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Gene Expression of Growth Differentiation Factors in the Developing Periodontium of Rat Molars
K. Sena1,*,
Y. Morotome1,
O. Baba2,
T. Terashima2,
Y. Takano2 and
I. Ishikawa1
1 Periodontology and
2 Biostructural Science, Department of Hard Tissue Engineering, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyou-ku, Tokyo 113-8549, Japan;
Correspondence: *corresponding author, sena.peri{at}tmd.ac.jp
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ABSTRACT
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Growth and differentiation factors (GDF) 5, 6, and 7 are known to play roles in tendon and ligament formation, and are therefore probably involved in the formation of periodontal ligament. In this study, we sought to determine temporal and spatial expression of GDF-5, -6, and -7 mRNA in developing periodontal tissue of rat molars using in situ hybridization. GDF gene expression in the periodontal ligament was first detected in cells associated with the initial process of periodontal ligament fiber bundle formation. Gene signals were also detected in cells located along the alveolar bone and cementum surfaces, the insertion sites of periodontal ligaments, during the course of root formation. GDF expression in these cells were down-regulated after completion of root formation. Our results appeared to suggest the involvement of GDF-5, -6, and -7 in the formation of the dental attachment apparatus.
Key Words: growth and differentiation factor (GDF) -5-6and -7 • in situ hybridization • developing periodontium
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INTRODUCTION
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Growth and differentiation factors (GDF) -5 (GDF-5/CDMP-1/BMP-14), -6 (GDF-6/CDMP-2/BMP-13), and -7 (GDF-7/CDMP-3/BMP-12) are members of the bone morphogenetic protein (BMP) family that forms part of the transforming growth factor-β (TGF-β) superfamily (Storm et al., 1994). BMP was originally discovered as a protein capable of inducing new bone formation when implanted subcutaneously in vivo (Urist, 1965). Subsequently, more than 10 additional BMPs have been identified based on sequence homology (Wozney et al., 1988; Celeste et al., 1990; Storm et al., 1994). Previous studies have shown that BMPs play important roles in developmental processes throughout early embryogenesis and organogenesis (Thesleff, 1995; Hogan, 1996). In particular, BMPs are essential for the differentiation of mesenchymal cells into various types of connective tissues, including cartilage, muscle, adipose tissue, tendons, and ligaments (Ahrens et al., 1993; Katagiri et al., 1994; Rosen et al., 1994; Wolfman et al., 1997).
Several studies have indicated the involvement of BMPs in early embryonic tooth development (Lyons et al., 1990; Vainio et al., 1993; Bègue-Kirn et al., 1994; Heikinheimo, 1994; Takahashi and Ikeda, 1996; Åberg et al., 1997; Helder et al., 1998). As for tissue repair, BMP-7 enhances the formation of reparative dentin (Rutherford et al., 1993) and dental cementum (Ripamonti et al., 1996). These findings support the contention that BMP family members act as signaling molecules in early tooth development and also represent effective stimulators of repair processes in mature dental tissues.
GDF-5, -6, and -7 were first identified by the degenerative polymerase chain-reaction (Storm et al., 1994). Ectopic implantation studies have demonstrated the ability of GDFs to induce tendon/ligament-like tissue formation in the subcutis (Wolfman et al., 1997) and muscle (Cox and Rosen, 1996). Ultrastructural and Northern blot analyses of ectopically induced tendon/ligament-like tissue have revealed the expression of several connective tissue markers characteristic of tendon and ligament tissues (Cox and Rosen, 1996; Wolfman et al., 1997). In addition, GDFs are reportedly expressed at the site of joint development in mouse embryos (Wolfman et al., 1997) and have the potential to augment tendon repair in chickens (Lou et al., 2001) and rats (Aspenberg and Forslund, 1999).
The dental attachment apparatus is a unique structure comprised of the periodontal ligament, which is rich in cellular and fibrous connective tissue, anchoring the root cementum to the alveolar bone proper. This structure has been referred to as a kind of joint (Schroeder, 1986). Morotome et al. (1998) demonstrated GDF-5, -6, and -7 gene expression in cells from bovine dental follicles surrounding the developing root at the root-forming stage, using reverse-transcriptase polymerase chain-reaction (RT-PCR). Given that the cementum, periodontal ligament, and alveolar bone proper are all derived from the dental follicle, GDFs may play key roles in the formation of the dental attachment apparatus. The patterns of in vivo expression of GDFs in developing periodontal tissues are unknown. The present study utilized in situ hybridization to investigate temporal changes in patterns of GDF-5, -6, and -7 mRNA expression in the developing periodontal tissues of rat molars, particularly during the formation of the dental attachment apparatus.
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MATERIALS & METHODS
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Tissue Preparation for in situ Hybridization
All experimental procedures were approved by the Institutional Committee of Animal Care and Use at the Tokyo Medical and Dental University, and were properly performed under the control of the Guidelines for Animal Experimentation at the Tokyo Medical and Dental University (approval number 0010302). In this study, male Wistar rats (2, 3, 8, 12, 20, and 36 wks old) were used. Rats were anesthetized by intraperitoneal injection of 8% chloral hydrate solution and fixed under perfusion from the left ventricle with 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PB; pH 7.4). Mandibles were dissected and further fixed in the same fixative at 4°C for 24 hrs. Tissues were decalcified in 8% ethylenediaminetetraacetate (EDTA; pH 7.4) until an acceptable radiographic end-point was achieved (1-10 wks), then embedded in paraffin. Four-micrometer-thick serial sections were cut in the buccal-lingual plane through the medial root of the first mandibular molar and processed for either in situ hybridization, hematoxylin-eosin staining, or histological observations by means of Azan staining.
Preparation of Probes
Rat GDF mRNA probes used for this study were generated from PCR product cloned into the modified pBluescript Sk+ vector (Oida et al., 1997; Morotome et al., 1998). The primer set for PCR included: 5'-TGGGACGACTGGATCATCGC-3' and 5'-CACCATGTCCTCATACTGCTT-3'. The nucleotide sequences and sizes of cDNAs are shown as a Table in the Appendix (www.dentalresearch.org). Probes were labeled with Digoxigenin (DIG)-11-UTP by means of a DIG RNA Labeling Kit (Roche Diagnostics GmbH, Mannheim, Germany).
In situ Hybridization
Sections were deparaffinized, hydrated, and digested with proteinase K (1 µg/mL) (Roche Diagnostics GmbH) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, for 15 min at 37°C. Post-fixation with 4% PFA in 0.1 M PB (pH 7.4) was performed for 10 min at room temperature. Sections were treated with 0.2 M HCl for 10 min at room temperature, and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine. The probe was diluted with hybridization solution to a final concentration of 1 µg/mL. This hybridization solution consisted of 50% formamide; 600 mM NaCl; 10 mM Tris-HCl (pH 7.6); 1 mM EDTA; 1x Denhardt’s solution; 0.2 µg/mL tRNA; 10% dextran sulphate; and 0.25% SDS. Hybridization was performed overnight at 45°C, followed by RNase A (5 µg/mL) treatment at 37°C in TNE buffer (10 mM Tris-HCl, pH 7.6; 500 mM NaCl; 1 mM EDTA) for 30 min, and a series of washes. For immunodetection of hybridized DIG-labeled probe, blocking reagent (Roche Diagnostics GmbH) in DIG buffer 1 (100 mM Tris-HCl, pH 7.5; 150 mM NaCl) was treated, and sections were reacted with a diluted anti-DIG Fab fragment conjugated with alkaline phosphatase (Roche Diagnostics GmbH) in DIG buffer 1 for 2 hrs at room temperature. We visualized hybridized signals by treating sections with nitroblue tetrazolium (NBT; Roche Diagnostics GmbH) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche Diagnostics GmbH) at room temperature until maximum signal-to-noise ratio was attained.
Undecalcified Sections
To locate sites in which mineralization of hard tissue was occurring, we injected some animals intraperitoneally with 15 mg/kg body weight of 3,3'-Bis [N,N-Bis (carboxymethyl) aminomethyl] fluorescein (Calcein, Dojindo, Kumamoto, Japan) 19 hrs before the animals’ death. Rats were perfusion-fixed with 4% PFA in 0.1 M PB (pH 7.4), and mandibular specimens were embedded in polyester resin (Rigolac, Nisshin EM, Tokyo, Japan). Embedded undecalcified specimens were sectioned in the buccal-lingual plane by means of a micro-cutting machine (EXAKT, Hamburg, Germany). Ground and polished sections were observed under confocal laser-scanning microscopy (Fluoview, Olympus, Tokyo, Japan).
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RESULTS
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Two-week-old Rats
At 2 wks, mandibular first molars (M1) of rats were located just before eruption into the oral cavity. Hertwig’s epithelial root sheath was observed at the apical end of the root dentin (Fig. 1A ). Fluorescent lines of labeled calcein were observed along the alveolar bone surface and dentin-mineralizing front (data not shown). Scattered thin collagen fibers stained blue by Azan staining were observed in the periodontal ligament space (Figs. 1B, 1C). The initial attachments of collagenous matrix fibers of acellular extrinsic fiber cementum were observed on the root surface, except in the regions covered by Hertwig’s epithelial root sheath. Collagen fibers in the periodontal ligament space were oriented obliquely to the alveolar bone surface, whereas fibers in the apical region were less specifically oriented. Cells expressing GDF-5, -6, and -7 genes were aligned on the inner aspect of the alveolar wall and scattered in the periodontal ligament space, except in the apical regions. Positive reactions for GDF genes were not detected in cells associated with thin layers of acellular cementum (Figs. 1D-1F, 1J-1L). Controls of in situ hybridization with sense probes showed no positive reactions (Figs. 1G-1I).
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Figure 1. Buccal-lingual sections of a two-week-old rat mandible through the first molar tooth germ (M1) stained with Azan (A-C) or treated for detecting GDF-5 (D,G,J), -6 (E,H,K), and -7 (F,I,L) transcripts. (A) Whole view of M1 before eruption. Small rectangles of B and C indicate the areas enlarged in B and C, respectively. (B) Obliquely oriented thin collagen fibers (stained blue) show initial signs of attachment to the root surface as matrix fibers of acellular extrinsic fiber cementum. (C) Collagen fibers are poorly oriented in the apical region. (D-F) Cells expressing the GDF genes (stained brown) are aligned on the surface of the alveolar bone (ab) and scattered in the periodontal ligament (arrowheads). (J-L) In the apical region, few GDF-positive cells are detectable only on the alveolar bone surface. (G-I) Control with sense probes showed no positive reactions. ab, alveolar bone; d, dentin; pul, pulp. Bar = 500 µm (A), 50 µm (B-L).
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Three-week-old Rats
M1 had erupted into the oral cavity (Fig. 2A) and deposition of cellular cementum had started in the apical region of the root (arrows, Fig. 2C). Fluorescent lines of calcein ran on the surface of alveolar bone and dentin-mineralizing front (data not shown). In the cervical region, collagenous matrix fibers of acellular extrinsic fiber cementum were continuous with periodontal ligament fiber bundles (Fig. 2B). These thick bundles of periodontal ligament were aligned, arranged, and extended obliquely from the acellular cementum surface toward the more occlusal portion of alveolar bone and connected both hard tissues. In the apical region, collagen fibers were still thin and poorly oriented (Fig. 2C). Expression of GDF-5, -6, and -7 genes was observed in cells on the surfaces of the alveolar wall, acellular and cellular cementum, and large numbers of cells in the periodontal ligament (Figs. 2D-2I). Cells of Hertwig’s epithelial root sheath did not express mRNA signals for GDFs. Controls with sense probes revealed no positive reactions (data not shown).
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Figure 2. Buccal-lingual sections of the M1 in three-week-old rat stained with Azan (A-C) or treated for detecting GDF-5 (D,G), -6 (E,H), and -7 (F,I) transcripts. Small rectangles of B and C (A) indicate the areas enlarged in B and C, respectively. (B) Collagenous matrix fibers of acellular extrinsic fiber cementum are shown to be continuous with thick periodontal ligament fiber bundles extending obliquely from acellular cementum toward the alveolar bone (ab). (C) Cellular cementum is deposited on the root dentin surface near the apical end (arrows). Only scattered thin collagen fibers are seen in this region. (D-I) GDFs mRNA expression in regions B and C indicated in Fig. 2A . GDF-positive cells are widely distributed in the periodontal ligament, on the surfaces of the alveolar bone and the cementum (arrowheads). ab, alveolar bone; d, dentin; pdl, periodontal ligament; pul, pulp. Bar = 500 µm (A), 50 µm (B-I).
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Eight-week-old Rats
In eight-week-old rats, the root had been elongated by apposition of both root dentin and cellular cementum (Fig. 3A). In the apical region, thick cellular cementum was observed on the surface of root dentin (Fig. 3D). Thicker and denser bundles of periodontal ligament connecting cementum and alveolar bone were observed in the periodontal ligament space (Figs. 3C, 3D). Fluorescent green lines of calcein were observed on the dentin-mineralizing front and on surfaces of the alveolar bone and cellular cementum (Fig. 3B). Expression of GDF-5, -6, and -7 genes were observed in cells on the surfaces of the cementum and alveolar wall and on those scattered in the periodontal ligament, although with less intensity than in three-week-old animals (Figs. 3E-3J). Controls using sense probes revealed no positive reactions (data not shown). Identical expression patterns of GDF transcripts were observed in the 12- and 20-week-old rats (data not shown).
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Figure 3. Buccal-lingual sections of the M1 in an eight-week-old rat stained with Azan (A,C,D) or treated for detecting GDF-5 (E,H), -6 (F,I), and -7 (G,J) transcripts. Small rectangles of C and D (A) indicate the areas enlarged in C and D, respectively. (C) Periodontal ligament fiber bundles appear thicker and denser than in the previous stage. (D) Thick cellular cementum (cc) on the root dentin surface. (B) Fluorescent green lines of labeled calcein follow the course of mineralizing fronts of dentin (d), alveolar bone (ab), and cellular cementum (cc) in the area outlined by the red rectangle in Fig. 3A. (E-J) Cells showing GDFs gene expression are observed in the cells on the surfaces of the cementum and the alveolar wall and also on those scattered in the periodontal ligament (pdl) (arrowheads). ab, alveolar bone; cc, cellular cementum; d, dentin; pdl, periodontal ligament; pul, pulp. Bar = 500 µm (A and B), 50 µm (C-J).
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36-week-old Rats
In 36-week-old rats, the root dentin was covered by thick cellular cementum. At the apical quarter of the root length, the root was fully organized by cellular cementum apposition (Fig. 4A). The fluorescent line of calcein was almost undetectable in the surrounding alveolar bone, dentin, and cementum (Fig. 4B). Signs of GDF gene expression were not visible throughout the cells in the cementum, periodontal ligament, and alveolar bone at this stage (Figs. 4E-4J). Controls with sense probes revealed no positive reactions (data not shown).
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Figure 4. Buccal-lingual sections of the M1 in a 36-week-old rat stained with Azan (A,C,D) or treated for detecting GDF-5 (E,H), -6 (F,I), and -7 (G,J) transcripts. (A) Apically, one-fourth of the root is fully covered by thick cellular cementum. Small rectangles of C and D indicate the areas enlarged in C and D, respectively. (B) The fluorescent green line of labeled calcein is almost undetectable in the alveolar bone (ab), dentin (d), and cementum (cc) in the area outlined by the red rectangle in Fig. 4A. (E,F) GDF-positive cells are not visible in the periodontal tissues in sections hybridized with the GDFs mRNA probe. ab, alveolar bone; cc, cellular cementum; d, dentin; pdl, periodontal ligament; pul, pulp. Bar = 500 µm (A and B), 50 µm (C-J).
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DISCUSSION
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This is the first study to show expression of GDF-5, -6, and -7 mRNAs in the growing dental tissues of rats in vivo. In this paper, we have successfully described the spatial and temporal expression of GDF mRNAs during the developmental process of M1 and associated periodontal tissues.
Analysis of our data demonstrates that cells expressing GDF-5, -6, and -7 mRNAs during root formation coincide with the cells involved in the formation of periodontal tissues such as cementum, periodontal ligament, and alveolar bone. Expression of GDF genes in cells on the surfaces of cementum and alveolar bone starts with the onset of formation of these two hard tissues and ends after completion of formation. In addition, cells expressing GDF mRNAs among periodontal ligament fibers appear to be associated with fiber bundle formation. GDF-5, -6, and -7 signals are undetectable in these cells in 36-week-old rats in which root formation appears to have been completed (Fig. 4). These results suggest the involvement of GDFs in the diverse development of periodontal cells into osteoblastic, cementoblastic, or ligament-forming cells. In the present study, obvious differences among expression patterns of GDF-5, -6, and -7 genes were not identified within the experimental period. However, fluctuations in the intensity of staining reactions in gene-expressing cells among different developmental stages allowed for the quantitative evaluation of messages for GDFs being expressed by individual cells. Cells involved in root- and ligament-forming stages exhibited significantly stronger signals compared with those in well-formed tissues, where low or no signals were observed.
Previously published reports have indicated that GDF-7 stimulates both proliferation and alkaline phosphatase expression of osteoblastic (ROS 17/2.8) cells (Furuya et al., 1999). GDF-5 and -6 are known to be bone- and cartilage-inducers, although less osteoinductive than the most closely related BMPs, such as BMP-5, -6, and -7 (Gruber et al., 2000). Involvement of GDFs in the differentiation of non-osteoblastic cell lineages is also suggested in several studies: GDF-5, -6, and -7 implantation studies demonstrate induction of tendon/ligament-like tissues in ectopic sites (Cox and Rosen, 1996; Wolfman et al., 1997); GDF-6, -7, and BMP-2 inhibit terminal differentiation of myoblast cell lines C2C12 and L-6, but GDF-6 and -7 do not induce cell differentiation into osteoblasts (Inada et al., 1996). Function studies of GDF-6 and -7 with the use of a chondrocyte cell line (MC615) also suggest that GDF-6 and -7 behave differently from other known members of the BMP family, such as BMP-2 and -4, well-known as osteogenic BMPs, in the context of osteoblastic differentiation (Valcourt et al., 1999). A GDF-7 transduced mesenchymal progenitor cell line is known to show no changes in alkaline phosphatase activity (Lou et al., 1999). Expression of GDF-5, -6, and -7 genes appears concomitant with the process of joint formation (Wolfman et al., 1997), where well-coordinated development of non-osteoblastic and osteoblastic cells is required. GDFs could synergistically regulate joint formation by influencing the differentiation of condensed mesenchymal cells into both osteoblastic and non-osteoblastic cells. Taken together, it appears reasonable to propose that GDFs play roles in the development of the attachment apparatus of periodontal tissues where pluripotent dental follicle cells differentiate into osteoblasts, cementoblasts, or periodontal fibroblasts. Further studies are required to explain exactly how these growth factors contribute to this process.
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ACKNOWLEDGMENTS
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Y. Morotome was a recipient of Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. This research was financially supported by JSPS (JSPS-Young Scientists No. 02514 and JSPS-RFTF96I00205).
The nucleotide sequence data reported in this paper appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession numbers: rat mRNA for growth and differentiation factor 5, partial cds AB087404; rat mRNA for growth and differentiation factor 6, partial cds AB087405; rat mRNA for growth and differentiation factor 7, partial cds AB087406.]
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication November 20, 2001.
Revision received November 1, 2002.
Accepted for publication November 7, 2002.
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Journal of Dental Research, Vol. 82, No. 3,
166-171 (2003)
DOI: 10.1177/154405910308200304
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ABSTRACT
INTRODUCTION