Transmission Electron Microscopic Characterization of Hypersensitive Human Radicular Dentin M. YOSHIYAMA, Y. NOIRI, K. OZAKI, A. UCHIDA, Y. ISHIKAWA', and H. ISHIDA' Departments of Operative Dentistry and 'Pharmacology, Tokushima University School of Dentistry, 3-18-15, Kuramoto-Cho, Tokushima 770, Japan
Transmission electron microscopy (TEM) and x-ray microanalysis (XMA) were used for the study of the ultrastructure of the lumens of dentinal tubules in superficial layers of dentin specimens obtained by use of a new biopsy technique from both hypersensitive and naturally desensitized areas of exposed root surfaces, in vivo. The TEM images showed clearly that the lumens of most of the tubules were occluded with mineral crystals in naturally desensitized areas, but such lumens were empty and surrounded with peritubular and intertubular dentin in hypersensitive areas. Moreover, electron-dense structures that lined peritubular dentin were observed in the empty lumens of dentinal tubules. J Dent Res 69(6):1293-1297, June, 1990
Introduction. The mechanisms of dentin hypersensitivity have been extensively investigated. The hydrodynamic theory of dentin sensitivity of Brannstrbm (1966) suggests that minute shifts of dentinal fluid or tubule contents occur in response to tactile, thermal, or osmotic stimuli. Pashley (1986) suggested that dentin hypersensitivity was associated with open dentinal tubules in hypersensitive areas. In a previous paper on a scanning electron microscopic (SEM) and microradiographic study of dentin specimens obtained by use of a new biopsy technique, we reported that the orifices of many dentinal tubules were open in hypersensitive areas but occluded with crystals in naturally desensitized areas on the same root surface (Yoshiyama et al., 1989). The purpose of the present study was to investigate the ultrastructure of the lumens of dentinal tubules in both hypersensitive and naturally desensitized areas of exposed radicular dentin, and to examine the intratubular crystals with x-ray mi-
cro-analysis.
use of a hollow cylindrical diamond bur (Shofu Inc., Japan) with a high-speed handpiece under water cooling (Fig. 1). TEM. -Eight pairs of dentin biopsy specimens from both hypersensitive and naturally desensitized areas were fixed in 4% glutaraldehyde solution, then post-fixed in 2% osmium tetroxide and embedded in epoxy resin (Epok 812), as described previously (Yoshiyama et al., 1989). Dentin sections with a thickness of 80 Axm were cut vertically to the exposed surfaces from embedded specimens by use of an Isomet lowspeed saw (Buehler Ltd., Evanston, IL). They were ground to a thickness of about 40 Axm with successively finer emery papers (from #600 to #1000). During a 20-minute exposure, the sections were radiographed at 10 kV and 3 mA with a contact microradiographer CMR-2 (Japan Softex, Japan). Kodak Spectroscopic emulsion type 649-0 (Eastman Kodak Co., Rochester, NY) was used for registration of the images. The film was developed for 50 min at 40C. Microradiograms were mounted on microscope slides and photographed. Dentin ground sections that had been microradiographed were embedded again in epoxy resin. This enabled ultrathin sections to be cut, thus showing transverse views of dentinal tubules in the superficial layer of exposed dentin. Ultrathin sections were cut with a diamond knife, and the sections were floated on a solution supersaturated with dentin powder, so that demineralization would be minimized. The sections were examined with a TEM
-
hypersensitive area
naturally-desensitized area Dentin biopsy of a cylindrical diamond point 0.2mm 1.0mm
Materials and methods. The subjects were eight adult patients who suffered from dentin hypersensitivity within wedge-shaped root dentin defects. The hypersensitive teeth were examined by stimulation of the exposed dentin surfaces within the wedge-shaped defects with each of three stimuli: (1) mechanical stimulation with a No. 23 dental explorer, (2) water at 20'C for five s, and (3) a compressed-air blast for five s. Hypersensitive and naturally desensitized areas of exposed dentin were distinguished by precise stimulation of the surface with an explorer. After performance of infiltration anesthesia and isolation of the tooth, cylindrical dentin specimens (with a diameter of 0.8 mm and a height of 0.5-0.8 mm) were obtained from the hypersensitive and adjacent naturally desensitized areas, respectively, with Received for publication July 11, 1989 Accepted for publication January 5, 1990
1.8mm 8 pairs of dentin biopsy specimens
Microradiography (MR)
Transmission electron microscopy (TEM)
X-ray microanalysis (XMA) Fig. 1-Schematic diagram of the method involved in the preparation of the specimens.
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Surface
(A)
Fig. 3-TEM image of a superficial layer in a dentin specimen obtained from a hypersensitive area. Most of the lumens of dentinal tubules (arrows) are empty. Bar = 2 Arm. TABLE 1 THE NUMBERS OF THE OCCLUDED DENTINAL TUBULES AND THE TOTAL DENTINAL TUBULES EXAMINED IN ALL TEM IMAGES OF HYPERSENSITIVE AND NATURALLY DESENSITIZED AREAS
(8)
Fig. 2-Microradiograms of exposed dentin surface areas of a hypersensitive and naturally desensitized area. (A) shows a dentin surface of a naturally desensitized area, and (B) shows a hypersensitive area.
(H-500, Hitachi Co., Tokyo, Japan) at an accelerating voltage of 75 kV. XMA. -For energy-dispersive analysis of the intratubular material of dentin specimens, an XMA (7000Q, Kevex, Foster, CA) attached to a TEM (H-500, Hitachi Co., Tokyo, Japan) was used. The ultrathin sections were examined after being carbon-coated under a vacuum. The system was operated as follows: accelerating voltage, 10 kV; spot size, 100 nm; counting time, 100 s.
Results. Prior to TEM, eight pairs of biopsy specimens were subjected to microradiography. The microradiographic observations showed that most of the dentinal tubules appeared to be occluded with radio-opaque materials in naturally desensitized areas, but that, in hypersensitive areas, most of the lumens of the dentinal tubules appeared to be empty, as shown in Fig. 2. TEM. -In all TEM images of ultrathin sections of the superficial layers in the dentin specimens from hypersensitive areas, a large number of the lumens of the dentinal tubules were empty, as shown in Fig. 3. The ratio of the occluded dentinal tubules to the total dentinal tubules found in all TEM images of eight specimens from hypersensitive areas was 15.0 + 8.4%, as shown in Table 1. The lumens had a diameter of 0.5-1.5 pm and were surrounded with a higher electron-dense zone than the intertubular dentin, which was thought to be
Hypersensitive
Naturally Desensitized
Areas
Areas
210 160 Number of total dentinal tubules 24 170 Number of occluded dentinal tubules 81.0 ± 8.1 15.0 ± 8.4 Ratio (%) The values of the ratios were the means of the percentages of occluded dentinal tubules to the total dentinal tubules examined ± standard errors.
peritubular dentin (Fig. 4). Electron-dense materials that lined the peritubular dentin were observed in some of the empty tubules (Fig. 5). On the other hand, in all TEM images of the superficial layers in the dentin specimens from naturally desensitized areas, a large percentage of the lumens of the dentinal tubules was occluded with high electron-dense materials, which appeared to be continuous with the peritubular dentin (Fig. 6). The ratio of the occluded dentinal tubules to the total dentinal tubules found in all TEM images of eight specimens from naturally desensitized areas was 81.0 ± 8.1% (Table 1). TEM images at higher magnification showed that the lumens of tubules were occluded with both peritubular dentin and with extremely fine crystals of sizes less than 0.1 Am, or with large rhombohedral crystals deposited in the central region (Fig. 7). In some tubules, the lumens were partly occluded with fine needle-like crystals that were deposited in the peripheral region along with the peritubular dentin (Fig. 8). Accordingly, these results suggested that the lumens of dentinal tubules in naturally desensitized areas were occluded with mainly three kinds of materials: (1) high electron-dense materials continuous with peritubular dentin , (2) fine crystals not continuous with peritubular dentin, and (3) a mixed form composed of high electron-dense materials and fine crystals. Moreover, it was confirmed that the inhibition of the occlusion of dentinal tubules was involved in the occurrence of dentin hypersensitivity. XMA. -The molar ratios of calcium (Ca) and phosphorus (P) in intertubular dentin, peritubular dentin, and the material
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Vol. 69 No. 6
TEM CHARACTERIZATION OF HYPERSENSITIVE DENTIN
Fig. 4-Higher magnification of a dentinal tubule of a hypersensitive area. The lumen of the tubule is surrounded with peritubular dentin, which is more highly mineralized (arrow) than the surrounding intertubular dentin. Bar = 1 WLm.
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Fig. 5-Higher magnification of an electron-dense structure in a lumen of a dentinal tubule of a hypersensitive area. The electron-dense structure (arrow) lines the peritubular dentin of the empty dentinal tubules. Bar = 1 WLm.
that occluded dentinal tubules in naturally desensitized areas were examined by XMA. The presence of Ca and P was clearly detected in the materials described above, and there were no significant differences among the mean values of the ratios (Table 2). The elements of Ca and P were not detected in the electron-dense structures within the lumens of hypersensitive areas.
Discussion. We previously reported that the orifices of many dentinal tubules were open in hypersensitive areas and occluded with crystals in naturally desensitized areas on the same dentin surface (Yoshiyama et al., 1989). In this paper, we used TEM to study the ultrastructure of the lumens of the dentinal tubules in superficial layers of dentin specimens from both hypersensitive and naturally desensitized dentin. The images of the lumens of the dentinal tubules showed clearly that most of the lumens of the tubules were also occluded with mineral crystals in naturally desensitized areas. These findings were in accordance with our previous results described above, and they are also supported by the results of the obturation of dentinal tubules on the root surface by mineral deposition (Hiatt and Johansen, 1972), the formation of intratubular deposition by the repetitive addition of minerals on the walls of tubules (Tronstad, 1973), and the occlusion of dentinal tubules with a continuous growth of peritubular dentin (Brinnstrdm and Garberoglio, 1980). It was also clarified that empty dentinal tubules were surrounded with both peritubular and intertubular dentin (Fig. 4). Moreover, electron-dense structures that lined
Fig. 6-TEM image of a superficial layer in a dentin specimen obtained from a naturally desensitized area. Most lumens of the dentinal tubules (arrows) are occluded with highly electron-dense materials. The material seems to be a continuous growth of peritubular dentin. Bar = 2 ,urm.
peritubular dentin were often observed in the empty lumens of dentinal tubules (Fig. 5). Fig. 5 reveals what appears to be some type of intratubular material covered with an electrondense, fuzzy coating. This is similar to the non-cellular material that Holland (1985) found in peripheral dentin. We could not detect by XMA the presence of Ca and P in these electrondense structures. The results suggest that the electron-dense structure consists of organic components and corresponds with
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Fig. 7-Higher magnification of a dentinal tubule of a naturally desensitized area. The lumen is occluded with extremely fine crystals (arrow A) and large rhombohedral crystals (arrow B). Bar = 1 pLm.
Fig. 8-Higher magnification of a partly occluded dentinal tubule of a naturally desensitized area. Fine needle-like crystals (arrow) are deposited in the peripheral region of a lumen. Bar = 1 pAm.
the partition structure previously observed by SEM in the same site. By TEM, Thomas and Carella (1984) observed electrondense structures lining human dentinal tubules, and termed them lamina limitans, not odontoblastic processes. This structure has previously been identified as the inner hypomineralized lining of peritubular dentin (Shroff et al., 1956; Takuma, 1967; Thomas, 1979). On the other hand, Pashley et al. (1982) suggested that the materials that had electron-dense structure might represent plasma protein that leaked from pulpal capillaries under exposed tubules, and they estimated that fibrinogen could be among those proteins and that it could convert to fibrin within the tubules (Pashley et al., 1984; Pashley, 1986). We used ultrathin sections of undecalcified dentin specimens for the present study. Therefore, further study is required for clarification of the intratubular materials by use of EDTA-decalcified
dentin specimens, and perhaps immunohistochemical probes that are specific for fibrinogen, fibrin, or glycoproteins of the lamina limitans. The results of this study support the hydrodynamic theory of dentin sensitivity (Brannstrdm, 1966) that postulates that sensitive dentin must have open tubules. It also supports a corollary to that theory which predicts that exposed, insensitive dentin would have tubules that are occluded. The prediction was borne out by the results.
TABLE 2 MOLAR RATIOS OF Ca AND P IN THE SUPERFICIAL LAYERS OF DENTIN SPECIMENS IN NATURALLY DESENSITIZED AREAS Areas Examined Molar Ratio (Ca/P) Intertubular dentin 1.51 - 0.09 Peritubular dentin 1.49 + 0.07 The materials that 1.43 - 0.05 occluded dentinal tubules Th molar ratios of calcium (Ca) and phosphorus (P) were examined with XMA, as described in "Materials and methods". The values were the means of the values of eight experiments - standard error. There were no significant differences in these data (p>0.1).
REFERENCES
BRANNSTROM, M. (1966): Sensitivity of Dentine, Oral Surg Oral Med Oral Pathol 21:517-529.
BRANNSTROM, M. and GARBEROGLIO, R. (1980): Occlusion of Dentinal Tubules under Superficial Attrited Dentine, Swed Dent J 4:87-91. HIATT, W.H. and JOHANSEN, E. (1972): Root Preparation I. Obturation of Dentinal Tubules in Treatment of Root Hypersensitivity, J Periodontol 43:373-380. HOLLAND, G.R. (1985): The Odontoblast Process: Form and Function, J Dent Res 64:499-514. PASHLEY, D.H. (1986): Dentin Permeability, Dentin Sensitivity, and Treatment through Tubule Occlusion, J Endodontics 12:465474.
PASHLEY, D.H.; GALLOWAY, S.E.; and STEWART, F.P. (1984): Effects of Fibrinogen in vivo on Dentin Permeability in the Dog, Ar-ch Oral Biol 29:725-728. PASHLEY, D.H.; NELSON, R.; and KEPLER, E.E. (1982): Effects of Plasma and Salivary Constituents on Dentin Permeability, J Dent Res 61:978-981. SHROFF, F.R.; WILLIAMSON, K.I.; BERTAUD, W.S.; and HALL,
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Vol. 69 No. 6
TEM CHARACTERIZATION OF HYPERSENSITIVE DENTIN
D.M. (1956): Further Electron Microscope Studies of Dentin. The Nature of the Odontoblast Process, Oral Surg Oral Med Oral Pathol 9:432-443. TAKUMA, S. (1967): Ultrastructure of Dentinogenesis. In: Structural and Chemical Organization of Teeth, Vol. 1, A.E.W. Miles, Ed., New York: Academic Press, pp. 325-370. THOMAS, H.F. (1979): The Extent of the Odontoblast Process in Human Dentin, J Dent Res 58:2207-2218. THOMAS, H.F. and CARELLA, P. (1984): Correlation of Scanning
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and Transmission Electron Microscopy of Human Dentinal Tubules, Arch Oral Biol 29:641-646. TRONSTAD, L. (1973): Scanning Electron Microscopy of Attrited Dentinal Surfaces and Subjacent Dentin in Human Teeth, Scand J Dent Res 81:112-122. YOSHIYAMA, M.; MASADA, J.; UCHIDA, A.; and ISHIDA, H. (1989): Scanning Electron Microscopic Characterization of Sensitive vs. Insensitive Human Radicular Dentin, J Dent Res 68:14981502.
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Transmission electron microscopy (TEM) and x-ray microanalysis (XMA) were used for the study of the ultrastructure of the lumens of dentinal tubules in superficial layers of dentin specimens obtained by use of a new biopsy technique from both hypersensitive and naturally desensitized areas of exposed root surfaces, in vivo. The TEM images showed clearly that the lumens of most of the tubules were occluded with mineral crystals in naturally desensitized areas, but such lumens were empty and surrounded with peritubular and intertubular dentin in hypersensitive areas. Moreover, electron-dense structures that lined peritubular dentin were observed in the empty lumens of dentinal tubules. J Dent Res 69(6):1293-1297, June, 1990
Introduction. The mechanisms of dentin hypersensitivity have been extensively investigated. The hydrodynamic theory of dentin sensitivity of Brannstrbm (1966) suggests that minute shifts of dentinal fluid or tubule contents occur in response to tactile, thermal, or osmotic stimuli. Pashley (1986) suggested that dentin hypersensitivity was associated with open dentinal tubules in hypersensitive areas. In a previous paper on a scanning electron microscopic (SEM) and microradiographic study of dentin specimens obtained by use of a new biopsy technique, we reported that the orifices of many dentinal tubules were open in hypersensitive areas but occluded with crystals in naturally desensitized areas on the same root surface (Yoshiyama et al., 1989). The purpose of the present study was to investigate the ultrastructure of the lumens of dentinal tubules in both hypersensitive and naturally desensitized areas of exposed radicular dentin, and to examine the intratubular crystals with x-ray mi-
cro-analysis.
use of a hollow cylindrical diamond bur (Shofu Inc., Japan) with a high-speed handpiece under water cooling (Fig. 1). TEM. -Eight pairs of dentin biopsy specimens from both hypersensitive and naturally desensitized areas were fixed in 4% glutaraldehyde solution, then post-fixed in 2% osmium tetroxide and embedded in epoxy resin (Epok 812), as described previously (Yoshiyama et al., 1989). Dentin sections with a thickness of 80 Axm were cut vertically to the exposed surfaces from embedded specimens by use of an Isomet lowspeed saw (Buehler Ltd., Evanston, IL). They were ground to a thickness of about 40 Axm with successively finer emery papers (from #600 to #1000). During a 20-minute exposure, the sections were radiographed at 10 kV and 3 mA with a contact microradiographer CMR-2 (Japan Softex, Japan). Kodak Spectroscopic emulsion type 649-0 (Eastman Kodak Co., Rochester, NY) was used for registration of the images. The film was developed for 50 min at 40C. Microradiograms were mounted on microscope slides and photographed. Dentin ground sections that had been microradiographed were embedded again in epoxy resin. This enabled ultrathin sections to be cut, thus showing transverse views of dentinal tubules in the superficial layer of exposed dentin. Ultrathin sections were cut with a diamond knife, and the sections were floated on a solution supersaturated with dentin powder, so that demineralization would be minimized. The sections were examined with a TEM
-
hypersensitive area
naturally-desensitized area Dentin biopsy of a cylindrical diamond point 0.2mm 1.0mm
Materials and methods. The subjects were eight adult patients who suffered from dentin hypersensitivity within wedge-shaped root dentin defects. The hypersensitive teeth were examined by stimulation of the exposed dentin surfaces within the wedge-shaped defects with each of three stimuli: (1) mechanical stimulation with a No. 23 dental explorer, (2) water at 20'C for five s, and (3) a compressed-air blast for five s. Hypersensitive and naturally desensitized areas of exposed dentin were distinguished by precise stimulation of the surface with an explorer. After performance of infiltration anesthesia and isolation of the tooth, cylindrical dentin specimens (with a diameter of 0.8 mm and a height of 0.5-0.8 mm) were obtained from the hypersensitive and adjacent naturally desensitized areas, respectively, with Received for publication July 11, 1989 Accepted for publication January 5, 1990
1.8mm 8 pairs of dentin biopsy specimens
Microradiography (MR)
Transmission electron microscopy (TEM)
X-ray microanalysis (XMA) Fig. 1-Schematic diagram of the method involved in the preparation of the specimens.
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YOSHIYAMA et al.
5Ojim
Surface
(A)
Fig. 3-TEM image of a superficial layer in a dentin specimen obtained from a hypersensitive area. Most of the lumens of dentinal tubules (arrows) are empty. Bar = 2 Arm. TABLE 1 THE NUMBERS OF THE OCCLUDED DENTINAL TUBULES AND THE TOTAL DENTINAL TUBULES EXAMINED IN ALL TEM IMAGES OF HYPERSENSITIVE AND NATURALLY DESENSITIZED AREAS
(8)
Fig. 2-Microradiograms of exposed dentin surface areas of a hypersensitive and naturally desensitized area. (A) shows a dentin surface of a naturally desensitized area, and (B) shows a hypersensitive area.
(H-500, Hitachi Co., Tokyo, Japan) at an accelerating voltage of 75 kV. XMA. -For energy-dispersive analysis of the intratubular material of dentin specimens, an XMA (7000Q, Kevex, Foster, CA) attached to a TEM (H-500, Hitachi Co., Tokyo, Japan) was used. The ultrathin sections were examined after being carbon-coated under a vacuum. The system was operated as follows: accelerating voltage, 10 kV; spot size, 100 nm; counting time, 100 s.
Results. Prior to TEM, eight pairs of biopsy specimens were subjected to microradiography. The microradiographic observations showed that most of the dentinal tubules appeared to be occluded with radio-opaque materials in naturally desensitized areas, but that, in hypersensitive areas, most of the lumens of the dentinal tubules appeared to be empty, as shown in Fig. 2. TEM. -In all TEM images of ultrathin sections of the superficial layers in the dentin specimens from hypersensitive areas, a large number of the lumens of the dentinal tubules were empty, as shown in Fig. 3. The ratio of the occluded dentinal tubules to the total dentinal tubules found in all TEM images of eight specimens from hypersensitive areas was 15.0 + 8.4%, as shown in Table 1. The lumens had a diameter of 0.5-1.5 pm and were surrounded with a higher electron-dense zone than the intertubular dentin, which was thought to be
Hypersensitive
Naturally Desensitized
Areas
Areas
210 160 Number of total dentinal tubules 24 170 Number of occluded dentinal tubules 81.0 ± 8.1 15.0 ± 8.4 Ratio (%) The values of the ratios were the means of the percentages of occluded dentinal tubules to the total dentinal tubules examined ± standard errors.
peritubular dentin (Fig. 4). Electron-dense materials that lined the peritubular dentin were observed in some of the empty tubules (Fig. 5). On the other hand, in all TEM images of the superficial layers in the dentin specimens from naturally desensitized areas, a large percentage of the lumens of the dentinal tubules was occluded with high electron-dense materials, which appeared to be continuous with the peritubular dentin (Fig. 6). The ratio of the occluded dentinal tubules to the total dentinal tubules found in all TEM images of eight specimens from naturally desensitized areas was 81.0 ± 8.1% (Table 1). TEM images at higher magnification showed that the lumens of tubules were occluded with both peritubular dentin and with extremely fine crystals of sizes less than 0.1 Am, or with large rhombohedral crystals deposited in the central region (Fig. 7). In some tubules, the lumens were partly occluded with fine needle-like crystals that were deposited in the peripheral region along with the peritubular dentin (Fig. 8). Accordingly, these results suggested that the lumens of dentinal tubules in naturally desensitized areas were occluded with mainly three kinds of materials: (1) high electron-dense materials continuous with peritubular dentin , (2) fine crystals not continuous with peritubular dentin, and (3) a mixed form composed of high electron-dense materials and fine crystals. Moreover, it was confirmed that the inhibition of the occlusion of dentinal tubules was involved in the occurrence of dentin hypersensitivity. XMA. -The molar ratios of calcium (Ca) and phosphorus (P) in intertubular dentin, peritubular dentin, and the material
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Vol. 69 No. 6
TEM CHARACTERIZATION OF HYPERSENSITIVE DENTIN
Fig. 4-Higher magnification of a dentinal tubule of a hypersensitive area. The lumen of the tubule is surrounded with peritubular dentin, which is more highly mineralized (arrow) than the surrounding intertubular dentin. Bar = 1 WLm.
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Fig. 5-Higher magnification of an electron-dense structure in a lumen of a dentinal tubule of a hypersensitive area. The electron-dense structure (arrow) lines the peritubular dentin of the empty dentinal tubules. Bar = 1 WLm.
that occluded dentinal tubules in naturally desensitized areas were examined by XMA. The presence of Ca and P was clearly detected in the materials described above, and there were no significant differences among the mean values of the ratios (Table 2). The elements of Ca and P were not detected in the electron-dense structures within the lumens of hypersensitive areas.
Discussion. We previously reported that the orifices of many dentinal tubules were open in hypersensitive areas and occluded with crystals in naturally desensitized areas on the same dentin surface (Yoshiyama et al., 1989). In this paper, we used TEM to study the ultrastructure of the lumens of the dentinal tubules in superficial layers of dentin specimens from both hypersensitive and naturally desensitized dentin. The images of the lumens of the dentinal tubules showed clearly that most of the lumens of the tubules were also occluded with mineral crystals in naturally desensitized areas. These findings were in accordance with our previous results described above, and they are also supported by the results of the obturation of dentinal tubules on the root surface by mineral deposition (Hiatt and Johansen, 1972), the formation of intratubular deposition by the repetitive addition of minerals on the walls of tubules (Tronstad, 1973), and the occlusion of dentinal tubules with a continuous growth of peritubular dentin (Brinnstrdm and Garberoglio, 1980). It was also clarified that empty dentinal tubules were surrounded with both peritubular and intertubular dentin (Fig. 4). Moreover, electron-dense structures that lined
Fig. 6-TEM image of a superficial layer in a dentin specimen obtained from a naturally desensitized area. Most lumens of the dentinal tubules (arrows) are occluded with highly electron-dense materials. The material seems to be a continuous growth of peritubular dentin. Bar = 2 ,urm.
peritubular dentin were often observed in the empty lumens of dentinal tubules (Fig. 5). Fig. 5 reveals what appears to be some type of intratubular material covered with an electrondense, fuzzy coating. This is similar to the non-cellular material that Holland (1985) found in peripheral dentin. We could not detect by XMA the presence of Ca and P in these electrondense structures. The results suggest that the electron-dense structure consists of organic components and corresponds with
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Fig. 7-Higher magnification of a dentinal tubule of a naturally desensitized area. The lumen is occluded with extremely fine crystals (arrow A) and large rhombohedral crystals (arrow B). Bar = 1 pLm.
Fig. 8-Higher magnification of a partly occluded dentinal tubule of a naturally desensitized area. Fine needle-like crystals (arrow) are deposited in the peripheral region of a lumen. Bar = 1 pAm.
the partition structure previously observed by SEM in the same site. By TEM, Thomas and Carella (1984) observed electrondense structures lining human dentinal tubules, and termed them lamina limitans, not odontoblastic processes. This structure has previously been identified as the inner hypomineralized lining of peritubular dentin (Shroff et al., 1956; Takuma, 1967; Thomas, 1979). On the other hand, Pashley et al. (1982) suggested that the materials that had electron-dense structure might represent plasma protein that leaked from pulpal capillaries under exposed tubules, and they estimated that fibrinogen could be among those proteins and that it could convert to fibrin within the tubules (Pashley et al., 1984; Pashley, 1986). We used ultrathin sections of undecalcified dentin specimens for the present study. Therefore, further study is required for clarification of the intratubular materials by use of EDTA-decalcified
dentin specimens, and perhaps immunohistochemical probes that are specific for fibrinogen, fibrin, or glycoproteins of the lamina limitans. The results of this study support the hydrodynamic theory of dentin sensitivity (Brannstrdm, 1966) that postulates that sensitive dentin must have open tubules. It also supports a corollary to that theory which predicts that exposed, insensitive dentin would have tubules that are occluded. The prediction was borne out by the results.
TABLE 2 MOLAR RATIOS OF Ca AND P IN THE SUPERFICIAL LAYERS OF DENTIN SPECIMENS IN NATURALLY DESENSITIZED AREAS Areas Examined Molar Ratio (Ca/P) Intertubular dentin 1.51 - 0.09 Peritubular dentin 1.49 + 0.07 The materials that 1.43 - 0.05 occluded dentinal tubules Th molar ratios of calcium (Ca) and phosphorus (P) were examined with XMA, as described in "Materials and methods". The values were the means of the values of eight experiments - standard error. There were no significant differences in these data (p>0.1).
REFERENCES
BRANNSTROM, M. (1966): Sensitivity of Dentine, Oral Surg Oral Med Oral Pathol 21:517-529.
BRANNSTROM, M. and GARBEROGLIO, R. (1980): Occlusion of Dentinal Tubules under Superficial Attrited Dentine, Swed Dent J 4:87-91. HIATT, W.H. and JOHANSEN, E. (1972): Root Preparation I. Obturation of Dentinal Tubules in Treatment of Root Hypersensitivity, J Periodontol 43:373-380. HOLLAND, G.R. (1985): The Odontoblast Process: Form and Function, J Dent Res 64:499-514. PASHLEY, D.H. (1986): Dentin Permeability, Dentin Sensitivity, and Treatment through Tubule Occlusion, J Endodontics 12:465474.
PASHLEY, D.H.; GALLOWAY, S.E.; and STEWART, F.P. (1984): Effects of Fibrinogen in vivo on Dentin Permeability in the Dog, Ar-ch Oral Biol 29:725-728. PASHLEY, D.H.; NELSON, R.; and KEPLER, E.E. (1982): Effects of Plasma and Salivary Constituents on Dentin Permeability, J Dent Res 61:978-981. SHROFF, F.R.; WILLIAMSON, K.I.; BERTAUD, W.S.; and HALL,
Downloaded from jdr.sagepub.com at Bobst Library, New York University on April 13, 2015 For personal use only. No other uses without permission.
Vol. 69 No. 6
TEM CHARACTERIZATION OF HYPERSENSITIVE DENTIN
D.M. (1956): Further Electron Microscope Studies of Dentin. The Nature of the Odontoblast Process, Oral Surg Oral Med Oral Pathol 9:432-443. TAKUMA, S. (1967): Ultrastructure of Dentinogenesis. In: Structural and Chemical Organization of Teeth, Vol. 1, A.E.W. Miles, Ed., New York: Academic Press, pp. 325-370. THOMAS, H.F. (1979): The Extent of the Odontoblast Process in Human Dentin, J Dent Res 58:2207-2218. THOMAS, H.F. and CARELLA, P. (1984): Correlation of Scanning
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