Cell Tissue Res (1991) 263:325-336
Cell and Tissue Research 9 Springer-Verlag 1991
Establishment of acellular extrinsic fiber cementum on human teeth A light- and electron-microscopic 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 present study describes for the first time the development of early acellular extrinsic fiber cementum (AEFC) until its establishment on human teeth. Precisely selected premolars with roots developed to 5 0 % 100% of their final length were prefixed in Karnovsky's fixative and most of them were decalcified in EDTA. Their roots were subdivided into about 10 blocks each, cut from the mesial and distal root surfaces. Following osmication, these blocks were embedded in Epon and sectioned for light- and transmission electron microscopy. Some blocks were cut non-demineralized. From semithin stained sections, the density of the collagenous fiber fringe protruding from the root surface was measured by using the Videoplan-system. After initiation of this fiber fringe and its attachment to the dentinal root surface followed by mineralization, the fringe gradually increased in length and subsequently became mineralized. Fringe elongation and the advancement of the mineralization front appeared to progress proportionally. Thus, in all stages of A E F C development, a short fiber fringe covered the mineralized AEFC. Its density remained constant, irrespective of A E F C thickness. The latter gradually increased and reached an early maximum of 15 20 ~tm in the cervical region. At this stage, the A E F C fringe appeared to fuse with the future dentogingival or other collagen fibers o f the tooth supporting apparatus. Mineralization of the fringe commenced with isolated, spherical or globular centers, which later fused with the mineralization front and became incorporated in AEFC.
In a preceding study, the initiation of acellular extrinsic fiber cementum (AEFC) has been described for human premolars with roots developed to about 50% of their final length (Bosshardt and Schroeder 1990 a). This initiation included the development of a short collagenous fiber fringe and its attachment to the underlying dentinal matrix. Newly formed fibers of this fringe directly intermingled with the non-mineralized matrix containing dentinal collagen fibrils, and this boundary determined the future dentino-cemental junction (DCJ). The external front of dentin mineralization did not advance to this zone until interdigitation of the two fibril populations had been established. It was suggested that this fiber fringe, covering the external dentinal surface, is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization. Although several studies dealing with the ultrastructure of A E F C on fully formed roots of human deciduous (Furseth 1967) and permanent (Herting 1962, 1964; Selvig 1965, 1967; Furseth 1974) teeth exist, there is no study describing the genesis of A E F C in man. Dynamic data of cementum apposition in deciduous teeth are available from one non-human primate (Bosshardt et al. 1989). The present report is a continuation of the above mentioned companion paper and attempts formally to describe the development of human AEFC, commencing with the beginning of mineralization o f the short fiber fringe and terminating with the establishment of a thin mineralized layer of A E F C on the root surface.
Key words: Cementum - Fiber fringe - Periodontal ligament fibers Dentino-cemental junction - Electron microscopy - H u m a n
Materials and methods
D. D. Bosshardt Abbreviations: A E F C acellular extrinsic fiber cementum; CIFC cellular intrinsic fiber cementum; C M S C cellular mixed stratified cementum; CEJ cemento-enamel junction; C M centers of mineralization; D dentin; D C J dentino-cemental junction; E D T A ethylene diaminetetraacetic acid; F F fiber fringe; GL glycogen storage granules; M F mineralization front; P L periodontal ligament; P L F periodontal ligament fibers
Thirty-five premolars (22 maxillary and 13 mandibular, 25 first and 10 second), extracted for orthodontic reasons from 17 females and 18 males, 9-27 years of age, were selected from a large collection of freshly extracted human teeth (mostly premolars, including some third molars). These teeth presented with either incomplete roots developed to 50%-90% of their final length or with fully developed roots. A total of 35 premolars was processed for lightand electron microscopy as described previously (Bosshardt and Schroeder 1991). For the present study, 205 demineralized Epon-
Offprint requests to:
326
200
x
E
150
E 9~100 U.
xx
x
x x NKXX:,Nl-Z
u.i 50
blocks originating from the mesial and distal sites of 32 premolars were used. From each block, 1 to 2-gin-thick sections were cut vertical to the root surface in a corono-apical direction, 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 root surface areas usually covered with pure AEFC. They comprized (1) regions remote (up to 3-6 mm) from the advancing edge of the root, and (2) regions close to the cemento-enamel junction (CEJ). This resulted in 13 selected areas from 10 teeth. In addition, some nondecalcified blocks were prepared for ultrasectioning. Ultrathin sections (60-80 nm) cut with a diamond knife and an LKB-Ultrotome III, were contrasted with uranyl acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Electron-microscopic examination and recording was carried out with a Philips 201 transmission electron microscope. In addition, two premolars (with roots developed to 50% of their final length) demonstrating above-average tissue preservation were selected to calculate the fiber density of the fringe as it develops along the root surface. From the roots of both teeth, 2 mesial blocks (one comprizing the apical and the other the coronal half of the root) were cut and stained as described above. The fiber density, along the entire root surface, extending from a point about 200 gm coronal to the advancing edge of the root to the CEJ, was counted and calculated for consecutive root surface fractions of 100 gm each in three independent l-gin-thick sections of every block. Measurements and calculations (number of fibers per mm of root surface) were performed using the Videoplan-system (Kontron/Zeiss, Zurich, Switzerland). All abbreviations used in this paper are summarized on the title page.
rr UJ
"
0
Results I
I
I
I
I
1,0
2,0
3,0
4,0
I
5,0 6,0
I
I
I
7,0
8,0
9,0
ROOT LENGTH (mm) 200
A 150 I XX XN ~ y E
XX
X
XX
..
~
XNNN< X XX
Ii)
~. 100
X X:
Fig. 3. Higher magnificationof the area outlined in Fig, 2b, demonstrating that longitudinallycut fringe fibers (FF) extend into the periodontal ligament(PL) space and partially enmesh adjacent connective-tissuecells. Cross- or obliquely-cutfibers are short and bend abruptly at adjacent cells. The latter are similar morphologically, exhibitinga large electron-lucentcytoplasm and an euchromatin-rich nucleus. Glycogenstorage granules (GL) are regularly seen. The fringefibrils terminatein an almost completelyobscured and electron-dense zone that represents the just-mineralized dentino-cementaljunction (DCJ). The equallyobscured mineralization centers, representing cross-cut fibers (arrows), seem to fuse with the external mineralization front (MF, arrowheads). D dentin. x 6700
329
330
331 As the fringe fibers entered the AEFC, their course was either straight and vertical to the root surface or interwoven (Figs. 7b, 8a). In the outer half of AEFC, A E F C fibers were dense, in the inner half less so. This resulted in high electron density in the outer, and moderate electron density in the inner layer of A E F C (Figs. 7 b, 8a). A similar impression was obtained from light-microscopic sections (Fig. 7a). At higher magnification (Fig. 8 a), A E F C fibers could often be followed throughout the AEFC, in particular within the loose inner layer. The collagen fibril structure appeared to be obscured in the outer layer, but very distinct in the inner layer (Fig. 8 b). Longitudinally cut fibers of the latter were seen to intermingle directly with the randomly oriented collagen fibrils of the dentinal matrix (Fig. 8 b).
Discussion
As a continuation of a preceeding paper describing the initiation of acellular extrinsic fiber cementum (AEFC) and its attachment to the dentinal matrix of the root surface (Bosshardt and Schroeder 1991), the present investigation provides new data on how A E F C develops on human teeth. These data and findings can be summarized in the following statements : (1) initiation and early establishment of A E F C occur mainly during tooth eruption, i.e., prior to the development of the tooth supporting fiber apparatus; (2) A E F C genesis commences prior to the beginning of any development o f cellular mixed stratified cementum (CMSC), and A E F C thickness gradually increases in the apico-coronal direction; (3) the average density of the collagenous fiber fringe, representing the early A E F C matrix, remains constant along the growing root surface, i.e., the base o f the fringe assumes its maximum density during the phase of A E F C initiation, within a zone of about 0.2 m m coronal to the advancing root edge; (4) this fiber fringe continues to exist at the root surface, even after A E F C development has begun, and remains short until the A E F C has attained a thickness of about 15-20 gm; (5) accordingly, the fiber fringe undergoes elongation in proportion to the advancement of the external A E F C mineralization front; (6) mineralization of the fiber fringe, i.e., of A E F C matrix, follows a globular pattern. These statements are based on static observations and measurements on specimens of human teeth selected
.
Cell and Tissue Research 9 Springer-Verlag 1991
Establishment of acellular extrinsic fiber cementum on human teeth A light- and electron-microscopic 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 present study describes for the first time the development of early acellular extrinsic fiber cementum (AEFC) until its establishment on human teeth. Precisely selected premolars with roots developed to 5 0 % 100% of their final length were prefixed in Karnovsky's fixative and most of them were decalcified in EDTA. Their roots were subdivided into about 10 blocks each, cut from the mesial and distal root surfaces. Following osmication, these blocks were embedded in Epon and sectioned for light- and transmission electron microscopy. Some blocks were cut non-demineralized. From semithin stained sections, the density of the collagenous fiber fringe protruding from the root surface was measured by using the Videoplan-system. After initiation of this fiber fringe and its attachment to the dentinal root surface followed by mineralization, the fringe gradually increased in length and subsequently became mineralized. Fringe elongation and the advancement of the mineralization front appeared to progress proportionally. Thus, in all stages of A E F C development, a short fiber fringe covered the mineralized AEFC. Its density remained constant, irrespective of A E F C thickness. The latter gradually increased and reached an early maximum of 15 20 ~tm in the cervical region. At this stage, the A E F C fringe appeared to fuse with the future dentogingival or other collagen fibers o f the tooth supporting apparatus. Mineralization of the fringe commenced with isolated, spherical or globular centers, which later fused with the mineralization front and became incorporated in AEFC.
In a preceding study, the initiation of acellular extrinsic fiber cementum (AEFC) has been described for human premolars with roots developed to about 50% of their final length (Bosshardt and Schroeder 1990 a). This initiation included the development of a short collagenous fiber fringe and its attachment to the underlying dentinal matrix. Newly formed fibers of this fringe directly intermingled with the non-mineralized matrix containing dentinal collagen fibrils, and this boundary determined the future dentino-cemental junction (DCJ). The external front of dentin mineralization did not advance to this zone until interdigitation of the two fibril populations had been established. It was suggested that this fiber fringe, covering the external dentinal surface, is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization. Although several studies dealing with the ultrastructure of A E F C on fully formed roots of human deciduous (Furseth 1967) and permanent (Herting 1962, 1964; Selvig 1965, 1967; Furseth 1974) teeth exist, there is no study describing the genesis of A E F C in man. Dynamic data of cementum apposition in deciduous teeth are available from one non-human primate (Bosshardt et al. 1989). The present report is a continuation of the above mentioned companion paper and attempts formally to describe the development of human AEFC, commencing with the beginning of mineralization o f the short fiber fringe and terminating with the establishment of a thin mineralized layer of A E F C on the root surface.
Key words: Cementum - Fiber fringe - Periodontal ligament fibers Dentino-cemental junction - Electron microscopy - H u m a n
Materials and methods
D. D. Bosshardt Abbreviations: A E F C acellular extrinsic fiber cementum; CIFC cellular intrinsic fiber cementum; C M S C cellular mixed stratified cementum; CEJ cemento-enamel junction; C M centers of mineralization; D dentin; D C J dentino-cemental junction; E D T A ethylene diaminetetraacetic acid; F F fiber fringe; GL glycogen storage granules; M F mineralization front; P L periodontal ligament; P L F periodontal ligament fibers
Thirty-five premolars (22 maxillary and 13 mandibular, 25 first and 10 second), extracted for orthodontic reasons from 17 females and 18 males, 9-27 years of age, were selected from a large collection of freshly extracted human teeth (mostly premolars, including some third molars). These teeth presented with either incomplete roots developed to 50%-90% of their final length or with fully developed roots. A total of 35 premolars was processed for lightand electron microscopy as described previously (Bosshardt and Schroeder 1991). For the present study, 205 demineralized Epon-
Offprint requests to:
326
200
x
E
150
E 9~100 U.
xx
x
x x NKXX:,Nl-Z
u.i 50
blocks originating from the mesial and distal sites of 32 premolars were used. From each block, 1 to 2-gin-thick sections were cut vertical to the root surface in a corono-apical direction, 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 root surface areas usually covered with pure AEFC. They comprized (1) regions remote (up to 3-6 mm) from the advancing edge of the root, and (2) regions close to the cemento-enamel junction (CEJ). This resulted in 13 selected areas from 10 teeth. In addition, some nondecalcified blocks were prepared for ultrasectioning. Ultrathin sections (60-80 nm) cut with a diamond knife and an LKB-Ultrotome III, were contrasted with uranyl acetate and lead citrate (Reynolds 1963; Frasca and Parks 1965). Electron-microscopic examination and recording was carried out with a Philips 201 transmission electron microscope. In addition, two premolars (with roots developed to 50% of their final length) demonstrating above-average tissue preservation were selected to calculate the fiber density of the fringe as it develops along the root surface. From the roots of both teeth, 2 mesial blocks (one comprizing the apical and the other the coronal half of the root) were cut and stained as described above. The fiber density, along the entire root surface, extending from a point about 200 gm coronal to the advancing edge of the root to the CEJ, was counted and calculated for consecutive root surface fractions of 100 gm each in three independent l-gin-thick sections of every block. Measurements and calculations (number of fibers per mm of root surface) were performed using the Videoplan-system (Kontron/Zeiss, Zurich, Switzerland). All abbreviations used in this paper are summarized on the title page.
rr UJ
"
0
Results I
I
I
I
I
1,0
2,0
3,0
4,0
I
5,0 6,0
I
I
I
7,0
8,0
9,0
ROOT LENGTH (mm) 200
A 150 I XX XN ~ y E
XX
X
XX
..
~
XNNN< X XX
Ii)
~. 100
X X:
Fig. 3. Higher magnificationof the area outlined in Fig, 2b, demonstrating that longitudinallycut fringe fibers (FF) extend into the periodontal ligament(PL) space and partially enmesh adjacent connective-tissuecells. Cross- or obliquely-cutfibers are short and bend abruptly at adjacent cells. The latter are similar morphologically, exhibitinga large electron-lucentcytoplasm and an euchromatin-rich nucleus. Glycogenstorage granules (GL) are regularly seen. The fringefibrils terminatein an almost completelyobscured and electron-dense zone that represents the just-mineralized dentino-cementaljunction (DCJ). The equallyobscured mineralization centers, representing cross-cut fibers (arrows), seem to fuse with the external mineralization front (MF, arrowheads). D dentin. x 6700
329
330
331 As the fringe fibers entered the AEFC, their course was either straight and vertical to the root surface or interwoven (Figs. 7b, 8a). In the outer half of AEFC, A E F C fibers were dense, in the inner half less so. This resulted in high electron density in the outer, and moderate electron density in the inner layer of A E F C (Figs. 7 b, 8a). A similar impression was obtained from light-microscopic sections (Fig. 7a). At higher magnification (Fig. 8 a), A E F C fibers could often be followed throughout the AEFC, in particular within the loose inner layer. The collagen fibril structure appeared to be obscured in the outer layer, but very distinct in the inner layer (Fig. 8 b). Longitudinally cut fibers of the latter were seen to intermingle directly with the randomly oriented collagen fibrils of the dentinal matrix (Fig. 8 b).
Discussion
As a continuation of a preceeding paper describing the initiation of acellular extrinsic fiber cementum (AEFC) and its attachment to the dentinal matrix of the root surface (Bosshardt and Schroeder 1991), the present investigation provides new data on how A E F C develops on human teeth. These data and findings can be summarized in the following statements : (1) initiation and early establishment of A E F C occur mainly during tooth eruption, i.e., prior to the development of the tooth supporting fiber apparatus; (2) A E F C genesis commences prior to the beginning of any development o f cellular mixed stratified cementum (CMSC), and A E F C thickness gradually increases in the apico-coronal direction; (3) the average density of the collagenous fiber fringe, representing the early A E F C matrix, remains constant along the growing root surface, i.e., the base o f the fringe assumes its maximum density during the phase of A E F C initiation, within a zone of about 0.2 m m coronal to the advancing root edge; (4) this fiber fringe continues to exist at the root surface, even after A E F C development has begun, and remains short until the A E F C has attained a thickness of about 15-20 gm; (5) accordingly, the fiber fringe undergoes elongation in proportion to the advancement of the external A E F C mineralization front; (6) mineralization of the fiber fringe, i.e., of A E F C matrix, follows a globular pattern. These statements are based on static observations and measurements on specimens of human teeth selected
.
