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Developmental Properties of the Hertwig’s Epithelial Root Sheath in Mice

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;

Correspondence: ^* corresponding author, hsjung{at}yumc.yonsei.ac.kr

	ABSTRACT

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Hertwig’s epithelial root sheath (HERS) plays an important role in tooth root formation. In this study, we examined root formation of the first molar in mice, focusing on cell proliferation, cell death, cell migration, and the expression patterns of the signaling molecules, including glycoproteins and proteoglycans between PN8 and PN26. The number of HERS cells decreased during root formation, although HERS retained total length until PN15. The migration of HERS cells did not occur during root formation. Moreover, the immunopositive reaction of laminin beta-3 and syndecan-1 in HERS indicates that both cell adhesion and cell proliferation are essential for HERS development. Bmp-2, Bmp-4, and Msx-2 were expressed in HERS cells during root formation. We also developed an in vitro culture system for investigating the periodontium and suggest that this system provides an excellent vehicle for full exploration, and hence improved understanding, of the development and regeneration of the periodontium. Together, our results provide a comprehensive model describing the morphogenesis of early root development in vertebrates.

Key Words: Hertwig’s epithelial root sheath • root formation • mice • embryogenesis

	INTRODUCTION

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Tooth development is believed to be a model system for the examination of organ development during embryogenesis. Although many studies on crown development have been reported (Vainio et al., 1991; Chen et al., 1996; Yoshiba et al., 1998, 2000; Jernvall and Thesleff, 2000; Lesot et al., 2000; Satokata et al., 2000; Zhang et al., 2000), there is little available information on root development in mice (Gurling and Sampson, 1985; Ohnishi et al., 2003; Yamashiro et al., 2003). After the bell stage in tooth development, inner enamel epithelium (IEE) connects with outer enamel epithelium (OEE) at the cervix. This structure is referred to as Hertwig’s Epithelial Root Sheath (HERS). Morphologically, the HERS bends inward at the early stage of root formation. The HERS cells do not grow toward the dental papilla during root formation and disappear at the completion of root formation. Gurling and Sampson (1985) have suggested that OEE cells migrate into the IEE. However, Kenney and Ramfjord (1969) did not observe the migration of OEE into the HERS. The detailed developmental process of the HERS is unclear.

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.

	MATERIALS & METHODS

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All experiments were performed according to the guidelines of the Yonsei University, College of Dentistry, Intramural Animal Use and Care Committee.

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.

View larger version (70K): [in this window] [in a new window]   Figure 1. Developmental analysis of HERS with the use of in vitro culture. (a) The numbers of IEE and OEE cells were counted. The dotted yellow line shows the HERS. The red and black numbers indicate the numbers of IEE and OEE cells, respectively. (b) The HERS length was measured between the arrows, from the connection point of the IEE and OEE to the end of the HERS. (c) DiI was injected at two points (arrowheads): the connection point of OEE and IEE, and the end of the HERS. (d) After dissecting the mandible, we removed the soft tissues, including the oral epithelium above the molar, and cut one-third from the underside of the mandibular bone, including the incisor. Bone from the distal root area was also removed. The arrows show the exposed distal root. (e) After preparation, the specimens were placed on the hole-making metal grid with the use of BGJb medium. (f,g) At PN15, the cell size in both IEE and OEE in the HERS increased compared with that at PN8. (h) However, the cell size of HERS decreased at PN20. The cell sizes at PN8 and PN20 were similar. The OEE cells were much wider than the IEE cells (arrows in g-h). Yellow dotted line: HERS. Bars: a, 15 µm; b, 40 µm; c,d, 1 mm; g,h, 15 µm).

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).

	RESULTS

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Numbers of Inner Enamel Epithelium and Outer Enamel Epithelium Cells, and Total HERS Length
To identify the normal developmental process of HERS in vivo, we quantified the cell numbers of IEE and OEE in HERS, as well as the total HERS length, using the H-E-stained sections in the different stages of root development (Table 1). 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).

View larger version (86K): [in this window] [in a new window]   Figure 2. Several signaling factors are mutually coordinated in HERS development. (a) At PN10, an immunopositive reaction for PCNA was observed, not only in many HERS cells, but also in the cells of the dental pulp and dental follicle. (b) At PN18, some HERS cells showed an immunopositive reaction. However, the number of immunopositive cells decreased compared with that at PN10. Moreover, clusters of 3-4 PCNA-positive cells could be seen in the HERS. (c) At PN10, no TUNEL-positive cells could be detected in the HERS. In the dental pulp and dental follicle, a few cells showed a positive reaction. (d) At PN18, although the numbers of IEE and OEE cells decreased dramatically, a small number of HERS cells showed a TUNEL-positive reaction. (e) In the underside view of the specimen, DiI labeling could be observed on the base (orange dotted line) and end (yellow dotted line) of the HERS (arrows) after 3 days of culture. (f) In this section, the HERS is indicated as a yellow dotted line. The DiI-labeled cells did not move during tooth root formation. The blue line shows dentin. (g) At PN10, an immunopositive reaction for laminin beta-3 localized in HERS cells. The dental pulp and dental follicle cells show a negative reaction. (h) At PN10, syndecan-1 immunoreactivity could be observed in the HERS cells, although the cells of the dental pulp and dental follicle did not show a positive reaction. (i) Bmp-2 was detected in the HERS at PN10. Positive signals were also observed in the cells of the dental pulp and dental follicle. (j) At PN10, Bmp-4 was expressed in the HERS. However, the expression was weak compared with that of the Bmp-2. (k) Compared with the mesenchyme, there was strong expression for Msx-2 in the HERS. Black dotted line, HERS; F, dental follicle, P, dental pulp. Bars: a-d, 15 µm; e, 100 µm; f, 50 µm; g-k, 15 µm.

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).

	DISCUSSION

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To understand the development of HERS in the mouse molar, we counted the cell numbers of IEE and OEE in HERS and measured the total HERS length. The results showed that the cell number of IEE was greater than that of OEE in HERS during root formation. Previous studies have reported that IEE cells are square, while OEE cells are rectangular (Owens, 1978; Gurling and Sampson, 1985). The difference in the numbers of cells between IEE and OEE may be due to cell shape. Moreover, this result was in contrast to previous results (Gurling and Sampson, 1985) in that the number of HERS cells decreased, although total HERS length increased from PN10 to PN15. It is well-known that IEE cells change their shape and size according to their function during crown formation (Katchburian and Holt, 1972; Yamamoto et al., 1998). This study showed that the sizes of both IEE and OEE cells in HERS changed during root formation. It is conceivable that the function of the HERS may alter during this process. Moreover, total HERS length decreased dramatically at PN20, which is the eruption time of the M1. Therefore, the eruption might cause a reduction in total HERS length.

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.

	ACKNOWLEDGMENTS

 
The authors acknowledge Dr. Y.-P. Chen for providing the Bmp-2, Bmp-4, Msx-1, and Msx-2 plasmids. This work was supported by grant number R13-2003-13 from the Basic Research Program of the Korea Science and Engineering Foundation.

Received for publication October 16, 2003. Revision received July 4, 2004. Accepted for publication July 7, 2004.

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Journal of Dental Research, Vol. 83, No. 9, 688-692 (2004)
DOI: 10.1177/154405910408300906


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