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Alteration of odontoblast osteonectin expression following dental cavity preparation
Archives of Oral Biology 46 (2001) 829– 834 www.elsevier.com/locate/archoralbio
Alteration of odontoblast osteonectin expression following dental cavity preparation Toshiyuki Itota a,*, Yoshihiro Nishitani a, Norio Sogawa b, Chiharu Sogawa b, Norifumi Konishi a, Yasuhiro Torii a a b
Department of Operati6e Dentistry, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Department of Dental Pharmacology, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Accepted 20 March 2001
Abstract Cavity preparation can increase the active synthesis and secretion of non-collagenous proteins by odontoblasts, thus resulting in the deposition of tertiary dentine. In this study, the effect of cavity preparation on osteonectin expression was examined in odontoblasts of the rat tooth pulp. A class V cavity was prepared in rat first molars to stimulate odontoblastic secretory activity, and the animals were killed at various intervals. In the normal pulp, osteonectin immunoreactivity was detected in odontoblasts but not other cells. At 1 day after cavity preparation, immunoreactivity had diminished beneath the cavity. At 3 days, strong immunoreactivity could be detected in odontoblasts beneath the cavity. Numerous round cells underlying the odontoblastic layer also demonstrated immunoreactivity. Thereafter, the intensity of osteonectin immunoreactivity in odontoblasts beneath tertiary dentine decreased gradually, and at 30 and 60 days, it was weaker than in normal pulp. These findings suggest that osteonectin is actively synthesized by odontoblasts underlying a cavity in the initial stage of tertiary dentine formation. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cavity preparation; Immunohistochemistry; Odontoblast; Osteonectin; Tertiary dentine
1. Introduction Osteonectin is a phosphorylated glycoprotein synthesized by osteoblasts and many other cells (Liao et al., 1998) and is associated with tissue development, remodelling, cell turnover and repair (Yan and Sage, 1999). In the tooth germ, osteonectin has been demonstrated in the majority of cells of the dental papilla, the enamel organ and their extracellular matrixes, with the exception of enamel (Bronckers et al., 1989). Based on the findings that bovine osteonectin binds to collagen and Abbre6iations: PBS, phosphate-buffered saline. * Corresponding author. Tel.: + 81-86-2356672; fax: + 8186-2356674. E-mail address: [email protected] (T. Itota).
has a high affinity for hydroxyapatite in vitro (StetlerStevenson and Veis, 1986), it has been suggested that osteonectin may have a role in linking matrix and mineral during connective tissue mineralization. The expression of osteonectin is more limited in adult tissues; in the tooth pulp, osteonectin is restricted to odontoblasts, and little is known of its function in adult tissues (Reichert et al., 1992; Takano-Yamamoto et al., 1994). Tertiary dentine is produced as a result of a number of stimuli, including cavity preparation. It can be classified as reactionary or reparative, depending on the intensity of the stimulus and the survival of the odontoblasts (Smith et al., 1995). In its initial stages, cavity preparation has been reported to result in tissue damage such as disruption of the odontoblastic layer and
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T. Itota et al. / Archi6es of Oral Biology 46 (2001) 829–834
aspiration of odontoblasts (Lilja et al., 1982). Cell death has also been suggested to occur in severely damaged odontoblasts (Bronckers et al., 1996; Gwinnett and Tay, 1998). Three days after cavity preparation, odontoblast-like cells differentiate and synthesize collagenous and non-collagenous proteins including dentine sialoprotein (Butler et al., 1992; Butler, 1995; D’Souza et al., 1995). The matrix proteins are secreted by odontoblasts into the predentine (Butler, 1995; Butler and Ritchie, 1995), and tertiary dentine begins to be produced rapidly and abundantly. The exact nature of the non-collagenous proteins involved in tertiary dentine formation is not fully understood, but it is possible that they participate in the regulation of odontoblastic metabolism, matrix deposition and assembly, and mineralization. In the present study, osteonectin immunoreactivity was examined, following cavity preparation, using an indirect immunofluorescence method.
2. Materials and methods
2.3. Immunohistochemical procedures To investigate osteonectin immunoreactivity, the sections were incubated with monoclonal anti-osteonectin antibody (1:1000; Haematologic Technologies Inc., USA) followed by incubation with fluorescein isothiocyanate-conjugated, donkey antimouse IgG (1:75; Jackson Immunoresearch Labs, USA). These antisera were diluted in PBS containing 0.1% bovine serum albumin (Nacalai tesque, Japan) and 0.75% Triton-X (Sigma, USA). All sections were mounted with Vectashield (Vector Laboratories, Inc., USA) to avoid photobleaching. The fluorescence images were captured immediately with a laser scanning confocal imaging system (Radiance 2000 and LaserSharp 2000; Bio-rad, USA). Then, sections were washed in PBS and stained with haematoxylin and eosin. To test the specificity of the anti-osteonectin antibody, the primary antibody was preabsorbed with human platelet osteonectin (10 mg/ml, Haematologic Technologies Inc.), and this was used to check for lack of specificity.
2.4. Quantitati6e densitometry 2.1. Operati6e procedures Forty-two adult male Sprague –Dawley rats (6 weeks old, 180 g body wt ) were used in this study. Animals were anaesthetized with intraperitoneal sodium pentobarbital at a dose of 1.0 ml/kg and placed in a restraining device. All cavity preparations were performed by one investigator. A class V cavity was prepared on the mesial aspect of the maxillary first molars to approximately half the distance through dentine with a no. 1/4 round bur. The depth and width of the cavity (from 0.4 to 0.6 mm) were nearly equal to the diameter of the bur, and the cavity was left unrestored. The animals were then allowed to recover. On 1, 3, 7, 14, 30 and 60 days after surgery, rats were anaesthetized with sodium pentobarbital and perfused intracardiacly with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The data were obtained from six animals per group. Six animals without cavity preparations were also used as a normal control group.
2.2. Tissue preparation Maxillary segments were carefully dissected and immersed in the same fixative overnight, then demineralized in 4.17% buffered disodium dihydrogen ethylenediamine tetraacetate dihydrate (pH 7.4) for 11 days. The segments were then immersed in 20% sucrose in PBS (pH 7.4) for 24 h, and cryostat sections at 30 mm thickness were cut in the parasagittal plane and mounted on silane-coated slides (Dako, Japan).
The density of osteonectin immunoreactivity in the odontoblastic layer was analysed with Scion image beta 3b (http://www.scioncorp.com/). Density was measured in the odontoblastic layer under the cavity. In control sections, the odontoblastic layer in the cervical region of the pulp was also analysed. In addition, central regions of the pulp were analysed to determine the background level. The ratio density of background:density of odontoblastic layer was used to ascertain the intensity of osteonectin immunoreactivity. Statistical analysis was performed using the Mann – Whitney U-test at the PB 0.05 level for differences in the density of osteonectin immunoreactivity in the odontoblastic layer between control and experimental groups.
3. Results No immunofluorescence was detected in sections incubated with the anti-osteonectin antibody preabsorbed with antigen (results not shown). Osteonectin immunoreactivity was localized to the cell body and process of odontoblasts in the normal tooth pulp (Fig. 1A –C). The odontoblast nucleus was always immunonegative (Fig. 1C). Immunoreactive odontoblast processes were detected within predentine but not primary or secondary dentine (Fig. 1C). The predentine, dentine, blood vessels and nerve fibres, and pulp cells were also immunonegative (Fig. 1B).
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At 1 day after cavity preparation, osteonectin-immunonegative regions had appeared in the odontoblastic layer beneath the cavity (Fig. 1D –F). The immunoreactivity appeared to be weak in some odontoblasts, whereas in others, it remained unchanged (Fig. 1F). Immunoreactivity in odontoblastic processes ap-
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peared to become weaker, and the length of immunoreactive processes remained unchanged. At 3 days, the odontoblasts beneath the cavity showed strong osteonectin immunoreactivity (Fig. 1G –I). The density of immunoreactivity in the odontoblastic layer of the experimental group at 3 days after cavity preparation
Fig. 1. Microphotographs of haematoxylin and eosin staining (A, D, G, J) and osteonectin immunoreactivity (B, C, E, F, H, I, K, L) in the normal pulp (A –C) and at 1 (D– F), 3 (G –I) and 7 (J – L) days after cavity preparation. (A) and (B), (D) and (E), (G) and (H) and (J) and (K) are from the same fields of view, respectively. Osteonectin immunoreactivity was localized to cell bodies and odontoblast processes in the normal rat (A – C). At 1 day after cavity preparation (D – F), immunonegative regions appeared near the predentine in the odontoblastic layer (arrowheads in F). In some odontoblasts, osteonectin immunoreactivity became weak beneath the cavity (arrows in F). At 3 days (G –I), odontoblasts beneath the cavity exhibited strong osteonectin immunoreactivity (H, I), whereas their processes were devoid of immunoreactivity (I). Numerous round cells beneath the odontoblastic layer also showed immunoreactivity (arrows in H, I). At 7 days (J – L), both cell bodies and processes of odontoblasts underlying tertiary dentine (* in J – L) were immunoreactive for osteonectin (K, L). An arrowhead in (L) indicates a round cell with osteonectin-immunoreactivity. Bars =100 mm (A) and 50 mm (C). (A), (B), (D), (E), (G), (H), (J), (K) and (C), (F), (I), (L) are at the same magnifications, respectively. CA (D, G, J) = cavity; OB (A, D, G, J) =odontoblastic layer.
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Fig. 2. Microphotographs of osteonectin immunoreactivity (A, B, D, E) and haematoxylin and eosin staining (C) at 14 (A), 30 (B) and 60 (C – E) days after cavity preparation. (C) and (D) are from the same fields of view. From 14 days, osteonectin immunoreactivity in odontoblasts beneath tertiary dentine (* in A – E) decreased gradually (A, B, D, E). At 60 days (C – E), odontoblasts had a more rounded appearance and showed weak osteonectin immunoreactivity (arrowheads in E). Bars =100 mm (A) and 50 mm (E). (A), (B), (C) and (D) are at the same magnification. CA (A, B, C) = cavity; OB (C) = odontoblastic layer.
(mean9S.E.M.=1.6590.05) was significantly greater than that of the normal control (1.42 90.04) (Fig. 3). Immunoreactivity was occasionally absent from odontoblastic processes (Fig. 1I). At this stage, numerous round cells appeared beneath the odontoblastic layer (Fig. 1G). These cells had a round nucleus and accumulated to a depth of 100 –150 mm from the odontoblastic layer (Fig. 1G). The round cells were also immunoreactive for osteonectin (Fig. 1H, I). At 7 days, tertiary dentine was observed beneath the cavity (thickness of tertiary dentine =20–40 mm) and the interface between tertiary dentine and predentine was irregular (Fig. 1J). Osteonectin immunoreactivity appeared to be weaker at this stage than at 3 days (Fig. 3). Osteonectin-immunoreactive round cells underlying the odontoblastic layer had decreased in number (Fig. 1K, L). From 14 days, the tertiary dentine became thicker, and the intensity of osteonectin immunoreactivity seemed to decrease gradually (Fig. 2A –E). At 14 days, immunoreactive processes could be detected within the predentine beneath the tertiary dentine. At 30 days, the density of immunoreactivity in odontoblasts had become much weaker (Fig. 3). At 60 days, odontoblasts beneath the cavity had a round appearance and showed only a faint osteonectin immunoreactivity (Fig. 2C – E). The differences between the normal control group and experimental group at 30 days (mean 9S.E.M.= 1.279 0.02) or 60 days (1.20 90.03) were statistically significant (Fig. 3). At all stages, the central portion of the tooth pulp was devoid of immunoreactivity.
4. Discussion We demonstrate that osteonectin expression in the odontoblastic layer was altered following cavity preparation. Osteonectin was localized to the cell bodies and processes of odontoblasts but not to other cells in the
Fig. 3. Change in osteonectin immunoreactivity in the odontoblastic layer after cavity preparation. The intensity of the immunoreactivity is shown as the ratio of density of the central portion in the tooth pulp:density of odontoblastic layer (mean 9S.E.M.). Immunoreactivity increased significantly (P B0.05) at 3 days and decreased gradually from 7 days. At 30 and 60 days, osteonectin immunoreactivity was weaker than in the normal control (*PB 0.05, Mann – Whitney Utest).
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normal rat pulp. This finding is consistent with those of Reichert et al. (1992) and Takano-Yamamoto et al. (1994). To the best of our knowledge, osteonectin expression following cavity preparation had not been examined before; the function of this protein during reparative dentinogenesis remains unclear. At 1 day after cavity preparation, osteonectin-immunonegative regions appeared in the odontoblastic layer. Cavity preparation is known to damage odontoblasts (Lilja et al., 1982). The breakdown of the junctional complexes between adjacent odontoblasts and the formation of large spaces, which often contain flocculent material, suggests that cell death occurs (Chiego, 1992; Bronckers et al., 1996; Gwinnett and Tay, 1998). The appearance of immunonegative regions may reflect the death of osteonectin-immunoreactive odontoblasts. Alternatively, cavity preparation may cause a decrease in protein expression in surviving odontoblasts. At 3 days, numerous round cells appeared beneath the odontoblastic layer, and these may represent the differentiation of odontoblast-like cells, because they demonstrate osteonectin immunoreactivity. This possibility is supported by earlier findings that odontoblast-like cells appear 5 days after cavity preparation (Inoue and Shimono, 1992). In our study, at this stage, the odontoblastic layer showed strong osteonectin immunoreactivity; thereafter, the intensity of the immunoreactivity appeared to decrease gradually. We observed the formation of tertiary dentine at 7 days after cavity preparation. From these results, it would appear that odontoblasts actively synthesize osteonectin until tertiary dentine formation begins. Osteonectin lacks hydroxyapatite nucleation activity at a high concentration in vitro (Hunter et al., 1996). The primary mechanism for inhibition of hydroxyapatite formation by osteonectin is the blocking of growth sites on nucleated calcium phosphates (Doi et al., 1989). However, this protein may inhibit mineralization at the initial stage of tertiary dentine formation after cavity preparation. Boskey (1995) has reported that dentine sialoprotein inhibits the formation and growth of apatite. In preliminary experiments, we have found that the distribution patterns of osteonectin and dentine sialoprotein immunoreactivities are very similar at all stages after cavity preparation (unpublished data). These findings suggest that osteonectin might inhibit the formation of tertiary dentine in the initial stages. At 30 and 60 days, tertiary dentine was fully formed, and odontoblasts showed only weak osteonectin immunoreactivity. From these results, we suggest that it is unlikely that osteonectin is synthesized actively after the formation of tertiary dentine, but further study is required to explore this.
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Acknowledgements We greatly thank Dr Butler at the Department of Biological Chemistry, University of Texas-Houston Health Science Center for the gift of the anti-dentine sialoprotein serum and Dr Yoshiyama, Chair and Professor of the Department of Operative Dentistry, Okayama University Dental School, for reviewing this manuscript. This research was supported by Grants in Aid for Scientific Research 10671790 to T.I. from The Ministry of Education, Science, and Culture of Japan, Tokyo, Japan.
References Boskey, A.L., 1995. Osteopontin and related phosphorylated sialoproteins: effects on mineralization. Ann. NY Acad. Sci. 760, 249 – 256. Bronckers, A.L.J.J., Lyaruu, D.M., Goei, W., Litz, M., Luo, G., Karsenty, G., Woltgens, J.H.M., D’Souza, R.N., 1996. Nuclear DNA fragmentation during postnatal tooth development of mouse and hamster and during dentin repair in the rat. Eur. J. Oral Sci. 104, 102 – 111. Bronckers, A.L.J.J., Lyaruu, D.M., Woltgens, J.H.M., 1989. Immunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis. Connect. Tissue Res. 22, 65 – 70. Butler, W.T., 1995. Dentin matrix proteins and dentinogenesis. Connect. Tissue Res. 33, 59 – 65. Butler, W.T., Bhown, M., Brunn, J.C., D’Souza, R.N., Farach-Carson, M.C., Happonen, R.-P., Schrohenloher, R.E., Seyer, J.M., Somerman, M.J., Foster, R.A., Tomana, M., van Dijk, S., 1992. Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP). Matrix 12, 343 – 351. Butler, W.T., Ritchie, H., 1995. The nature and functional significance of dentin extracellular matrix proteins. Int. J. Dev. Biol. 39, 169 – 179. Chiego, D.J. Jr., 1992. An ultrastructural and autoradiographic analysis of primary and replacement odontoblasts following cavity preparation and wound healing in the rat molar. Proc. Finn. Dent. Soc. 88 (Suppl. 1), 243 – 256. Doi, Y., Okuda, R., Takezawa, Y., Shibata, S., Moriwaki, Y., Wakamatsu, N., Shimizu, N., Moriyama, K., Shimokawa, H., 1989. Osteonectin inhibiting de novo formation of apatite in the presence of collagen. Calcif. Tissue Int. 44, 200 – 208. D’Souza, R.N., Bachman, T., Baumgardner, K.R., Butler, W.T., Litz, M., 1995. Characterization of cellular responses involved in reparative dentinogenesis in rat molars. J. Dent. Res. 74, 702 – 709. Gwinnett, A.J., Tay, F.R., 1998. Early and intermediate time response of the dental pulp to an acid etch technique in vivo. Am. J. Dent. 11, S35 – S44 special issue. Hunter, G.K., Hauschka, P.V., Poole, A.R., Rosenberg, L.C., Goldberg, H.A., 1996. Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem. J. 317, 59 – 64.
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Inoue, T., Shimono, M., 1992. Repair dentinogenesis following transplantation into normal and germ-free animals. Proc. Finn. Dent. Soc. 88 (suppl. 1), 183 –194. Liao, H., Brandsten, C., Lundmark, C., Christersson, C., Wurtz, T., 1998. Osteonectin RNA and collagen a1(I) RNA in the developing rat maxilla. Eur. J. Oral Sci. 106 (Suppl. 1), 418 – 423. Lilja, J., Nordenvall, K.J., Branstrom, M., 1982. Dentin sensitivity, odontoblasts and nerves under desiccated or infected experimental cavities. A clinical, light microscopic and ultrastructural investigation. Swed. Dent. J. 6, 93 – 103. Reichert, T., Storkel, S., Becker, K., Fisher, L.W., 1992. The role of osteonectin in human tooth development: an immunohistological study. Calcif. Tissue Int. 50, 468 –472.
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Smith, A.J., Cassidy, N., Perry, H., Begue-Kirn, C., Ruch, J-V., Lesot, H., 1995. Reactionaly dentinogenesis. Int. J. Dev. Biol. 39, 273 – 280. Stetler-Stevenson, W.G., Veis, A., 1986. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif. Tissue Int. 38, 135 – 141. Takano-Yamamoto, T., Takemura, T., Kitamura, Y., Nomura, S., 1994. Site-specific expression of mRNAs for osteonectin, osteocalcin, and osteopontin revealed by in situ hybridization in rat periodontal ligament during physiological tooth movement. J. Histochem. Cytochem. 42, 885 – 896. Yan, Q., Sage, E.H., 1999. SPARC, a matricellular glycoprotein with important biological functions. J. Histochem. Cytochem. 47, 1495 – 1506.
Alteration of odontoblast osteonectin expression following dental cavity preparation Toshiyuki Itota a,*, Yoshihiro Nishitani a, Norio Sogawa b, Chiharu Sogawa b, Norifumi Konishi a, Yasuhiro Torii a a b
Department of Operati6e Dentistry, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Department of Dental Pharmacology, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Accepted 20 March 2001
Abstract Cavity preparation can increase the active synthesis and secretion of non-collagenous proteins by odontoblasts, thus resulting in the deposition of tertiary dentine. In this study, the effect of cavity preparation on osteonectin expression was examined in odontoblasts of the rat tooth pulp. A class V cavity was prepared in rat first molars to stimulate odontoblastic secretory activity, and the animals were killed at various intervals. In the normal pulp, osteonectin immunoreactivity was detected in odontoblasts but not other cells. At 1 day after cavity preparation, immunoreactivity had diminished beneath the cavity. At 3 days, strong immunoreactivity could be detected in odontoblasts beneath the cavity. Numerous round cells underlying the odontoblastic layer also demonstrated immunoreactivity. Thereafter, the intensity of osteonectin immunoreactivity in odontoblasts beneath tertiary dentine decreased gradually, and at 30 and 60 days, it was weaker than in normal pulp. These findings suggest that osteonectin is actively synthesized by odontoblasts underlying a cavity in the initial stage of tertiary dentine formation. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cavity preparation; Immunohistochemistry; Odontoblast; Osteonectin; Tertiary dentine
1. Introduction Osteonectin is a phosphorylated glycoprotein synthesized by osteoblasts and many other cells (Liao et al., 1998) and is associated with tissue development, remodelling, cell turnover and repair (Yan and Sage, 1999). In the tooth germ, osteonectin has been demonstrated in the majority of cells of the dental papilla, the enamel organ and their extracellular matrixes, with the exception of enamel (Bronckers et al., 1989). Based on the findings that bovine osteonectin binds to collagen and Abbre6iations: PBS, phosphate-buffered saline. * Corresponding author. Tel.: + 81-86-2356672; fax: + 8186-2356674. E-mail address: [email protected] (T. Itota).
has a high affinity for hydroxyapatite in vitro (StetlerStevenson and Veis, 1986), it has been suggested that osteonectin may have a role in linking matrix and mineral during connective tissue mineralization. The expression of osteonectin is more limited in adult tissues; in the tooth pulp, osteonectin is restricted to odontoblasts, and little is known of its function in adult tissues (Reichert et al., 1992; Takano-Yamamoto et al., 1994). Tertiary dentine is produced as a result of a number of stimuli, including cavity preparation. It can be classified as reactionary or reparative, depending on the intensity of the stimulus and the survival of the odontoblasts (Smith et al., 1995). In its initial stages, cavity preparation has been reported to result in tissue damage such as disruption of the odontoblastic layer and
0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 1 ) 0 0 0 4 1 - 3
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T. Itota et al. / Archi6es of Oral Biology 46 (2001) 829–834
aspiration of odontoblasts (Lilja et al., 1982). Cell death has also been suggested to occur in severely damaged odontoblasts (Bronckers et al., 1996; Gwinnett and Tay, 1998). Three days after cavity preparation, odontoblast-like cells differentiate and synthesize collagenous and non-collagenous proteins including dentine sialoprotein (Butler et al., 1992; Butler, 1995; D’Souza et al., 1995). The matrix proteins are secreted by odontoblasts into the predentine (Butler, 1995; Butler and Ritchie, 1995), and tertiary dentine begins to be produced rapidly and abundantly. The exact nature of the non-collagenous proteins involved in tertiary dentine formation is not fully understood, but it is possible that they participate in the regulation of odontoblastic metabolism, matrix deposition and assembly, and mineralization. In the present study, osteonectin immunoreactivity was examined, following cavity preparation, using an indirect immunofluorescence method.
2. Materials and methods
2.3. Immunohistochemical procedures To investigate osteonectin immunoreactivity, the sections were incubated with monoclonal anti-osteonectin antibody (1:1000; Haematologic Technologies Inc., USA) followed by incubation with fluorescein isothiocyanate-conjugated, donkey antimouse IgG (1:75; Jackson Immunoresearch Labs, USA). These antisera were diluted in PBS containing 0.1% bovine serum albumin (Nacalai tesque, Japan) and 0.75% Triton-X (Sigma, USA). All sections were mounted with Vectashield (Vector Laboratories, Inc., USA) to avoid photobleaching. The fluorescence images were captured immediately with a laser scanning confocal imaging system (Radiance 2000 and LaserSharp 2000; Bio-rad, USA). Then, sections were washed in PBS and stained with haematoxylin and eosin. To test the specificity of the anti-osteonectin antibody, the primary antibody was preabsorbed with human platelet osteonectin (10 mg/ml, Haematologic Technologies Inc.), and this was used to check for lack of specificity.
2.4. Quantitati6e densitometry 2.1. Operati6e procedures Forty-two adult male Sprague –Dawley rats (6 weeks old, 180 g body wt ) were used in this study. Animals were anaesthetized with intraperitoneal sodium pentobarbital at a dose of 1.0 ml/kg and placed in a restraining device. All cavity preparations were performed by one investigator. A class V cavity was prepared on the mesial aspect of the maxillary first molars to approximately half the distance through dentine with a no. 1/4 round bur. The depth and width of the cavity (from 0.4 to 0.6 mm) were nearly equal to the diameter of the bur, and the cavity was left unrestored. The animals were then allowed to recover. On 1, 3, 7, 14, 30 and 60 days after surgery, rats were anaesthetized with sodium pentobarbital and perfused intracardiacly with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The data were obtained from six animals per group. Six animals without cavity preparations were also used as a normal control group.
2.2. Tissue preparation Maxillary segments were carefully dissected and immersed in the same fixative overnight, then demineralized in 4.17% buffered disodium dihydrogen ethylenediamine tetraacetate dihydrate (pH 7.4) for 11 days. The segments were then immersed in 20% sucrose in PBS (pH 7.4) for 24 h, and cryostat sections at 30 mm thickness were cut in the parasagittal plane and mounted on silane-coated slides (Dako, Japan).
The density of osteonectin immunoreactivity in the odontoblastic layer was analysed with Scion image beta 3b (http://www.scioncorp.com/). Density was measured in the odontoblastic layer under the cavity. In control sections, the odontoblastic layer in the cervical region of the pulp was also analysed. In addition, central regions of the pulp were analysed to determine the background level. The ratio density of background:density of odontoblastic layer was used to ascertain the intensity of osteonectin immunoreactivity. Statistical analysis was performed using the Mann – Whitney U-test at the PB 0.05 level for differences in the density of osteonectin immunoreactivity in the odontoblastic layer between control and experimental groups.
3. Results No immunofluorescence was detected in sections incubated with the anti-osteonectin antibody preabsorbed with antigen (results not shown). Osteonectin immunoreactivity was localized to the cell body and process of odontoblasts in the normal tooth pulp (Fig. 1A –C). The odontoblast nucleus was always immunonegative (Fig. 1C). Immunoreactive odontoblast processes were detected within predentine but not primary or secondary dentine (Fig. 1C). The predentine, dentine, blood vessels and nerve fibres, and pulp cells were also immunonegative (Fig. 1B).
T. Itota et al. / Archi6es of Oral Biology 46 (2001) 829–834
At 1 day after cavity preparation, osteonectin-immunonegative regions had appeared in the odontoblastic layer beneath the cavity (Fig. 1D –F). The immunoreactivity appeared to be weak in some odontoblasts, whereas in others, it remained unchanged (Fig. 1F). Immunoreactivity in odontoblastic processes ap-
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peared to become weaker, and the length of immunoreactive processes remained unchanged. At 3 days, the odontoblasts beneath the cavity showed strong osteonectin immunoreactivity (Fig. 1G –I). The density of immunoreactivity in the odontoblastic layer of the experimental group at 3 days after cavity preparation
Fig. 1. Microphotographs of haematoxylin and eosin staining (A, D, G, J) and osteonectin immunoreactivity (B, C, E, F, H, I, K, L) in the normal pulp (A –C) and at 1 (D– F), 3 (G –I) and 7 (J – L) days after cavity preparation. (A) and (B), (D) and (E), (G) and (H) and (J) and (K) are from the same fields of view, respectively. Osteonectin immunoreactivity was localized to cell bodies and odontoblast processes in the normal rat (A – C). At 1 day after cavity preparation (D – F), immunonegative regions appeared near the predentine in the odontoblastic layer (arrowheads in F). In some odontoblasts, osteonectin immunoreactivity became weak beneath the cavity (arrows in F). At 3 days (G –I), odontoblasts beneath the cavity exhibited strong osteonectin immunoreactivity (H, I), whereas their processes were devoid of immunoreactivity (I). Numerous round cells beneath the odontoblastic layer also showed immunoreactivity (arrows in H, I). At 7 days (J – L), both cell bodies and processes of odontoblasts underlying tertiary dentine (* in J – L) were immunoreactive for osteonectin (K, L). An arrowhead in (L) indicates a round cell with osteonectin-immunoreactivity. Bars =100 mm (A) and 50 mm (C). (A), (B), (D), (E), (G), (H), (J), (K) and (C), (F), (I), (L) are at the same magnifications, respectively. CA (D, G, J) = cavity; OB (A, D, G, J) =odontoblastic layer.
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Fig. 2. Microphotographs of osteonectin immunoreactivity (A, B, D, E) and haematoxylin and eosin staining (C) at 14 (A), 30 (B) and 60 (C – E) days after cavity preparation. (C) and (D) are from the same fields of view. From 14 days, osteonectin immunoreactivity in odontoblasts beneath tertiary dentine (* in A – E) decreased gradually (A, B, D, E). At 60 days (C – E), odontoblasts had a more rounded appearance and showed weak osteonectin immunoreactivity (arrowheads in E). Bars =100 mm (A) and 50 mm (E). (A), (B), (C) and (D) are at the same magnification. CA (A, B, C) = cavity; OB (C) = odontoblastic layer.
(mean9S.E.M.=1.6590.05) was significantly greater than that of the normal control (1.42 90.04) (Fig. 3). Immunoreactivity was occasionally absent from odontoblastic processes (Fig. 1I). At this stage, numerous round cells appeared beneath the odontoblastic layer (Fig. 1G). These cells had a round nucleus and accumulated to a depth of 100 –150 mm from the odontoblastic layer (Fig. 1G). The round cells were also immunoreactive for osteonectin (Fig. 1H, I). At 7 days, tertiary dentine was observed beneath the cavity (thickness of tertiary dentine =20–40 mm) and the interface between tertiary dentine and predentine was irregular (Fig. 1J). Osteonectin immunoreactivity appeared to be weaker at this stage than at 3 days (Fig. 3). Osteonectin-immunoreactive round cells underlying the odontoblastic layer had decreased in number (Fig. 1K, L). From 14 days, the tertiary dentine became thicker, and the intensity of osteonectin immunoreactivity seemed to decrease gradually (Fig. 2A –E). At 14 days, immunoreactive processes could be detected within the predentine beneath the tertiary dentine. At 30 days, the density of immunoreactivity in odontoblasts had become much weaker (Fig. 3). At 60 days, odontoblasts beneath the cavity had a round appearance and showed only a faint osteonectin immunoreactivity (Fig. 2C – E). The differences between the normal control group and experimental group at 30 days (mean 9S.E.M.= 1.279 0.02) or 60 days (1.20 90.03) were statistically significant (Fig. 3). At all stages, the central portion of the tooth pulp was devoid of immunoreactivity.
4. Discussion We demonstrate that osteonectin expression in the odontoblastic layer was altered following cavity preparation. Osteonectin was localized to the cell bodies and processes of odontoblasts but not to other cells in the
Fig. 3. Change in osteonectin immunoreactivity in the odontoblastic layer after cavity preparation. The intensity of the immunoreactivity is shown as the ratio of density of the central portion in the tooth pulp:density of odontoblastic layer (mean 9S.E.M.). Immunoreactivity increased significantly (P B0.05) at 3 days and decreased gradually from 7 days. At 30 and 60 days, osteonectin immunoreactivity was weaker than in the normal control (*PB 0.05, Mann – Whitney Utest).
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normal rat pulp. This finding is consistent with those of Reichert et al. (1992) and Takano-Yamamoto et al. (1994). To the best of our knowledge, osteonectin expression following cavity preparation had not been examined before; the function of this protein during reparative dentinogenesis remains unclear. At 1 day after cavity preparation, osteonectin-immunonegative regions appeared in the odontoblastic layer. Cavity preparation is known to damage odontoblasts (Lilja et al., 1982). The breakdown of the junctional complexes between adjacent odontoblasts and the formation of large spaces, which often contain flocculent material, suggests that cell death occurs (Chiego, 1992; Bronckers et al., 1996; Gwinnett and Tay, 1998). The appearance of immunonegative regions may reflect the death of osteonectin-immunoreactive odontoblasts. Alternatively, cavity preparation may cause a decrease in protein expression in surviving odontoblasts. At 3 days, numerous round cells appeared beneath the odontoblastic layer, and these may represent the differentiation of odontoblast-like cells, because they demonstrate osteonectin immunoreactivity. This possibility is supported by earlier findings that odontoblast-like cells appear 5 days after cavity preparation (Inoue and Shimono, 1992). In our study, at this stage, the odontoblastic layer showed strong osteonectin immunoreactivity; thereafter, the intensity of the immunoreactivity appeared to decrease gradually. We observed the formation of tertiary dentine at 7 days after cavity preparation. From these results, it would appear that odontoblasts actively synthesize osteonectin until tertiary dentine formation begins. Osteonectin lacks hydroxyapatite nucleation activity at a high concentration in vitro (Hunter et al., 1996). The primary mechanism for inhibition of hydroxyapatite formation by osteonectin is the blocking of growth sites on nucleated calcium phosphates (Doi et al., 1989). However, this protein may inhibit mineralization at the initial stage of tertiary dentine formation after cavity preparation. Boskey (1995) has reported that dentine sialoprotein inhibits the formation and growth of apatite. In preliminary experiments, we have found that the distribution patterns of osteonectin and dentine sialoprotein immunoreactivities are very similar at all stages after cavity preparation (unpublished data). These findings suggest that osteonectin might inhibit the formation of tertiary dentine in the initial stages. At 30 and 60 days, tertiary dentine was fully formed, and odontoblasts showed only weak osteonectin immunoreactivity. From these results, we suggest that it is unlikely that osteonectin is synthesized actively after the formation of tertiary dentine, but further study is required to explore this.
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Acknowledgements We greatly thank Dr Butler at the Department of Biological Chemistry, University of Texas-Houston Health Science Center for the gift of the anti-dentine sialoprotein serum and Dr Yoshiyama, Chair and Professor of the Department of Operative Dentistry, Okayama University Dental School, for reviewing this manuscript. This research was supported by Grants in Aid for Scientific Research 10671790 to T.I. from The Ministry of Education, Science, and Culture of Japan, Tokyo, Japan.
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