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RESEARCH REPORT |
1 Division in Anatomy and Developmental Biology, Department of Oral Biology, Research Center for Orofacial Hard Tissue Regeneration, Oral Science Research Center, College of Dentistry, Brain Korea 21 project for Medical Science, Yonsei Center of Biotechnology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea; and
2 Department of Oral Anatomy, School of Dentistry, Iwate Medical University, 1-3-27 Chuo-dori, Morioka city, Iwate 090-8505, Japan;
* corresponding author, hsjung{at}yumc.yonsei.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Hertwig’s epithelial root sheath • root formation • mice • embryogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have reported that signaling molecules and growth factors are involved in tooth development (Chen et al., 1996; Peters and Balling, 1999; Jernvall and Thesleff, 2000; Lesot et al., 2000; Satokata et al., 2000; Zhang et al., 2000). Moreover, it is generally believed that proteins such as glycoproteins and proteoglycans play an important role in early tooth formation (Vainio et al., 1991; Yoshiba et al., 1998, 2000). However, there are few available reports on the expression of these molecules and proteins during tooth root development (Yamashiro et al., 2003). Therefore, the precise form of signaling expression in HERS cells during root formation is unclear.
An organ culture is a useful technique for examining tooth development. Many studies have used tooth germ culture and clarified the tooth crown developmental process, including gene expression. However, studies using ordinary tooth germ culture methods have failed to observe root formation (Bernstein et al., 1990), due to the presence of calcified alveolar bone in the developing mandible.
In this study of the developmental process of HERS in vivo, the numbers of HERS cells and total HERS length were quantified in mice at post-natal (PN) days 8 to 26. Moreover, cell proliferation and apoptosis in the HERS were examined. To investigate the migration of the HERS cells during root formation, we injected DiI (1,1- dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchloride; molecular probes, D-282) into the HERS cells, using a novel mandible organ culture. In addition, proteoglycan, glycoprotein, and gene expression were examined. Improved understanding of the development of the periodontium could lead to new approaches for regeneration.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
The first molars (M1) of ICR mice (Mus musculus) of PN days 8, 10, 15, 18, 20, and 26 (5 mice each; total, 30 mice) were examined.
Tissue Preparation
Samples from PN8, 10, 15, 20, and 26 mice were fixed by perfusion with 4% paraformaldehyde under diethyl ether anesthesia. After decalcification with 10% di-sodium EDTA (EDTA-2Na), the specimens were embedded in paraffin wax. We prepared 5-µm frontal serial sections from the mesial surface of the M1. For immunohistochemistry and in situ hybridization, we prepared 10-µm frontal frozen sections from PN10 and PN18 mice.
Counting the Cell Numbers of Inner and Outer Enamel Epithelium and Measuring HERS Length
The paraffin sections were stained with hematoxylin-eosin. Every fifth section was placed on a glass slide. The cell numbers of IEE and OEE were counted in 3 sections per slide (Fig. 1a
). Using an ocular graticule (Olympus, Tokyo, Japan), we measured total HERS length from the connection point (base) of IEE and OEE, without the stratum intermedium and the stellate reticulum, to the end of HERS (Fig. 1b). Data were expressed as mean ± SD. For statistical analysis, we used Student’s t test.
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). The mandible culture method was improved on that described in a previous study (Fujiwara, 1997), as follows: From the mandibles, we removed the soft tissue, bone from the bottom of the mandible, and buccal bone from the distal root of M1 (Fig. 1d). We placed each specimen on a hole-making metal frame so that it could be clearly viewed through a dissecting microscope (Fig. 1e). As a culture medium, we used a serum-free and chemically defined BGJb medium (Sigma-Aldrich Co., St. Louis, MO, USA) that included 20 IU/mL penicillin-streptomycin, 100 mg/L ascorbic acid, and ITSTM pre-mix (5 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/mL selenious acid; Sigma-Aldrich Co., St. Louis, MO, USA). The mandibles were cultured for 3 days in an incubation chamber, with the medium being changed every 24 hrs.
Immunohistochemistry
Frozen sections were processed by the avidin-biotin complex (ABC) method (Hsu et al., 1981). Goat polyclonal anti-laminin beta-3 antibodies (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and goat polyclonal anti-syndecan-1 antibodies (1:100, Santa Cruz Biotechnology, Inc.) were used as the primary antibodies. Mouse monoclonal anti-proliferation cell nuclear antigen (PCNA) antibody (1:200, Neomarkers; Lab Vision Co., Fremont, CA, USA) was used to analyze cell proliferation. An In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used to detect the apoptotic cells. Normal serum, instead of the primary antibody, was used as a control.
In situ Hybridization
In situ hybridization of the digoxigenin-labeled probes was performed according to the method described by Schaeren-Wiemers and Gerfin-Moser (1993).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The numbers of IEE and OEE cells decreased gradually from PN8 to PN26. In a comparison of IEE and OEE, the number of IEE cells was higher than that of OEE. The HERS retained its length from PN8 to PN15, although total HERS length decreased temporarily at PN10. In addition, total HERS length decreased dramatically from PN15 to PN20. The cell size of both IEE and OEE at PN8 was smaller than that at PN15 (Figs. 1f, 1g); however, the size decreased at PN20 (Fig. 1h). Moreover, the width of the OEE cells was higher than that of IEE cells throughout the period of root development (Figs. 1f–1h).
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). The total HERS length as well as the numbers of cells in both IEE and OEE decreased gradually in both three- and five-day cultures. Moreover, the number of IEE cells was greater than that of OEE. This trend was similar to that observed in vivo.
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). In addition, the cells of the dental pulp and dental follicle showed an immunopositive reaction. By comparison at PN10, the immunopositive cell number for PCNA was lower at PN18 (statistical data not shown; P < 0.01) (Fig. 2b). However, immunoreaction was observed in both IEE and OEE. Moreover, the PCNA-immunopositive cells in HERS were generally found as clusters of 3 or 4 cell groups. The neighboring cells of the dental pulp and dental follicle showed PCNA immunoreactivity.
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). Rarely, TUNEL-positive cells could be detected in HERS at PN18 (Fig. 2d).
Cell Migration of HERS Cells
To clarify the extent of cell migration in the HERS, we injected DiI at both the bases and the ends of the HERS cells in the PN15 mice. Mandibles were cultured for 3 days. In the underside view of the specimen, DiI labeling was observed at the base and at the end of the HERS after three-day culture (Fig. 2e). In frozen sagittal sections of the same specimens, the DiI-labeled cells remained at the base and at the end of the HERS (Fig. 2f).
Laminin Beta-3 and Syndecan-1 Expression of HERS
To understand the characteristics of HERS cells, we performed immunohistochemistry for laminin and syndecan. An immunopositive reaction for laminin beta-3 was observed in both IEE and OEE of the HERS at PN10 (Fig. 2g). In contrast, no positive reaction was observed in dental pulp and dental follicle. An immunopositive reaction for syndecan-1 was detected in both IEE and OEE of the HERS only at PN10 (Fig. 2h). There was no signal when normal serum was used as a control for PCNA, laminin beta-3, and syndecan-1 immunohistochemistry (data not shown).
Expression Patterns of Bmp-2, Bmp-4, and Msx-2 in HERS
To analyze the genetic mechanisms of HERS development, we examined the expression of Bmp-2, Bmp-4, Msx-1, and Msx-2. Bmp-2 and Bmp-4 were detected not only in the cells of the dental pulp and dental follicle, but also in both IEE and OEE at PN10 (Fig. 2i). However, Bmp-4 expression was weaker than Bmp-2 expression (Fig. 2j). Msx-1 was not detected in the HERS cells, although a strong positive reaction was observed in the dental pulp and dental follicle (data not shown). In contrast, strong Msx-2 expression was detected in the HERS cells compared with the reaction of the dental pulp and dental follicle (Fig. 2k).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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An in vitro organ culture is a useful technique for the investigation of tooth development. However, we found it difficult to create an in vitro culture to examine root formation, because the tooth root grows in a special environment surrounded by calcified alveolar bone (Bernstein et al., 1990). Using the new culture system, we were able to adopt conditions approaching those in vivo. Our improved novel in vitro culture system for HERS development may be valuable in future investigations of root formation.
During the development of HERS, both cell proliferation and cell death in the HERS are important. In this study, the cells of IEE and OEE reacted positively to PCNA at both PN10 and PN18, although few TUNEL-positive cells were observed in the HERS, which was also reported previously (Kaneko et al., 1999; Suzuki et al., 2002). The PCNA-positive cells in HERS made a cluster and were arranged in 3 or 4 cell groups. It is well-known that the epithelial cell rests of Malassez are derived from fragmentation of HERS during tooth root formation. It is conceivable that the HERS may fragment in the PCNA-negative area.
One of the crucial questions regarding HERS is whether the HERS cells migrate. They remained intact during root formation in our in vitro organ culture for 3 days after the DiI injection. This suggests that no special growth point exists in the HERS. This study showed that both IEE and OEE cells do not migrate during root formation.
The HERS cells secrete enamel matrix proteins (Lindskog and Hammarström, 1982). Based on this finding, investigators have used enamel matrix proteins to regenerate the periodontium, and this therapy successfully produced periodontal tissues. In contrast, the properties of IEE in the HERS are quite similar to those of IEE in the enamel-free area, because they do not differentiate into ameloblasts, but secrete enamel proteins (Yamamoto et al., 1997). Moreover, they had an immunopositive reaction for laminin beta-3 (Yoshiba et al., 2000). These findings suggest that immunoreactivity for laminin beta-3 may be related to the secretion of enamel proteins. Syndecan is a type of cell-surface proteoglycan. Syndecan has different functions in the epithelium and mesenchyme (Vainio et al., 1991). An immunopositive reaction for syndecan-1 was observed in the HERS cells, which showed an immunopositive reaction for PCNA, but not in the mesenchymal cells during tooth root formation. The developing apical root tip showed an immunopositive reaction for syndecan-1 (Worapamorn et al., 2001). These findings suggest that syndecan-1 might be related to cell proliferation in HERS and might have different functions in the epithelium and mesenchyme, depending on the stages during root formation.
Recently, it was reported that Msx-2 was expressed in the root sheath, although Bmps were not observed in the HERS during root formation (Yamashiro et al., 2003). On the contrary, our results showed that Bmp-2 and Bmp-4 were expressed in the HERS cells. The enamel knot expresses many signaling molecules—such as Bmp, Msx, Shh, and Wnt—and regulates the morphogenesis of the complex shape of the tooth (Jernvall and Thesleff, 2000). The mouse molar is multi-rooted, and formation of its shape has not been fully elucidated. Moreover, no special signaling center, such as the enamel knot, has been found during root formation. Therefore, it is arguable that the HERS cells act as the signaling center, which is consistent with HERS cells expressing Bmp-2, Bmp-4, and Msx-2.
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ACKNOWLEDGMENTS |
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Received October 16, 2003; Last revision July 4, 2004; Accepted July 7, 2004
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REFERENCES |
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