. 5a). Consequently, the demarcation of C I F C from predentin varied in distinctiveness and was mainly based on structural differences between the more r a n d o m fibril orientation and the homogeneous pattern of the dentinal matrix and the loosely arranged or partly oriented C I F C - m a t r i x elements (Fig. 5 ~ c ) . Concurrently with the appearance of C I F C - m a t r i x fibrils at the root-related cementoblast-surface, a matrix rich in collagen fibrils was observed on the opposite side of the cementoblasts, thus enclosing these cells (Fig. 3 b). Neighbouring cementoblasts were therefore either separated from each other by a narrow intercellular space or by variable amounts of C I F C - m a t r i x (Figs. 3 a, 4a). Collagen fibrils f r o m the central regions of such accumulated masses of C I F C - m a t r i x appeared to be arranged randomly (Fig. 4c), whereas collagen fibrils of
peripheral regions were loosely bundled (Fig. 4b). These fibril aggregates were located in indentations of the cementoblast-surface, which occurred at various sites around the cementoblasts (arrowheads in Fig. 4a). The aggregated fibrils demonstrated great variation in diameter. At the aggregate periphery, fibrils were intimately associated with narrow cytoplasmic processes cut in the same cross-sectional plane as the surrounding extracellular collagen fibrils (Fig. 4b, CP1). Following this initial and rapid CIFC-production, the cementum layer began to establish itself (Fig. 2c). Cementocytes became embedded in the C I F C - m a t r i x in varying numbers. They revealed numerous, broadbased, triangular cytoplasmic processes projecting into the surrounding matrix (CP2, insets in Fig. 6, Fig. 7). This C I F C - m a t r i x c o m m o n l y exhibited a heterogeneous pattern composed of randomly oriented collagen fibrils and small bundles of such fibrils cut in cross (stars in Fig. 6), oblique or tangential planes of sectioning (Figs. 6, 8a, b, 9a). In comparison to more apical regions, the cementum matrix was less-well demarcated from the dentinal matrix (Fig. 6). This was particularly true after the D C J had been obscured by the mineralization process. On the established cementum surface, cementoblasts often formed a continuous covering. They were variable in shape (Figs. 6, 8a, b, 9a) and had a fine-structural appearance similar to those cells located more apically, although their rough endoplasmic reticulum appeared
Fig. 3 a-e. Electron-microscopic view (a) of the advancing root edge (ARE) at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. The area outlined in a corresponds to h, that in b to e. Cementoblasts (CB) form a continuous covering on the external root surface, i.e., the predentin (PD in a). Immediately coronal to the ARE, numerous cytoplasmic extensions (CE) project towards the dentinal matrix and penetrate between and contact the dentinaI collagen fibrils (inset in a). About 50 gm from the ARE, a particular CB is surrounded by CIFC (b). The root-related CB-matrix interface shows numerous slender and fingerlike cytoplasmic processes (CP~) originating from the CB surface and interdigitating with collagen fibrils of the cementum matrix (e). Neither these CPs nor collagen fibrils are preferably oriented. The interrupted line in b corresponds to the dentino-cemental junction (DCJ). a x 3000, b • e • 6700
Fig. 5. The photomicrographs in a-c were aligned to allow the dentino-cemental junction (interrupted line) to form a straight Dine. With increasing distance from the advancing root edge (a=40 pm, b=60 pm, e=100 gm), the initially formed matrix of cellular intrinsic fiber cementum (CIFC) increases rapidly in thickness in the apico-coronal direction. The collagen fibrils of CIFC-matrix show an irregular orientation (a-e) or are occasionally bundled (a). Consequently, these fibrils are more or less clearly demarcated from the randomly oriented collagen fibrils of the not yet mineralized dentinal matrix (PD). In more coronal root regions, i.e., on established CIFC-surfaces, cementoblasts (CB) reveal many surface indentations that contain cross-sectional, slender cytoplasmic processes (CP~) and engulf cross-sectioned collagen fibrils of homogeneous diameter (d, e). Longitudinally cut collagen fibrils appear to radiate perpendicularly from the CBs plasma membrane (f). PD Predentin. a-c x 8000, d-f x 27000
Fig. 4a-e. Electron-microscopic view (a) of the external root surface at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2year-old boy. The left lower area outlined in a corresponds to b, the left upper area in a to c, and the right rectangular area in a to the left inset in a. Cementoblasts (CB) located approximately 50 btm from the advancing root edge (ARE), show well-developed rough endoplasmic reticulum (a). Numerous surface indentations are seen at various sites around the CB's (arrowheads in a). Surface recesses surround bundles of cross-sectioned collagen fibrils in intimate association with slender and fingerlike cytoplasmic processes (CPI in b). These fibrils are very heterogeneous in diameter (b). Collagen fibrils from central regions of CIFC-matrix show no preferable orientation (e). Towards the dentino-cemental junction (DCJ, interrupted line), many slender CPs originating from the CB surface interdigitate with the collagen fibrils of CIFC (CP~, inset in a). PD Predentin. a x 5400, b, e x 27000; inset: x 21000
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Fig. 4 a - c
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Fig. 5 a - f
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Fig. 7. Large cementocyte not distant from the cementum surface and near the advancing root edge, embedded in cementoid. This cell revealing well-developed rough endoplasmic reticulum, shows
two broad-based cytoplasmic processes (CP2) extending into the mineralized matrix of cellular intrinsic fiber cementum (CIFC). • 9000
less dense. Their nuclei were euchromatin-rich. These cementoblasts showed irregular profiles along the cementum surface, caused by numerous surface indentations (arrowheads in Figs. 6 and 8 a, b). The latter were filled with cross-cut and bundled collagen fibrils that were surrounded, in part, by variably long, broad-based cytoplasmic processes (CP2, Fig. 5d, e). These surface indentations intimately engulfed cross-sectioned collagen fibrils of similar diameter as well as cross-sectioned, narrow and fingerlike cytoplasmic processes distributed among the collagen fibrils (CP1, Fig. 5d, e; inset in Fig. 8). In tangential planes of sectioning, collagen fibrils 9 appeared to radiate perpendicularly from the cytoplas-
mic membrane of cementoblasts (Fig. 5f). Frequently, the slender and fingerlike cytoplasmic processes (Fig. 9 e, f and large inset) and their cytoplasmic bases (Fig. 9 b) enclosed one (Fig. 9e, f a n d large inset) or more (Fig. 9b) collagen fibrils. Such collagen fibrils were also seen within the cementoblast cytoplasm in close proximity to the plasma membrane (small inset in Fig. 9). Both collagen fibrils within slender cytoplasmic processes and those located near the cell periphery were enclosed in membrane-bounded compartments and were oriented parallel to the bundles of extracellular collagen fibrils. The cross-sectional diameter of the membrane-bounded collagen fibrils and their electron density were comparable to that of extracellular collagen fibrils. In more central portions of the cementoblast cytoplasm, single or small groups of collagen fibrils were observed in both longitudinal and cross-sectional planes of sectioning (Fig. 9c, d). A limiting membrane was usually observed between these fibrils and the cytoplasm, suggesting that such collagen fibrils were also enclosed within a membranebounded compartment. The latter appeared electron-lucent and, as seen in longitudinal sections, formed large intracytoplasmic channels (Fig. 9d). The fibrillar content of these channels was morphologically similar to the collagen fibrils in the extracellular space, showing a typical 64-nm pattern. Occasionally, intracytoplasmic channels contained moderately dense spherules along the course of the fibrils (Fig. 9d). Varicosities occurring
Fig. 6. Electron-microscopic view of the established, 30-50 gm-
thick, layer of cellular intrinsic fiber cementum (C1FC) that originated from the distal aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-oldboy. Note that 151~200 pm coronal to the advancing root edge, cementoblasts (CB) form a continuous covering over the established cementum surface. They show an irregular profile along the cementum-matrix, caused by numerous cementoblastsurface indentations (arrowheads). Cementocytes (CC and insets) are embedded in the CIFC-matrix. The CCs have numerous broadbased cytoplasmic processes (CP2) and are completely surrounded by collagen fibrils (insets). The matrix of CIFC is not well demarcated from the dentinal matrix (PD). PD Predentin. • 3000; small inset: x 5600, large inset: • 6700
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331 along the course of the membrane profiles and amorphous, electron-dense material suggestive of collagenolytic activity were not observed within these intracytoplasmic (collagen-containing) compartments. Intracytoplasmic channels containing collagen fibrils were also observed in perinucelar regions of the cementoblasts (not shown). Cementoblasts containing membrane-bounded collagen fibrils were seen in all parts of developing CIFC. In regions of its initiation, cementoblasts included single fibrils, while those cells covering the established CIFCsurface contained both single and groups of membranebounded collagen fibrils, the latter confined to intracytoplasmic channels.
Discussion
In continuation of two preceding papers describing the initiation and establishment of acellular extrinsic fiber cementum (AEFC) on human teeth (Bosshardt and Schroeder 1991 a, b), the present investigation provides, for the first time, detailed information on how cellular intrinsic fiber cementum (CIFC) develops on growing human roots. The findings can be summarized as follows: (1) Hertwig's root sheath did not cover the newly formed root dentin but immediately deviated from the advancing root edge. Consequently, connective tissue cells had access to the external root surface up to the ARE. (2) CIFC-formation commenced almost directly at the advancing root edge and resulted in a rapid cementum thickening along the first 100-200 gm coronal to this edge. (3) This rapid thickening terminated approximately at level with the mineralized edge of the growing root. However, CIFC continued to grow, although more slowly. (4) Large, basophilic and RER-rich cementoblasts, which rapidly became cementocytes, were responsible for fast CIFC-formation, while further cementum
Fig. 8 a, b. Electron-microscopicviews of the periodontal ligament-
related root surface at the mesial aspect of a mandibular first premolar (with a root developed to 75% of its final length), extracted from a 121/2-year-old boy. These regions are located 0.5 mm (a) and 1.0 mm (b) coronal to the advancing root edge. The boundary between cementoblasts (CB) and the established layer of cellular intrinsic fiber cementum (CIFC) shows irregular profiles caused by numerous cross-sectioned cell surface indentations (arrowheads in a, b). These indentations are filled with bundled collagen fibrils that are surrounded, in part, by broad-based cytoplasmic processes. Other very slender processes (CP1, inset) are seen among the bundled collagen fibrils. The latter are newly added to the CIFC-matrix that commonly exhibits a heterogeneous pattern. The mineralization front is uneven, and numerous calcified globules are present in the cementoid (a, b). PL Periodontal ligament, a, b x 3000; inset: x 27000
growth was due to superficial cementoblasts which covered most of the newly established CIFC-surface. (5) The CIFC-matrix was composed entirely of intrinsic collagen fibrils which, at the dentino-cemental junction, initially intermingled with the non-mineralized dentinal collagen fibrils. (6) Developing bundles of collagen fibrils were intimately associated with slender cytoplasmic processes and cementoblast surface indentations. (7) Intracytoplasmic, membrane-bounded collagen fibrils were a common finding within many cementoblasts. Although the light- and electron-microscopic observations were based on demineralized sections only, the front of mineralization could be detected indirectly by noting the light- and ultrastructural changes accompanying calcification. Furthermore, the results described were derived from static observations. Unlike AEFC, which is formed very regularly and very slowly (Nalbandian 1978; Schroeder 1986; Bosshardt etal. 1989), initial CIFC-formation is a very irregular and a rapid process (Bosshardt et al. 1989; Bosshardt and Schroeder 1990). This difference in cementum apposition amounts to the fact that initial CIFC-formation may be up to 30-fold faster than AEFC-formation, as revealed by fluorescence labelling lines in developing deciduous teeth of a non-human primate (Bosshardt et al. 1989). The high rate of apposition renders a developmental description of CIFC, being based on static observations, much more difficult than that of AEFC, the gradual development of which was possible to be shown step-by-step over a long period of time and distance (Bosshardt and Schroeder 199l a, b). The fact that all 8 premolars included in this study were developed to about 75% of their final root length indicates that initial CIFC-formation occurs and is subject to investigation at this particular stage of premolar development. Our findings confirm the high rate of initial CIFCapposition found in the deciduous teeth of a non-human primate (Bosshardt et al. 1989), and also corroborate the assumption of a multipolar mode of CIFC-matrix production observed in human teeth (Bosshardt and Schroeder 1990). In addition, this rapid and multipolar mode of matrix synthesis appears to be the reason for the incorporation of cementocytes, as already suggested by Paynter and Pudy (1958), Formicola et al. (1971) and Bosshardt and Schroeder (1990). A rather dense cementocyte distribution may, therefore, not only be found in the initially formed CIFC-matrix described in this study, but whenever this rapid form of intrinsic fiber cementum formation occurs, e.g., in resorption lacunae. On the other hand, the more coronally located cementoblasts and their cell-matrix interaction resembled the slow, unipolar, acellular intrinsic fiber cementum (A1FC) production described recently (Bosshardt and Schroeder 1990). However, there is still a significant difference between the slow-rate cementum apposition leading to A I F C and the type of cementum formation at the established CIFC-surface as observed in this study: In the latter case, the cementoblasts formed a
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333 continuous but not strictly unicellular layer on the cementum-surface and the collagenous matrix fibers produced were less well organized. Thus, AIFC probably shows the highest degree of fibril bundle organization, whereas initially produced CIFC-matrix reflects the least degree. However, all cells engaged in CIFC-formation were remarkably large, had euchromatin-rich, i.e., activated nuclei, and revealed a cytoplasmic composition typical for actively protein synthesizing cells (Furseth 1969). Based on their morphological features and their size, these cells are undoubtedly cementoblasts. They are identical with the cementoblasts observed by Furseth (1969) and by Bosshardt and Schroeder (/990) and strongly resemble osteoblasts (Marks and P o p o f f 1988), but they are unlike connective tissue cells (presumably fibroblasts) responsible for AEFC-formation (Beertsen and Everts 1990; Bosshardt and Schroeder 1991a, b). Nevertheless, the mechanism of the initial attachment of matrix fibrils to dentin is the same for both A E F C and CIFC: collagen fibrils of the two populations interdigitate in a transitional zone defining the future dentino-cemental junction before the mineralization front has reached this boundary. Since CIFC is rather similar to bone morphologically, the present observations offer an opportunity to compare the formal genesis of these two tissues. Marks and P o p o f f (1988) stated: "Protein secretion is generally polarized toward the bone surface, but at regular intervals along the surface of newly forming bone an osteoblast will secrete matrix away from the surface, eventually surrounding itself to become an osteocyte". In an electron-microscopic autoradiographic study, Frank and Frank (1969) showed that the osteoblast is a polarized cell with an alternating protein secretory activity, i.e., directed either toward the osteoid layer or to the opposite side. A similar alternating secretory activity might be true for the cementoblast producing CIFC-matrix in a multipolar fashion. Further investigations such as electron-microscopic autoradiography are needed to deter
