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Correlation of scanning and transmission electron microscopy of human dentinal tubules
0003-9969/84$3.00+ 0.00
~rchs oral Viol. Vol. 29, No. 8, pp. 641-646, 1984 Copyright c
Printed in Great Britain. All rights reserved
1984 Pergamon Press Ltd
CORRELATION OF SCANNING AND TRANSMISSION ELECTRON MICROSCOPY OF HUMAN DENTINAL TUBULES H. F. THOMAS and P. CARELLA Department
of Oral Biology, School of Dental Medicine, University of Connecticut Health Centre,
Farmington, CT 06032, U.S.A. Summary-Areas of human dentine were examined first with the scanning electron microscope (SEM) and subsequently with the transmission electron microscope (TEM). Structures observed in dentinal tubules from outer dentine by SEM were identified by TEM as electron-dense structures lining the tubules and not as odontoblast processes. These structures, termed lamina limitans, correspond to the previously described inner hypomineralized lining of dentinal tubules. INTRODUCTION
lars from individuals aged between 19 and 27 yr were obtained immediately after extraction. A groove was Transmission electron microscopy (TEM) (Holland, 1976; Thomas, 1979,1983; Thomas and Payne, 1983) cut around the tooth below the cementum-enamel has shown that odontoblast processes are limited to junction with a water-cooled tungsten-carbide bur in a high-speed handpiece. A chisel was placed in the inner dentine. Thomas (1979) demonstrated that, following demineralization and subsequent loss of groove, tapped with a hammer and the roots removed. Crowns with coronal pulp attached were peritubular dentine, electron-dense structures appear treated in one of the two following ways. Five within dentinal tubules. These structures were clearly distinguishable from odontoblast processes when the specimens were grooved mesio-distally and split, as above into buccal and lingual halves. The remaining two were seen together. The electron-dense structures were present in all dentinal tubules from the pre- five specimens were mounted on blocks and representative slices from the inner, middle and outer thirds dentine junction to the dentine-cnamel junction. of dentine were cut in a occluso-cervical plane with Thomas (1979) concluded that these structures corresponded to the inner hypomineralized lining of peri- a water-cooled diamond blade. These two methods tubular dentine (Shroff et al., 1956; Bradford, 1967; provided specimens in which dentinal tubules were Johansen, 1967; Takuma, 1967). Those who have cut either longitudinally or in cross-section. Each used scanning electron microscopy (SEM) do not all specimen was placed in Karnovsky fixative (Karnovsky, 1965) for 4 h. Specimens were rinsed in 0.1 M agree as to the extent of the odontoblast process; sodium cacodylate buffer, pH 7.4, and demineralized some (Brannstrom and Garberoglio, 1972; Tidmarsh, 1981; Thomas and Carella, 1983) have shown the in a formic-citric acid solution (Morse, 1945). All teeth were post-fixed in 1 per cent osmium tetroxide processes to be limited to inner dentine, others (Kelfor 4 h, rinsed in buffer (0.1 M sodium cacodylate, pH ley, Bergenholtz and Cox, 1981; Crooks, O’Reilly and 7.4) and placed in a 1 per cent saturated aqueous Owens, 1983; Grossman and Austin, 1983; Maniasolution of thiocarbohydrazide (Polysciences, Wartopoulos and Smith, 1983) claim to show odontoblast rington, PA 18976, U.S.A.) for 20 min at room processes within tubules in the outer dentine. Furtemperature (Kelley, Dekker and Bluemink, 1973). thermore, Yamada et al. (1983) and Gunji and Kobayashi (1983), using demineralization and col- Additional rinsing in buffer (0.1 M sodium cacodylate, pH 7.4) was followed by re-osmication, and lagenase digestion of the dentine matrix, have shown structures identified as odontoblast processes extenddehydration through a graded ethanol series. Specimens were critical point dried in ethanol-CO,, mouning throughout the entire thickness of dentine. One possible explanation for this discrepancy may ted on aluminium stubs and examined with an Hitreside in the failure of the SEM studies to recognize achi 3010 SEM (Hitachi Co., Tokyo, Japan) at 20 kV. the inner hypomineralized lining of dentinal tubules; Representative areas were photographed. Following a propylene oxide wash, all specimens previously this lining has been noted in some studies (Isokawa, Todo and Kubota, 1970; Isokawa et al., 1972; scanned were embedded in Epon. Sections ranging from 60 to 80nm were cut from the surface preThomas and Carella, 1983). viously scanned, stained with uranyl acetate and lead Our purpose was to examine areas of human citrate and examined with an Hitachi 300 TEM at dentine first by SEM and then subsequently by TEM 80 kV. Areas of interest which corresponded to areas to determine whether the structures seen by SEM previously scanned were again photographed. within outer dentinal tubules were cellular or whether they correspond to the inner hypomineralized lining of peritubular dentine. RESULTS MATERIALSAND Ten unerupted
METHODS
but fully-formed human third mo-
Odontoblasts were seen by SEM adjacent to a layer of predentine (Fig. 1). Odontoblast processes were observed leaving the cell bodies and entering the
H. F. THOMAS and P. CARELLA
642
predentine layer. In corresponding areas seen by TEM (Fig. 2) odontoblast processes were present in predentine. Degeneration of some odontoblast processes was visible, and some processes appeared to have shrunk away from the predentine matrix. However, no intermediate structures could be seen between the cell membranes of the odontoblast processes and the predentine matrix. Odontoblast processes were not found in dentinal tubules in SEM from the inner third of dentine (Fig. 3). The tubules were lined by an amorphous sheet-like structure which was separated from the tubule walls in some areas. Corresponding TEM (Fig. 4) showed odontoblast processes within dentinal tubules. However, in contrast to the appearance seen in predentine (Fig. 2), electron-dense structures that were clearly distinguishable from the cell membrane of the odontoblast process lined all tubules. Areas of peritubular dentine formation were visible and, where they occurred, were separated from the lumena of tubules by electron-dense structures (Fig, 4). All tubules from the middle and outer third of crown dentine examined by SEM (Fig. 5) contained sheet-like structures. A space was present between this structure and the intertubular dentine matrix. This space, termed the peritubular matrix space, resulted from loss of peritubular dentine during demineralization. Corresponding TEM (Fig. 6) showed electron-dense structures within each tubule which corresponded directly to the sheet-like structures seen in SEM. A space, the peritubular matrix space, was present between the electron-dense structure and the intertubular dentine matrix. Similar structures were seen in dentinal tubules cut in cross-section which were representative of those teeth which were sliced occluso-cervically. In tubules from the inner third of dentine (Fig. 7), solid structures were present within each tubule by SEM separated from tubule walls by a space. In corresponding TEM (Fig. 8), each dentinal tubule contained an electron-dense structure separated from the tubule wall by a space. Odontoblast processes were not seen in any sections. Higher magnification of tubules from the inner third of crown dentine (Figs 9 and 10) showed, in some areas, a fibrillar component in the peritubular matrix. These fibrils appeared to connect the sheet-like structure to the intertubular dentine matrix (Fig. 9). Dentinal tubules from mid and outer thirds of dentine (Figs 11 and 12) were similar in appearance to those from the inner third. SEM (Fig. 1 I) showed within each dentinal tubule a sheet-like structure, whereas TEM (Fig. 12) showed that these structures corresponded directly with the electron-dense structures which lined all tubules. No odontoblast processes were seen in any tubules examined. DISCUSSION
The study shows that the structures observed throughout dentinal tubules by SEM correspond directly with electron-dense structures seen by TEM in demineralized dentinal tubules. This electron-dense structure has previously been identified as the inner hypomineralized lining of peritubular dentine (Shroff ef al., 1956; Takuma, 1967; Thomas, 1979). The
structure seen within dentinal tubules by SEM is not, therefore, the odontoblast process but rather the inner hypomineralized lining of peritubular dentine in accord with the views of others (see Introduction). Isokawa et al. (1972) described a tubular membraneous structure which appeared on the surface of fractured dentine following etching with a weak acid. Thomas and Carella (1983) showed by SEM that all tubules are lined by such structures throughout their lengths. In accord with terminology already established for a similar structure observed in bone and mineralizing cartilage (Scherft, 1972; Shepard and Mitchell, 198 I), Thomas and Carella (1983) proposed that this structure be called the lamina limitans. The lamina limitans is not the odontoblast process; it does not possess a trilaminar structure and is too thick to represent a cell membrane. Additionally, when both the lamina limitans and odontoblast process are seen together, they are clearly distinguishable (Fig. 4). The separation between the lamina limitans and intertubular dentine increases as the distance from the pulp increases. This is due to the increase in amount of peritubular dentine formation, which is minimal in inner dentine (Fig. 4) and increases in mid and outer denture (Fig. 12; Atkinson and Harcourt, 1961). We did not see odontoblast processes in any tubules where there was more than minimal peritubular dentine formation, confirming previous observations (Thomas, 1979). In view of the presence of the lamina limitans in each dentinal tubule, care must be exercised in the identification of structures within dentinal tubules by SEM. An alternative interpretation for structures identified as odontoblast processes in outer dentine by SEM by previous workers (Kelley, Bergenholtz and Cox, 1981; Crooks et al.. 1983: Grossman and Austin, 1983; Maniatopoulos and Smith, 1983) is that they are not odontoblast processes but rather the lamina limitans. Furthermore, as the lamina limitans is thought to be composed primarily of glycosaminoglycans (Thomas and Carella, 1983), studies utilizing demineralization and collagenase digestion of dentine (Yamada et al., 1983; Gunji and Kobayashi. 1983) would also leave laminae limitantes lying on the exposed surface. Therefore, to be able to identify by SEM a structure as the odontoblast process, concurrent TEM should demonstrate a cellular process, a cell membrane and intracellular organelles. The technique employed here seems well suited for the examination of dentine. The combination of osmication of tissue followed by thiocarbohydrazide treatment coated the surface of the tissue sufficiently to allow SEM observations to be made. The resulting SEM image was not as good as that of tissue treated by conventional gold coating; lack of contrast was a particular deficiency. However, subsequent embedding of the tissue in Epon and thin-sectioning provided sections of sufficient quality for visualization by TEM. In tubules seen in cross-section by SEM, the lamina limitans appeared to be a solid structure. As corresponding TEM show that lamina limitans is in fact patent, we believe that the solid appearance represents an artifact of slicing the tooth and results from debris accumulating within the lamina limitans.
SEM/TEM of human dentinal tubules Transmission electron micrographs fail to reveal the extent of the organic matrix of peritubular dentine. Scanning electron micrographs, however, showed that the matrix is variable, and in certain areas (Fig. 9), can be considerable. The fibrillar component of this matrix appears to connect the lamina limitans to the matrix of intertubular dentine. The lumena of some tubules contained granular material. This material may result from processing of the tissue, and possibly be due to some modification of the lamina limitans following demineralization or thiocarbohydraxide treatment. As the lamina limitans probably contains glycomacromolecules, this modification would not be surprising because thiocarbohydrazide is used for the ultracytochemical demonstration of glycomacromolecules (Pearse, 1972). We previously proposed (Thomas and Carella, 1983) based on histochemical studies (Symons, 1967; Wislocki, Singer and Waldo, 1948), that the lamina limitans contains glycosaminoglycans and glycoproteins and may play a role in the control of deposition of peritubular dentine. We also believe that the lamina limitans corresponds to the fibrils of soft tissue first described within dentinal tubules by Tomes (1856). REFERENCES
Atkinson H. F. and Harcourt J. K. (1961) Some observations on the peritubular translucent zones in human dentine. Aust. dent. J. 6, 194197. Bradford E. W. (1967) Microanatomv and Histochemistrv of Dentine. In; Strthmzl and Chemical Organization oj Teeth (Edited by Miles A. E. W.) Vol. 2, pp. 3-34. Academic Press, New York. Brannstrom M. and Garberoglio R. (1972) The dentinal tubules and the odontoblast processes. Acta odont. stand. 30, 291-311.
Crooks P. V., O’Reilly C. B. and Owens P. D. A. (1983) Microscopy of the dentine of enamel-free areas of rat molar teeth. Archs oral Biol. Zs, 167-175. Grossman E. S. and Austin J. C. (1983) Scanning electron microscope observations on the tubule content of freeze-
fractured peripheral vervet monkey dentine (cercopithecus pyserythrus).
Archs oral Biol. 28, 279-281.
Gunji T. and Kobayashi S. (1983) Distribution and organization of odontoblast processes in human dentin. Arch. Hist. Jap. 46, 213-219.
Holland G. R. (1976) The extent of the odontoblast process in the cat. J. Anat. 121, 133-149. Isokawa S., Toda R. and Kubota K. (1970) A scanning electron microscopic observation of etched human peritubular dentin. Archs oral Biol. 15, 1303-1306. Isokawa S., Yoshida M., Komura A. and Iwatake Y. (1972) A preliminary study on the peritubular structure of human dentinal tubules by scanning electron microscopy. J. Nihon Univ. Sch. Dent., 14, 122-125. Johansen E. (1967) Ultrastructure of dentin. In: Structural
643
and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 2, pp. 3>74. Academic Press, New York. Karnovsky M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A-138A. Kelly K. W., Bergenholtz G. and Cox C. F. (1981) The extent of the odontoblast process in Rhesus monkeys (Macaca mulatta) as observed by scanning electron microscopy. Archs oral Biol. 26, 893-897. Kellv R. 0.. Dekker R. A. F. and Bluemink J. G. (1973) L;gand-mediated osmium binding: Its application in coat: ing biological specimens from scanning electron microscopy. J. Ultrastruci. Res. 45, 254-258. Maniatopoulos C. and Smith D. C. (1983) A scanning electron microscopic study of the odontoblast process in human coronal dentine. Archs oral Biol. 28, 701-710. Morse A. (1945) Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J. dent. Res. 24, 143-153. Pearse A. G. E. (1972) Histochemistry. Theoretical and Applied, Vol. 2, 3rd edn, pp. 1273-1274. Churchill Livingstone, London. Scherft J. P. (1972) The lamina limitans of the organic . matrix of calcified cartilage and bone. J. Ultrastruct. Res. 38, 318-331.
Shepard N. and Mitchell N. (1981) Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification. Histochemistry 70, 107-l 14. Shroff F. R., Williamson K. I., Bertaud W. S. and Hall D. M. (1956) Further electron microscope studies of dentin. The nature of the odontoblast process. Oral Surg. 9, 432443. Symons N. B. B. (1967) The microanatomy and histochemistry of dentinogenesis. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, pp. 285-324. Academic Press, New York. Takuma S. (1967) Ultrastructure of dentinogenesis. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, pp. 325-370. Academic Press,
New York. Thomas H. F. (1979)The extent of the odontoblast process in human dentin. J. dent. Res. 58, 2207-2218. Thomas H. F. (1983) The effect of various fixatives on the extent of the odontoblast process in human dentine. Archs oral Biol. 28, 465-469.
Thomas H. F. and Payne R. C. (1983) The ultrastructure of dentinal tubules from erupted human premolar teeth. J. dent. Res. 62, 532-536.
Thomas H. F. and Carella P. (1983) A scanning electron microscope study of dentinal tubules from human teeth. Archs oral Biol. 28, 1125-I 130.
Tidmarsh B. G. (1981) Contents of human dentinal tubules. ht. endo. J. 14, 191-196. Tomes J. (1856) On the presence of fibrils of soft tissue in the dentinal tubes. Phil. Trans. 146, 515-522. Wislocki G. B., Singer M. and Waldo C. M. (1948) Some histochemical reactions of mucopolysaccharides, giycogen, lipids and other substances in teeth. Anat. Rec. 101, 487-5 13. Yamada T., Nakamura K., Iwaku M. and Fusayama T. (1983) The extent of the odontoblast process in normal and carious human dentin. J. dent. Res. 62, 798-802.
Plates 1 and 2 overleaf.
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644
Plate
I
Fig. I. SEM view of predentine. Odontoblast process (OP) can be seen leaving the odontoblast (0) and entering a layer of predentine (PD).
cell body
Fig. 2. TEM view of predentine. Odontoblast processes (OP) can be seen within a layer of predentine (PD). No intermediate structure can be observed between the cell membrane of the odontoblast process and the predentine matrix. Fig. 3. SEM view of tubules from inner third of dentine. In some areas (+) separation of the sheet-like structure from the tubule wall can be seen indicating areas of early peritubular dentine formation. ID = intertubular dentine. Fig. 4. TEM tubules. Each membrane of (PTM),
view of tubules from inner third of dentine. Odontoblast processes (OP) can be seen within tubule is lined by an electron-dense structure (LS) which is distinguishable from the cell the odontoblast process (+ t). Early areas of peritubular dentine formation are visible the electron-dense structure is visible on the luminal side of the peritubular matrix.
Fig. 5. SEM of tubules from mid dentine. Intertubular dentine (ID) is visible between each tubule. tubule contains a sheet-like structure (LL). No odontoblast processes are visible.
Each
Fig. 6. TEM of tubules from mid dentine. The electron-dense structure lining all tubules (LS) corresponds directly with the sheet-like structure seen by SEM. A space (PTM) is present between the electron-dense structure (LS) and intertubular dentine (ID). This space corresponds to peritubular dentine which has been lost through demineralization. Plate 2. Fig. 7. SEM of cross-sectioned tubules from inner dentine. Each tubule contains a solid structure which is separated from the intertubular dentine (ID) by a space (PTM). Fig. 8. Corresponding
TEM view of an area seen in Fig. 7. Each tubule contains structure (LS). No odontoblast processes are visible.
an electron-dense
Fig. 9. Higher magnification of tubule from inner dentine viewed by SEM. The solid structure appears to be connected to the intertubular dentine (ID) by a fibrillar material. Fig. 10. Corresponding Fig. Il.
TEM view of Fig. 9. The electron-dense structure the solid structure seen by SEM.
(LS) corresponding
SEM view of tubules from mid third of dentine. Each tubule contains a solid structure is separated from intertubular dentine (ID) by a space (PTM).
Fig. 12. Corresponding electron-dense structure
(LL)
(LL)
directly with (LL) which
TEM view of tubules from mid third dentine. Each tubule contains (LS) which is separated from intertubular dentine (ID) by a space (PTM). odontoblast processes are visible.
an No
SEM/TEM
of human
Plate
dentinal
1.
tubules
645
646
H. F. THOMAS and P. CARELLA
Plate 2
~rchs oral Viol. Vol. 29, No. 8, pp. 641-646, 1984 Copyright c
Printed in Great Britain. All rights reserved
1984 Pergamon Press Ltd
CORRELATION OF SCANNING AND TRANSMISSION ELECTRON MICROSCOPY OF HUMAN DENTINAL TUBULES H. F. THOMAS and P. CARELLA Department
of Oral Biology, School of Dental Medicine, University of Connecticut Health Centre,
Farmington, CT 06032, U.S.A. Summary-Areas of human dentine were examined first with the scanning electron microscope (SEM) and subsequently with the transmission electron microscope (TEM). Structures observed in dentinal tubules from outer dentine by SEM were identified by TEM as electron-dense structures lining the tubules and not as odontoblast processes. These structures, termed lamina limitans, correspond to the previously described inner hypomineralized lining of dentinal tubules. INTRODUCTION
lars from individuals aged between 19 and 27 yr were obtained immediately after extraction. A groove was Transmission electron microscopy (TEM) (Holland, 1976; Thomas, 1979,1983; Thomas and Payne, 1983) cut around the tooth below the cementum-enamel has shown that odontoblast processes are limited to junction with a water-cooled tungsten-carbide bur in a high-speed handpiece. A chisel was placed in the inner dentine. Thomas (1979) demonstrated that, following demineralization and subsequent loss of groove, tapped with a hammer and the roots removed. Crowns with coronal pulp attached were peritubular dentine, electron-dense structures appear treated in one of the two following ways. Five within dentinal tubules. These structures were clearly distinguishable from odontoblast processes when the specimens were grooved mesio-distally and split, as above into buccal and lingual halves. The remaining two were seen together. The electron-dense structures were present in all dentinal tubules from the pre- five specimens were mounted on blocks and representative slices from the inner, middle and outer thirds dentine junction to the dentine-cnamel junction. of dentine were cut in a occluso-cervical plane with Thomas (1979) concluded that these structures corresponded to the inner hypomineralized lining of peri- a water-cooled diamond blade. These two methods tubular dentine (Shroff et al., 1956; Bradford, 1967; provided specimens in which dentinal tubules were Johansen, 1967; Takuma, 1967). Those who have cut either longitudinally or in cross-section. Each used scanning electron microscopy (SEM) do not all specimen was placed in Karnovsky fixative (Karnovsky, 1965) for 4 h. Specimens were rinsed in 0.1 M agree as to the extent of the odontoblast process; sodium cacodylate buffer, pH 7.4, and demineralized some (Brannstrom and Garberoglio, 1972; Tidmarsh, 1981; Thomas and Carella, 1983) have shown the in a formic-citric acid solution (Morse, 1945). All teeth were post-fixed in 1 per cent osmium tetroxide processes to be limited to inner dentine, others (Kelfor 4 h, rinsed in buffer (0.1 M sodium cacodylate, pH ley, Bergenholtz and Cox, 1981; Crooks, O’Reilly and 7.4) and placed in a 1 per cent saturated aqueous Owens, 1983; Grossman and Austin, 1983; Maniasolution of thiocarbohydrazide (Polysciences, Wartopoulos and Smith, 1983) claim to show odontoblast rington, PA 18976, U.S.A.) for 20 min at room processes within tubules in the outer dentine. Furtemperature (Kelley, Dekker and Bluemink, 1973). thermore, Yamada et al. (1983) and Gunji and Kobayashi (1983), using demineralization and col- Additional rinsing in buffer (0.1 M sodium cacodylate, pH 7.4) was followed by re-osmication, and lagenase digestion of the dentine matrix, have shown structures identified as odontoblast processes extenddehydration through a graded ethanol series. Specimens were critical point dried in ethanol-CO,, mouning throughout the entire thickness of dentine. One possible explanation for this discrepancy may ted on aluminium stubs and examined with an Hitreside in the failure of the SEM studies to recognize achi 3010 SEM (Hitachi Co., Tokyo, Japan) at 20 kV. the inner hypomineralized lining of dentinal tubules; Representative areas were photographed. Following a propylene oxide wash, all specimens previously this lining has been noted in some studies (Isokawa, Todo and Kubota, 1970; Isokawa et al., 1972; scanned were embedded in Epon. Sections ranging from 60 to 80nm were cut from the surface preThomas and Carella, 1983). viously scanned, stained with uranyl acetate and lead Our purpose was to examine areas of human citrate and examined with an Hitachi 300 TEM at dentine first by SEM and then subsequently by TEM 80 kV. Areas of interest which corresponded to areas to determine whether the structures seen by SEM previously scanned were again photographed. within outer dentinal tubules were cellular or whether they correspond to the inner hypomineralized lining of peritubular dentine. RESULTS MATERIALSAND Ten unerupted
METHODS
but fully-formed human third mo-
Odontoblasts were seen by SEM adjacent to a layer of predentine (Fig. 1). Odontoblast processes were observed leaving the cell bodies and entering the
H. F. THOMAS and P. CARELLA
642
predentine layer. In corresponding areas seen by TEM (Fig. 2) odontoblast processes were present in predentine. Degeneration of some odontoblast processes was visible, and some processes appeared to have shrunk away from the predentine matrix. However, no intermediate structures could be seen between the cell membranes of the odontoblast processes and the predentine matrix. Odontoblast processes were not found in dentinal tubules in SEM from the inner third of dentine (Fig. 3). The tubules were lined by an amorphous sheet-like structure which was separated from the tubule walls in some areas. Corresponding TEM (Fig. 4) showed odontoblast processes within dentinal tubules. However, in contrast to the appearance seen in predentine (Fig. 2), electron-dense structures that were clearly distinguishable from the cell membrane of the odontoblast process lined all tubules. Areas of peritubular dentine formation were visible and, where they occurred, were separated from the lumena of tubules by electron-dense structures (Fig, 4). All tubules from the middle and outer third of crown dentine examined by SEM (Fig. 5) contained sheet-like structures. A space was present between this structure and the intertubular dentine matrix. This space, termed the peritubular matrix space, resulted from loss of peritubular dentine during demineralization. Corresponding TEM (Fig. 6) showed electron-dense structures within each tubule which corresponded directly to the sheet-like structures seen in SEM. A space, the peritubular matrix space, was present between the electron-dense structure and the intertubular dentine matrix. Similar structures were seen in dentinal tubules cut in cross-section which were representative of those teeth which were sliced occluso-cervically. In tubules from the inner third of dentine (Fig. 7), solid structures were present within each tubule by SEM separated from tubule walls by a space. In corresponding TEM (Fig. 8), each dentinal tubule contained an electron-dense structure separated from the tubule wall by a space. Odontoblast processes were not seen in any sections. Higher magnification of tubules from the inner third of crown dentine (Figs 9 and 10) showed, in some areas, a fibrillar component in the peritubular matrix. These fibrils appeared to connect the sheet-like structure to the intertubular dentine matrix (Fig. 9). Dentinal tubules from mid and outer thirds of dentine (Figs 11 and 12) were similar in appearance to those from the inner third. SEM (Fig. 1 I) showed within each dentinal tubule a sheet-like structure, whereas TEM (Fig. 12) showed that these structures corresponded directly with the electron-dense structures which lined all tubules. No odontoblast processes were seen in any tubules examined. DISCUSSION
The study shows that the structures observed throughout dentinal tubules by SEM correspond directly with electron-dense structures seen by TEM in demineralized dentinal tubules. This electron-dense structure has previously been identified as the inner hypomineralized lining of peritubular dentine (Shroff ef al., 1956; Takuma, 1967; Thomas, 1979). The
structure seen within dentinal tubules by SEM is not, therefore, the odontoblast process but rather the inner hypomineralized lining of peritubular dentine in accord with the views of others (see Introduction). Isokawa et al. (1972) described a tubular membraneous structure which appeared on the surface of fractured dentine following etching with a weak acid. Thomas and Carella (1983) showed by SEM that all tubules are lined by such structures throughout their lengths. In accord with terminology already established for a similar structure observed in bone and mineralizing cartilage (Scherft, 1972; Shepard and Mitchell, 198 I), Thomas and Carella (1983) proposed that this structure be called the lamina limitans. The lamina limitans is not the odontoblast process; it does not possess a trilaminar structure and is too thick to represent a cell membrane. Additionally, when both the lamina limitans and odontoblast process are seen together, they are clearly distinguishable (Fig. 4). The separation between the lamina limitans and intertubular dentine increases as the distance from the pulp increases. This is due to the increase in amount of peritubular dentine formation, which is minimal in inner dentine (Fig. 4) and increases in mid and outer denture (Fig. 12; Atkinson and Harcourt, 1961). We did not see odontoblast processes in any tubules where there was more than minimal peritubular dentine formation, confirming previous observations (Thomas, 1979). In view of the presence of the lamina limitans in each dentinal tubule, care must be exercised in the identification of structures within dentinal tubules by SEM. An alternative interpretation for structures identified as odontoblast processes in outer dentine by SEM by previous workers (Kelley, Bergenholtz and Cox, 1981; Crooks et al.. 1983: Grossman and Austin, 1983; Maniatopoulos and Smith, 1983) is that they are not odontoblast processes but rather the lamina limitans. Furthermore, as the lamina limitans is thought to be composed primarily of glycosaminoglycans (Thomas and Carella, 1983), studies utilizing demineralization and collagenase digestion of dentine (Yamada et al., 1983; Gunji and Kobayashi. 1983) would also leave laminae limitantes lying on the exposed surface. Therefore, to be able to identify by SEM a structure as the odontoblast process, concurrent TEM should demonstrate a cellular process, a cell membrane and intracellular organelles. The technique employed here seems well suited for the examination of dentine. The combination of osmication of tissue followed by thiocarbohydrazide treatment coated the surface of the tissue sufficiently to allow SEM observations to be made. The resulting SEM image was not as good as that of tissue treated by conventional gold coating; lack of contrast was a particular deficiency. However, subsequent embedding of the tissue in Epon and thin-sectioning provided sections of sufficient quality for visualization by TEM. In tubules seen in cross-section by SEM, the lamina limitans appeared to be a solid structure. As corresponding TEM show that lamina limitans is in fact patent, we believe that the solid appearance represents an artifact of slicing the tooth and results from debris accumulating within the lamina limitans.
SEM/TEM of human dentinal tubules Transmission electron micrographs fail to reveal the extent of the organic matrix of peritubular dentine. Scanning electron micrographs, however, showed that the matrix is variable, and in certain areas (Fig. 9), can be considerable. The fibrillar component of this matrix appears to connect the lamina limitans to the matrix of intertubular dentine. The lumena of some tubules contained granular material. This material may result from processing of the tissue, and possibly be due to some modification of the lamina limitans following demineralization or thiocarbohydraxide treatment. As the lamina limitans probably contains glycomacromolecules, this modification would not be surprising because thiocarbohydrazide is used for the ultracytochemical demonstration of glycomacromolecules (Pearse, 1972). We previously proposed (Thomas and Carella, 1983) based on histochemical studies (Symons, 1967; Wislocki, Singer and Waldo, 1948), that the lamina limitans contains glycosaminoglycans and glycoproteins and may play a role in the control of deposition of peritubular dentine. We also believe that the lamina limitans corresponds to the fibrils of soft tissue first described within dentinal tubules by Tomes (1856). REFERENCES
Atkinson H. F. and Harcourt J. K. (1961) Some observations on the peritubular translucent zones in human dentine. Aust. dent. J. 6, 194197. Bradford E. W. (1967) Microanatomv and Histochemistrv of Dentine. In; Strthmzl and Chemical Organization oj Teeth (Edited by Miles A. E. W.) Vol. 2, pp. 3-34. Academic Press, New York. Brannstrom M. and Garberoglio R. (1972) The dentinal tubules and the odontoblast processes. Acta odont. stand. 30, 291-311.
Crooks P. V., O’Reilly C. B. and Owens P. D. A. (1983) Microscopy of the dentine of enamel-free areas of rat molar teeth. Archs oral Biol. Zs, 167-175. Grossman E. S. and Austin J. C. (1983) Scanning electron microscope observations on the tubule content of freeze-
fractured peripheral vervet monkey dentine (cercopithecus pyserythrus).
Archs oral Biol. 28, 279-281.
Gunji T. and Kobayashi S. (1983) Distribution and organization of odontoblast processes in human dentin. Arch. Hist. Jap. 46, 213-219.
Holland G. R. (1976) The extent of the odontoblast process in the cat. J. Anat. 121, 133-149. Isokawa S., Toda R. and Kubota K. (1970) A scanning electron microscopic observation of etched human peritubular dentin. Archs oral Biol. 15, 1303-1306. Isokawa S., Yoshida M., Komura A. and Iwatake Y. (1972) A preliminary study on the peritubular structure of human dentinal tubules by scanning electron microscopy. J. Nihon Univ. Sch. Dent., 14, 122-125. Johansen E. (1967) Ultrastructure of dentin. In: Structural
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and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 2, pp. 3>74. Academic Press, New York. Karnovsky M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137A-138A. Kelly K. W., Bergenholtz G. and Cox C. F. (1981) The extent of the odontoblast process in Rhesus monkeys (Macaca mulatta) as observed by scanning electron microscopy. Archs oral Biol. 26, 893-897. Kellv R. 0.. Dekker R. A. F. and Bluemink J. G. (1973) L;gand-mediated osmium binding: Its application in coat: ing biological specimens from scanning electron microscopy. J. Ultrastruci. Res. 45, 254-258. Maniatopoulos C. and Smith D. C. (1983) A scanning electron microscopic study of the odontoblast process in human coronal dentine. Archs oral Biol. 28, 701-710. Morse A. (1945) Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J. dent. Res. 24, 143-153. Pearse A. G. E. (1972) Histochemistry. Theoretical and Applied, Vol. 2, 3rd edn, pp. 1273-1274. Churchill Livingstone, London. Scherft J. P. (1972) The lamina limitans of the organic . matrix of calcified cartilage and bone. J. Ultrastruct. Res. 38, 318-331.
Shepard N. and Mitchell N. (1981) Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification. Histochemistry 70, 107-l 14. Shroff F. R., Williamson K. I., Bertaud W. S. and Hall D. M. (1956) Further electron microscope studies of dentin. The nature of the odontoblast process. Oral Surg. 9, 432443. Symons N. B. B. (1967) The microanatomy and histochemistry of dentinogenesis. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, pp. 285-324. Academic Press, New York. Takuma S. (1967) Ultrastructure of dentinogenesis. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, pp. 325-370. Academic Press,
New York. Thomas H. F. (1979)The extent of the odontoblast process in human dentin. J. dent. Res. 58, 2207-2218. Thomas H. F. (1983) The effect of various fixatives on the extent of the odontoblast process in human dentine. Archs oral Biol. 28, 465-469.
Thomas H. F. and Payne R. C. (1983) The ultrastructure of dentinal tubules from erupted human premolar teeth. J. dent. Res. 62, 532-536.
Thomas H. F. and Carella P. (1983) A scanning electron microscope study of dentinal tubules from human teeth. Archs oral Biol. 28, 1125-I 130.
Tidmarsh B. G. (1981) Contents of human dentinal tubules. ht. endo. J. 14, 191-196. Tomes J. (1856) On the presence of fibrils of soft tissue in the dentinal tubes. Phil. Trans. 146, 515-522. Wislocki G. B., Singer M. and Waldo C. M. (1948) Some histochemical reactions of mucopolysaccharides, giycogen, lipids and other substances in teeth. Anat. Rec. 101, 487-5 13. Yamada T., Nakamura K., Iwaku M. and Fusayama T. (1983) The extent of the odontoblast process in normal and carious human dentin. J. dent. Res. 62, 798-802.
Plates 1 and 2 overleaf.
H. F. THOMAS and P. CARELLA
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Plate
I
Fig. I. SEM view of predentine. Odontoblast process (OP) can be seen leaving the odontoblast (0) and entering a layer of predentine (PD).
cell body
Fig. 2. TEM view of predentine. Odontoblast processes (OP) can be seen within a layer of predentine (PD). No intermediate structure can be observed between the cell membrane of the odontoblast process and the predentine matrix. Fig. 3. SEM view of tubules from inner third of dentine. In some areas (+) separation of the sheet-like structure from the tubule wall can be seen indicating areas of early peritubular dentine formation. ID = intertubular dentine. Fig. 4. TEM tubules. Each membrane of (PTM),
view of tubules from inner third of dentine. Odontoblast processes (OP) can be seen within tubule is lined by an electron-dense structure (LS) which is distinguishable from the cell the odontoblast process (+ t). Early areas of peritubular dentine formation are visible the electron-dense structure is visible on the luminal side of the peritubular matrix.
Fig. 5. SEM of tubules from mid dentine. Intertubular dentine (ID) is visible between each tubule. tubule contains a sheet-like structure (LL). No odontoblast processes are visible.
Each
Fig. 6. TEM of tubules from mid dentine. The electron-dense structure lining all tubules (LS) corresponds directly with the sheet-like structure seen by SEM. A space (PTM) is present between the electron-dense structure (LS) and intertubular dentine (ID). This space corresponds to peritubular dentine which has been lost through demineralization. Plate 2. Fig. 7. SEM of cross-sectioned tubules from inner dentine. Each tubule contains a solid structure which is separated from the intertubular dentine (ID) by a space (PTM). Fig. 8. Corresponding
TEM view of an area seen in Fig. 7. Each tubule contains structure (LS). No odontoblast processes are visible.
an electron-dense
Fig. 9. Higher magnification of tubule from inner dentine viewed by SEM. The solid structure appears to be connected to the intertubular dentine (ID) by a fibrillar material. Fig. 10. Corresponding Fig. Il.
TEM view of Fig. 9. The electron-dense structure the solid structure seen by SEM.
(LS) corresponding
SEM view of tubules from mid third of dentine. Each tubule contains a solid structure is separated from intertubular dentine (ID) by a space (PTM).
Fig. 12. Corresponding electron-dense structure
(LL)
(LL)
directly with (LL) which
TEM view of tubules from mid third dentine. Each tubule contains (LS) which is separated from intertubular dentine (ID) by a space (PTM). odontoblast processes are visible.
an No
SEM/TEM
of human
Plate
dentinal
1.
tubules
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H. F. THOMAS and P. CARELLA
Plate 2