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Differential gene expression of bone-related proteins in epithelial and fibroblastic cells derived from human periodontal ligament
Cell Biology International 27 (2003) 519–524
Cell Biology International www.elsevier.com/locate/cellbi
Differential gene expression of bone-related proteins in epithelial and fibroblastic cells derived from human periodontal ligament Yoshihiro Mouri, Hideki Shiba*, Noriyoshi Mizuno, Takuji Noguchi, Tetsuji Ogawa, Hidemi Kurihara Department of Periodontal Medicine, Division of Frontier Medical Science, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan Received 5 September 2002; revised 27 January 2003; accepted 11 March 2003
Abstract Hertwig’s epithelial root sheath (HERS) is involved in the differentiation of cementoblasts. The cells of epithelial rests of Malassez (ERM) may contribute to that process. However, little is known about the role of these epithelial cells in cementum repair. In the present study, we investigated the expression of alkaline phosphatase (ALPase), osteopontin (OPN), bone morphogenetic protein (BMP)-2 and BMP-4 in epithelial cells (E cells) and fibroblastic cells (F cells) derived from the same human periodontal ligament. E cells were identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. Reverse transcription–polymerase chain reaction analysis showed that E cells have lower ALPase and BMP-4 mRNA levels than F cells. On the other hand, the expression of OPN mRNA in E cells was stronger than in F cells. No significant difference was observed in BMP-2 expression between E and F cells. Thus, they have different expression patterns of ALPase, BMP-4 and OPN, suggesting that ERM and mesenchymal cells in periodontal ligament may be cooperatively involved in cementum repair. Furthermore, E cell cultures will be useful in elucidating the role of ERM. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Epithelial rests of Malassez; Epithelial cells; Fibroblastic cells; Periodontal ligament; Cementum repair
1. Introduction The cells of epithelial rests of Malassez (ERM) are the only odontogenic epithelial cells that are present in periodontal ligament. A number of functions of ERM have been discussed, such as protection against root resorption, formation of apical cyst, maintenance of the periodontal ligament space and prevention of ankylosis (Grant and Bernick, 1969; Lindskog et al., 1998; Loe¨ and Waerhaug, 1961; Ten Cate, 1972; Waerhaug, 1958). * Corresponding author. Tel.: +81-82-257-5663; fax: +81-82-257-5664 E-mail address: [email protected] (H. Shiba). Abbreviations: ALPase, alkaline phosphatase; BMP, bone morphogenetic protein; DMEM, Dulbecco’s modified Eagle’s medium; ERM, epithelial rests of Malassez; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HERS, Hertwig’s epithelial root sheath; OPN, osteopontin; RT–PCR, reverse transcription–polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
It has also been suggested that ERM is involved in cementoblast differentiation (Ten Cate, 1996). This function is of particular interest, because elucidation of its mechanism could lead to a novel method of obtaining new attachments involving cementum regeneration. Cell culture systems are very useful for analyzing cell functions. In fact, the precise functions of the cells of ERM remain largely unknown, although the functions of periodontal ligament fibroblasts have been thoroughly investigated. ALPase is thought to be essential for biomineralization (Whyte et al., 1987). Periodontal ligament fibroblastic cells have high levels of ALPase activity and can differentiate into osteoblasts or cementoblasts (McCulloch and Bordin, 1991; Nohutcu et al., 1997; Somerman et al., 1990a). OPN, a noncollagenous protein expressed in osteogenic and nonosteogenic cells, performs multiple functions (Sodek et al., 1992). It is present at the root surface during the initiation of cementogenesis in development (Bronckers
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00075-1
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et al., 1994; Somerman et al., 1990b; Somerman et al., 1992). BMP-2 and BMP-4 are involved in epithelial– mesenchymal interaction (Thesleff et al., 1995; Vainio et al., 1993), which plays an important role in cell differentiation. Therefore, these bone-related proteins are thought to play a role in cementum repair. In the present study, we isolated and cultured epithelial cells derived from human periodontal ligament by enzymatic digestion. Furthermore, we tried to elucidate the role of ERM cells during cementum repair by comparing the expression of ALPase, OPN, BMP-2 and BMP-4 in epithelial cells derived from periodontal ligament with those in fibroblastic cells derived from the same tissue.
2. Materials and methods 2.1. Preparation and culture of cells Three periodontally healthy and non-carious premolars (premolars 1, 2 and 3) extracted in the course of orthodontic treatment were collected from three patients with their informed consent. The periodontal ligament tissue attached to the mid-third of each root was incubated at 37 (C for 20 min in DMEM supplemented with 0.2% collagenase (Wako Pure Chemical Industries, Osaka, Japan), 0.25% trypsin (Difco Laboratories, Detroit, MI, USA), 100 units/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B. For the isolation of E cells, a partial cell suspension was centrifuged at 800 rpm for 5 min. The pellet was suspended in MCDB medium (pH 7.4) (SIGMA, St Louis, MO, USA) containing 10 µg/ml insulin, 5 µg/ml transferrin, 10 µM 2-mercaptoethanol, 10 µM 2-aminoethanol, 10 nM sodium selenite, 100 units/ml penicillin, 100 µg/ml streptomycin and 50 ng/ml amphotericin B (medium A) (Myoken et al., 1995; Sato et al., 1987). The cells were seeded in 35 mm plastic tissue culture dishes coated with type I collagen, and incubated in 5% CO2/95% air at 37 (C. When the E cells reached subconfluence, they were harvested and subcultured in medium A containing 50 µg/ml bovine brain extract (medium B). For the isolation of F cells, the residual cell suspension was centrifuged at 800 rpm for 5 min and the pellet was suspended in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B (medium C) and the cells were seeded in 35 mm plastic tissue culture dishes coated with type I collagen, and incubated in 5% CO2/95% air at 37 (C until confluent cell monolayers were formed. E-1 and F-1 cells, E-2 and F-2 cells, and E-3 and F-3 cells were obtained from premolars 1, 2 and 3, respectively. When the cells became confluent, they were harvested and subcultured.
2.2. Preparation of periodontal ligament tissue Two periodontally healthy and non-carious premolars (premolars 4 and 5) extracted in the course of orthodontic treatment were collected from two patients with their informed consent. The periodontal ligament tissue attached to the mid-third of each root was isolated. 2.3. Western blots Cytokeratin 5 and 8 are expressed by cells of EMR (Gao et al., 1988). Therefore, E cells are identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. E-1 and F-1 cells in cultures at the third passage were harvested, seeded at a density of 1104 cells/9 mm plastic tissue culture well coated with type I collagen, and cultured in 0.2 ml mixed medium (1:1 (vol/vol) medium B:C) containing 50 µg/ml ascorbic acid for 4 days. Then, cells were lysed with Laemmli’s buffer (Laemmli, 1970) and boiled for 3 min. Samples (15 µl) were resolved on a 15% SDS–polyacrylamide gel by electrophoresis (SDS–PAGE) under nonreducing conditions, and electrophoretically transferred on to nitrocellulose membranes. The membranes were blocked with 3% non-fat dried milk for 1.5 h, and reacted with mouse anti-human cytokeratin 5 and 8 monoclonal antibodies (2 µg/ml) (Chemicon, Temecula, CA, USA) at 37 (C for 1 h. The membranes were incubated with goat antirabbit IgG horseradish peroxidase conjugated antibody (Chemicon) for 1 h at 37 (C. Immunodetection was performed with Chemi-luminescence Reagent Plus (NEN Life Science Products, Boston, MA, USA), following the manufacturer’s directions. 2.4. RNA isolation E-1, -2 and -3 cells and F-1, -2 and -3 cells at the third passage were harvested, seeded at a density of 27104 cells/35 mm plastic tissue culture dish coated with type I collagen, and maintained in 2 ml mixed medium (1:1 (vol/vol) medium B: C) containing 50 µg/ml ascorbic acid. Total RNA was extracted from the cultures using ISOGEN (Wako Pure Chemical Industries) on day 4 and quantified by spectrometry at 260 and 280 nm. In addition, total RNA was purified from the periodontal ligament tissues of premolars 4 and 5. 2.5. RT–PCR and sequencing PCR products Total RNA was reverse-transcribed using oligo (dT) 12–18 (Boehringer Mannheim Corp., IN, USA). RT reaction was performed for 60 min at 42 (C and stopped by heat inactivation for 5 min at 99 (C. Specific primers for OPN, ALPase, BMP-2, BMP-4 and GAPDH were designed according to the sequence data (see Table 1) (Kawamoto et al., 1998; Kiefer et al., 1989; Kishi et al.,
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3. Results 3.1. Morphology of E cells and F cells The primary cultures of E cells were morphologically polygonal by microscopic examination (Fig. 1A). On the other hand, F cells were spindle-shaped (Fig. 1B). 3.2. Keratin expression in E cells and F cells E cells, but not F cells, showed a positive reaction to immunoblotting with anti-cytokeratin 5 and 8 antibody (Fig. 2). 3.3. mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in periodontal ligament tissues RT–PCR analyses showed periodontal ligament tissues expressed mRNA for OPN, ALPase, BMP-2 and BMP-4 (Fig. 3). The DNA sequence of each PCR product was 100% identical to the previously reported sequences of the OPN, ALPase, BMP-2 and BMP-4 genes (Kiefer et al., 1989; Kishi et al., 1989; Wozney et al., 1988). 3.4. mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in E cells and F cells
Fig. 1. Phase-contrast micrographs of the primary culture of E and F cells. The pictures show the morphology of E-1 cells (A) and F-1 cells (B). The scale bar indicates 25 µm.
Fig. 4 shows the mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in E cells and F cells. The level of ALPase and BMP-4 mRNAs in E cells was lower than in F cells. On the other hand, E cells showed greater expression of OPN than F cells. No difference was observed in BMP-2 expression between E and F cells. These expression patterns were consistently observed in the three different cultures of each cell type. 4. Discussion
Fig. 2. Immunoblotting of keratin 5 and 8 in E and F cells. E-1 cells and F-1 cells were seeded and maintained as described in Materials and Methods. The experiment was repeated twice with similar results.
1989; Wozney et al., 1988). The PCR reaction and cycling conditions are described in the Table 1. The PCR products were subcloned into the plasmid vector pGEM-T (Promega, Madison, WI, USA) and identified and confirmed via sequencing by dideoxy chain termination.
Previous studies have shown that cultured epithelial cells derived from porcine periodontal ligaments produce fibronectin, glycosaminoglycans and bone resorbing factor (Birek et al., 1983; Merrilees et al., 1983; Uitto et al., 1992). Recently, epithelial cells from human periodontal ligament were shown to express fibroblast growth factor receptors in vitro (Yamanaka et al., 2000). However, the expression of bone-related proteins, such as ALPase, BMP-2, BMP-4 or OPN, in epithelial cells from periodontal ligament has not been investigated. In the present study, we were the first to succeed in isolating and culturing both E and F cells from the same human periodontal ligament by enzymatic digestion. We also examined bone-related proteins in cultures of E and F cells. OPN appears on the root surface at the same time as the initiation of cementogenesis and plays an important role in this mechanism (Uitto et al., 1992; Yamanaka et al., 2000). Some studies have suggested that OPN is
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Fig. 3. RT–PCR analysis of mRNA expression of OPN, ALPase, BMP-2 and BMP-4 in human periodontal tissues. RNA was extracted from periodontal tissues-1 and -2, prepared from premolars-4 and -5, respectively. PCR products, as described in Materials and Methods, were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. M, 100-bp ladder.
Fig. 4. RT–PCR analysis of mRNA expression of OPN, ALPase, BMP-2 and BMP-4 in cultures of E cells and F cells. E-1, -2 and -3 cells and F-1, -2 and -3 cells were seeded and maintained as described in Materials and Methods. RNA was extracted on day 4. PCR products, as described in the Materials and Methods, were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. M, 100-bp ladder.
Table 1 Oligonucleotide primers used for RT–PCR determining bone-related proteins Gene
Annealing temperature ((C)
Sequence
GAPDH (613 bp)
53
OPN (532 bp)
53
ALPase (382 bp)
54
BMP-2 (440 bp)
54
BMP-4 (339 bp)
48
F: 5#-GTCAAGGCTGAGAACGGGAA-3# R: 5#-GCTTCACCACCTTCTTGATG-3# F: 5#-CACCTGTGCCATACCAGTTAAAC-3# R: 5#-GGTGATGTCCTCGTCTGTAGCATC-3# F: 5#-GTACTGGCGAGACCAAGCG-3# R: 5#-GGCCCAGCGCAGGATGGAGG-3# F: 5#-CCGCTGTCTTCTAGCGTTGC-3# R: 5#-CCTGAAGCTCTGCTGAGGTG-3# F: 5#-GTCCTGCTAGGAGGCGCGAG-3# R: 5#-GTTCTCCAGATGTTCTTCG-3#
Denaturing, annealing and elongation conditions were 94 (C, 30 s, the temperature as described above, 1 min and 72 (C, 1 min, respectively. The cycling condition was a total of 30 cycles. F, forward primer; R, reverse primer.
Y. Mouri et al. / Cell Biology International 27 (2003) 519–524
produced by dental mesenchymal cells (Lekic et al., 1996; Ramakrishnan et al., 1995), and others by the cells of HERS (Bosshardt and Nanci, 1998; Lekic et al., 1996; Ramakrishnan et al., 1995). The present study showed that E cells, but not F cells, strongly express OPN mRNA. Therefore, cells of ERM may secrete OPN on to the root surface during the early stages of cementum repair. The present study showed that BMP-2 and BMP-4 mRNAs are expressed in E cells and F cells. BMP-2 and BMP-4 modulate tooth development through epithelial– mesenchymal interaction (Thesleff et al., 1995; Vainio et al., 1993). These findings suggest that interaction occurs between cells of ERM and undifferentiated mesenchymal cells in periodontal ligament during cementum repair. It is widely accepted that the mesenchymal cells of dental follicles differentiate into cementoblasts during cementum development (Cho and Garant, 1996). However, it has also been suggested that HERS cells may undergo epithelial–mesenchymal transformation to differentiate into cementoblasts (MacNeil and Thomas, 1993). The present study showed that F cells, but not E cells, have high levels of ALPase mRNAs, suggesting that mesenchymal cells in periodontal ligament tissue have the capacity to differentiate into cementoblasts during cementum repair. However the possibility still remains that E cells can transform into F cells. Enamel matrix proteins are involved in the formation of cementum (Hammarstro¨m, 1997). HERS cells express amelin mRNA in vivo (Fong et al., 1996), and also produce amelogenin (Hamamoto et al., 1996). Thus, HERS is thought to play an important role in cementum development through the production of enamel matrix proteins. There is a stong possibility that cells of ERM produce enamel matrix protein during cementum repair. In conclusion, bone-related proteins show different mRNA expression patterns in E cells and F cells. Furthermore, this culture model should be useful for elucidating the role of ERM during cementum repair.
Acknowledgements This work was supported in part by the Satake Foundation for Hiroshima University, plus Grant-inAid for Scientific Research (A) (No. 12307051) and Grant-in-Aid for Scientific Research (C) (No. 12897020) from the Japan Society for the Promotion of Science, Japan. References Birek C, Heersche JNM, Jez D, Brunette DM. Secretion of a bone resorbing factor by epithelial cells cultured from porcine rests of Malassez. J Periodont Res 1983;18:75–81.
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Bosshardt DD, Nanci A. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells. J Hist & Cytochem 1998;46:135–42. Bronckers AL, Farach-Carson MC, Van Waveren EV, Butler WT. Immunolocalization of osteopontin, osteonectin, and dentin sialoprotein during dental root formation and early cemento-genesis in the rat. J Bone Miner Res 1994;9:833–41. Cho MI, Garant PR. Expression and role of epidermal growth factor receptors during differentiation of cementoblasts, osteoblasts, and periodontal ligament fibroblasts in the rat. Anat Rec 1996; 245:342–60. Fong CD, Slaby I, Hammarstro¨m L. Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath. J Bone Miner Res 1996;11:892–8. Gao Z, Mackenzie IC, Williams DM, Cruchley AT, Leigh I, Lane EB. Patterns of keratin-expression in rests of Malassez and periapical lesions. J Oral Pathol 1988;17:178–85. Grant DA, Bernick S. A possible continuity between the epithelial rests and epithelial attachment in miniature swine. J Periodont 1969;40:87–95. Hamamoto Y, Nakajima T, Ozawa H, Uchida T. Production of amelogenin by enamel epithelium of Hertwig’s root sheath. Oral Surg Oral Med Oral pathol Oral Radiol Endod 1996;81:703–9. Hammarstro¨m L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997;24:658–68. Kawamoto T, Pan H, Yan W, Ishida H, Usui E, Oda R et al. Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes. Eur J Biochem 1998;256:503–9. Kiefer MC, Bauer DM, Barr PJ. The cDNA and derived amino acid sequence for human osteopontin. Nucleic Acids Res 1989;17:3306. Kishi F, Matsuura S, Kajii T. Nucleotide sequence of the human liver-type alkaline-phosphatase cDNA. Nucleic Acids Res 1989; 17:2129. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970;227:680–5. Lekic P, Sodek J, McCulloch CAG. Osteopontin and bone sialoprotein expression in regenerating rat periodontal ligament and alveolar bone. Anatom Rec 1996;244:50–8. Lindskog S, Blomlo¨f L, Hammarstro¨m L. Evidence for a role of odontogenic epithelium in maintaining the periodontal space. J Clin Periodont 1998;15:371–3. Loe¨ H, Waerhaug J. Experimental replantation of teeth in dogs and monkeys. Archs Oral Biol 1961;3:176–84. MacNeil RL, Thomas HF. Development of murine periodontium. II. Role of the epithelial root sheath in formation of the periodontal attachment. J Periodontol 1993;64:285–91. McCulloch CAG, Bordin S. Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodontal Res 1991; 26:144–54. Merrilees MJ, Sodek J, Aubin JE. Effects of cells of epithelial rests of Malassez and endothelial cells on synthesis of glycosaminoglycans by periodontal ligament fibroblasts in vitro. Dev Biol 1983; 97:146–53. Myoken Y, Myoken Y, Okamoto T, Sato JD, Takada K. Effect of fibroblast growth factor-1 on the three-dimensional growth and morphogenesis of human salivary gland epithelial cells embedded in collagen gels. In Vitro Cell Dev Biol 1995;31:84–6. Nohutcu RM, McCauley LK, Koh AJ, Somerman MJ. Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro. J Periodontol 1997;68:320–7. Ramakrishnan PR, Lin WL, Sodek J, Cho MI. Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro. Calcif Tissue Int 1995;57:52–9. Sato JD, Kawamoto T, Okamoto T. Cholesterol requirement of P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro. J Exp Med 1987;165:1761–6.
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Y. Mouri et al. / Cell Biology International 27 (2003) 519–524
Sodek J, Chen J, Kasugai S, Nagata T, Zhang Q, Shapiro HS, Wrana JL, Goldberg HA. Sialoproteins in bone remodeling. In: Davidovitch Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation, pp. 127–136. Somerman MJ, Young MF, Foster RA, Moehring JM, Imm G, Sauk JJ. Characteristics of human periodontal ligament cells in vitro. Archs Oral Biol 1990a;35:241–7. Somerman MJ, Shroff B, Argraves WS, Morrison G, Craig AM, Denhardt DT et al. Expression of attachment proteins during cementogenesis. J Biol Buccale 1990b;18:207–14. Somerman MJ, Shroff B, Foster RA, Butler WT, Sauk JJ. Mineralassociated adhesion proteins are linked to root formation. Proc Finn Dent Soc 1992;88(suppl I):451–61. Ten Cate AR. The epithelial cell rests of Malassez and the genesis of the dental cyst. Oral Surg 1972;34:956–64. Ten Cate AR. The role of epithelium in the development, structure and function of the tissues of tooth support. Oral Dis 1996;2:55–62. ´ berg T. EpithelialThesleff I, Vaahtokari A, Kettunen P, A mesenchymal signaling during tooth development. Connect Tissue Res 1995;32:9–15.
Uitto VJ, Larjava H, Peltonen J, Brunette DM. Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells. J Dent Res 1992;71:1203–11. Vainio S, Karavanova I, Jowett A, Thesleff I. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 1993; 75:45–58. Waerhaug J. Effect of C-avitaminosis on the supporting structure of teeth. J Periodont 1958;29:87–97. Whyte MP, Rettinger SD, Vrabel LA. Infantile hypophosphatasia: Enzymatic detect explored with alkaline phosphatase-deficient skin fibroblasts in culture. Calcif Tissue Int 1987;40:244–52. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW et al. Novel regulators bone formation: molecular clones and activities. Science 1988;242:1528–34. Yamanaka T, Sakamoto A, Tanaka Y, Zhang Y, Hayashido Y, Toratani S et al. Isolation and culture of epithelial cells derived from ERM in human periodontal ligament. In Vitro Cell Dev Biol 2000;36:548–53.
Cell Biology International www.elsevier.com/locate/cellbi
Differential gene expression of bone-related proteins in epithelial and fibroblastic cells derived from human periodontal ligament Yoshihiro Mouri, Hideki Shiba*, Noriyoshi Mizuno, Takuji Noguchi, Tetsuji Ogawa, Hidemi Kurihara Department of Periodontal Medicine, Division of Frontier Medical Science, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan Received 5 September 2002; revised 27 January 2003; accepted 11 March 2003
Abstract Hertwig’s epithelial root sheath (HERS) is involved in the differentiation of cementoblasts. The cells of epithelial rests of Malassez (ERM) may contribute to that process. However, little is known about the role of these epithelial cells in cementum repair. In the present study, we investigated the expression of alkaline phosphatase (ALPase), osteopontin (OPN), bone morphogenetic protein (BMP)-2 and BMP-4 in epithelial cells (E cells) and fibroblastic cells (F cells) derived from the same human periodontal ligament. E cells were identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. Reverse transcription–polymerase chain reaction analysis showed that E cells have lower ALPase and BMP-4 mRNA levels than F cells. On the other hand, the expression of OPN mRNA in E cells was stronger than in F cells. No significant difference was observed in BMP-2 expression between E and F cells. Thus, they have different expression patterns of ALPase, BMP-4 and OPN, suggesting that ERM and mesenchymal cells in periodontal ligament may be cooperatively involved in cementum repair. Furthermore, E cell cultures will be useful in elucidating the role of ERM. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Epithelial rests of Malassez; Epithelial cells; Fibroblastic cells; Periodontal ligament; Cementum repair
1. Introduction The cells of epithelial rests of Malassez (ERM) are the only odontogenic epithelial cells that are present in periodontal ligament. A number of functions of ERM have been discussed, such as protection against root resorption, formation of apical cyst, maintenance of the periodontal ligament space and prevention of ankylosis (Grant and Bernick, 1969; Lindskog et al., 1998; Loe¨ and Waerhaug, 1961; Ten Cate, 1972; Waerhaug, 1958). * Corresponding author. Tel.: +81-82-257-5663; fax: +81-82-257-5664 E-mail address: [email protected] (H. Shiba). Abbreviations: ALPase, alkaline phosphatase; BMP, bone morphogenetic protein; DMEM, Dulbecco’s modified Eagle’s medium; ERM, epithelial rests of Malassez; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HERS, Hertwig’s epithelial root sheath; OPN, osteopontin; RT–PCR, reverse transcription–polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
It has also been suggested that ERM is involved in cementoblast differentiation (Ten Cate, 1996). This function is of particular interest, because elucidation of its mechanism could lead to a novel method of obtaining new attachments involving cementum regeneration. Cell culture systems are very useful for analyzing cell functions. In fact, the precise functions of the cells of ERM remain largely unknown, although the functions of periodontal ligament fibroblasts have been thoroughly investigated. ALPase is thought to be essential for biomineralization (Whyte et al., 1987). Periodontal ligament fibroblastic cells have high levels of ALPase activity and can differentiate into osteoblasts or cementoblasts (McCulloch and Bordin, 1991; Nohutcu et al., 1997; Somerman et al., 1990a). OPN, a noncollagenous protein expressed in osteogenic and nonosteogenic cells, performs multiple functions (Sodek et al., 1992). It is present at the root surface during the initiation of cementogenesis in development (Bronckers
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00075-1
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Y. Mouri et al. / Cell Biology International 27 (2003) 519–524
et al., 1994; Somerman et al., 1990b; Somerman et al., 1992). BMP-2 and BMP-4 are involved in epithelial– mesenchymal interaction (Thesleff et al., 1995; Vainio et al., 1993), which plays an important role in cell differentiation. Therefore, these bone-related proteins are thought to play a role in cementum repair. In the present study, we isolated and cultured epithelial cells derived from human periodontal ligament by enzymatic digestion. Furthermore, we tried to elucidate the role of ERM cells during cementum repair by comparing the expression of ALPase, OPN, BMP-2 and BMP-4 in epithelial cells derived from periodontal ligament with those in fibroblastic cells derived from the same tissue.
2. Materials and methods 2.1. Preparation and culture of cells Three periodontally healthy and non-carious premolars (premolars 1, 2 and 3) extracted in the course of orthodontic treatment were collected from three patients with their informed consent. The periodontal ligament tissue attached to the mid-third of each root was incubated at 37 (C for 20 min in DMEM supplemented with 0.2% collagenase (Wako Pure Chemical Industries, Osaka, Japan), 0.25% trypsin (Difco Laboratories, Detroit, MI, USA), 100 units/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B. For the isolation of E cells, a partial cell suspension was centrifuged at 800 rpm for 5 min. The pellet was suspended in MCDB medium (pH 7.4) (SIGMA, St Louis, MO, USA) containing 10 µg/ml insulin, 5 µg/ml transferrin, 10 µM 2-mercaptoethanol, 10 µM 2-aminoethanol, 10 nM sodium selenite, 100 units/ml penicillin, 100 µg/ml streptomycin and 50 ng/ml amphotericin B (medium A) (Myoken et al., 1995; Sato et al., 1987). The cells were seeded in 35 mm plastic tissue culture dishes coated with type I collagen, and incubated in 5% CO2/95% air at 37 (C. When the E cells reached subconfluence, they were harvested and subcultured in medium A containing 50 µg/ml bovine brain extract (medium B). For the isolation of F cells, the residual cell suspension was centrifuged at 800 rpm for 5 min and the pellet was suspended in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin B (medium C) and the cells were seeded in 35 mm plastic tissue culture dishes coated with type I collagen, and incubated in 5% CO2/95% air at 37 (C until confluent cell monolayers were formed. E-1 and F-1 cells, E-2 and F-2 cells, and E-3 and F-3 cells were obtained from premolars 1, 2 and 3, respectively. When the cells became confluent, they were harvested and subcultured.
2.2. Preparation of periodontal ligament tissue Two periodontally healthy and non-carious premolars (premolars 4 and 5) extracted in the course of orthodontic treatment were collected from two patients with their informed consent. The periodontal ligament tissue attached to the mid-third of each root was isolated. 2.3. Western blots Cytokeratin 5 and 8 are expressed by cells of EMR (Gao et al., 1988). Therefore, E cells are identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. E-1 and F-1 cells in cultures at the third passage were harvested, seeded at a density of 1104 cells/9 mm plastic tissue culture well coated with type I collagen, and cultured in 0.2 ml mixed medium (1:1 (vol/vol) medium B:C) containing 50 µg/ml ascorbic acid for 4 days. Then, cells were lysed with Laemmli’s buffer (Laemmli, 1970) and boiled for 3 min. Samples (15 µl) were resolved on a 15% SDS–polyacrylamide gel by electrophoresis (SDS–PAGE) under nonreducing conditions, and electrophoretically transferred on to nitrocellulose membranes. The membranes were blocked with 3% non-fat dried milk for 1.5 h, and reacted with mouse anti-human cytokeratin 5 and 8 monoclonal antibodies (2 µg/ml) (Chemicon, Temecula, CA, USA) at 37 (C for 1 h. The membranes were incubated with goat antirabbit IgG horseradish peroxidase conjugated antibody (Chemicon) for 1 h at 37 (C. Immunodetection was performed with Chemi-luminescence Reagent Plus (NEN Life Science Products, Boston, MA, USA), following the manufacturer’s directions. 2.4. RNA isolation E-1, -2 and -3 cells and F-1, -2 and -3 cells at the third passage were harvested, seeded at a density of 27104 cells/35 mm plastic tissue culture dish coated with type I collagen, and maintained in 2 ml mixed medium (1:1 (vol/vol) medium B: C) containing 50 µg/ml ascorbic acid. Total RNA was extracted from the cultures using ISOGEN (Wako Pure Chemical Industries) on day 4 and quantified by spectrometry at 260 and 280 nm. In addition, total RNA was purified from the periodontal ligament tissues of premolars 4 and 5. 2.5. RT–PCR and sequencing PCR products Total RNA was reverse-transcribed using oligo (dT) 12–18 (Boehringer Mannheim Corp., IN, USA). RT reaction was performed for 60 min at 42 (C and stopped by heat inactivation for 5 min at 99 (C. Specific primers for OPN, ALPase, BMP-2, BMP-4 and GAPDH were designed according to the sequence data (see Table 1) (Kawamoto et al., 1998; Kiefer et al., 1989; Kishi et al.,
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3. Results 3.1. Morphology of E cells and F cells The primary cultures of E cells were morphologically polygonal by microscopic examination (Fig. 1A). On the other hand, F cells were spindle-shaped (Fig. 1B). 3.2. Keratin expression in E cells and F cells E cells, but not F cells, showed a positive reaction to immunoblotting with anti-cytokeratin 5 and 8 antibody (Fig. 2). 3.3. mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in periodontal ligament tissues RT–PCR analyses showed periodontal ligament tissues expressed mRNA for OPN, ALPase, BMP-2 and BMP-4 (Fig. 3). The DNA sequence of each PCR product was 100% identical to the previously reported sequences of the OPN, ALPase, BMP-2 and BMP-4 genes (Kiefer et al., 1989; Kishi et al., 1989; Wozney et al., 1988). 3.4. mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in E cells and F cells
Fig. 1. Phase-contrast micrographs of the primary culture of E and F cells. The pictures show the morphology of E-1 cells (A) and F-1 cells (B). The scale bar indicates 25 µm.
Fig. 4 shows the mRNA expression of ALPase, OPN, BMP-2 and BMP-4 in E cells and F cells. The level of ALPase and BMP-4 mRNAs in E cells was lower than in F cells. On the other hand, E cells showed greater expression of OPN than F cells. No difference was observed in BMP-2 expression between E and F cells. These expression patterns were consistently observed in the three different cultures of each cell type. 4. Discussion
Fig. 2. Immunoblotting of keratin 5 and 8 in E and F cells. E-1 cells and F-1 cells were seeded and maintained as described in Materials and Methods. The experiment was repeated twice with similar results.
1989; Wozney et al., 1988). The PCR reaction and cycling conditions are described in the Table 1. The PCR products were subcloned into the plasmid vector pGEM-T (Promega, Madison, WI, USA) and identified and confirmed via sequencing by dideoxy chain termination.
Previous studies have shown that cultured epithelial cells derived from porcine periodontal ligaments produce fibronectin, glycosaminoglycans and bone resorbing factor (Birek et al., 1983; Merrilees et al., 1983; Uitto et al., 1992). Recently, epithelial cells from human periodontal ligament were shown to express fibroblast growth factor receptors in vitro (Yamanaka et al., 2000). However, the expression of bone-related proteins, such as ALPase, BMP-2, BMP-4 or OPN, in epithelial cells from periodontal ligament has not been investigated. In the present study, we were the first to succeed in isolating and culturing both E and F cells from the same human periodontal ligament by enzymatic digestion. We also examined bone-related proteins in cultures of E and F cells. OPN appears on the root surface at the same time as the initiation of cementogenesis and plays an important role in this mechanism (Uitto et al., 1992; Yamanaka et al., 2000). Some studies have suggested that OPN is
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Fig. 3. RT–PCR analysis of mRNA expression of OPN, ALPase, BMP-2 and BMP-4 in human periodontal tissues. RNA was extracted from periodontal tissues-1 and -2, prepared from premolars-4 and -5, respectively. PCR products, as described in Materials and Methods, were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. M, 100-bp ladder.
Fig. 4. RT–PCR analysis of mRNA expression of OPN, ALPase, BMP-2 and BMP-4 in cultures of E cells and F cells. E-1, -2 and -3 cells and F-1, -2 and -3 cells were seeded and maintained as described in Materials and Methods. RNA was extracted on day 4. PCR products, as described in the Materials and Methods, were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. M, 100-bp ladder.
Table 1 Oligonucleotide primers used for RT–PCR determining bone-related proteins Gene
Annealing temperature ((C)
Sequence
GAPDH (613 bp)
53
OPN (532 bp)
53
ALPase (382 bp)
54
BMP-2 (440 bp)
54
BMP-4 (339 bp)
48
F: 5#-GTCAAGGCTGAGAACGGGAA-3# R: 5#-GCTTCACCACCTTCTTGATG-3# F: 5#-CACCTGTGCCATACCAGTTAAAC-3# R: 5#-GGTGATGTCCTCGTCTGTAGCATC-3# F: 5#-GTACTGGCGAGACCAAGCG-3# R: 5#-GGCCCAGCGCAGGATGGAGG-3# F: 5#-CCGCTGTCTTCTAGCGTTGC-3# R: 5#-CCTGAAGCTCTGCTGAGGTG-3# F: 5#-GTCCTGCTAGGAGGCGCGAG-3# R: 5#-GTTCTCCAGATGTTCTTCG-3#
Denaturing, annealing and elongation conditions were 94 (C, 30 s, the temperature as described above, 1 min and 72 (C, 1 min, respectively. The cycling condition was a total of 30 cycles. F, forward primer; R, reverse primer.
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produced by dental mesenchymal cells (Lekic et al., 1996; Ramakrishnan et al., 1995), and others by the cells of HERS (Bosshardt and Nanci, 1998; Lekic et al., 1996; Ramakrishnan et al., 1995). The present study showed that E cells, but not F cells, strongly express OPN mRNA. Therefore, cells of ERM may secrete OPN on to the root surface during the early stages of cementum repair. The present study showed that BMP-2 and BMP-4 mRNAs are expressed in E cells and F cells. BMP-2 and BMP-4 modulate tooth development through epithelial– mesenchymal interaction (Thesleff et al., 1995; Vainio et al., 1993). These findings suggest that interaction occurs between cells of ERM and undifferentiated mesenchymal cells in periodontal ligament during cementum repair. It is widely accepted that the mesenchymal cells of dental follicles differentiate into cementoblasts during cementum development (Cho and Garant, 1996). However, it has also been suggested that HERS cells may undergo epithelial–mesenchymal transformation to differentiate into cementoblasts (MacNeil and Thomas, 1993). The present study showed that F cells, but not E cells, have high levels of ALPase mRNAs, suggesting that mesenchymal cells in periodontal ligament tissue have the capacity to differentiate into cementoblasts during cementum repair. However the possibility still remains that E cells can transform into F cells. Enamel matrix proteins are involved in the formation of cementum (Hammarstro¨m, 1997). HERS cells express amelin mRNA in vivo (Fong et al., 1996), and also produce amelogenin (Hamamoto et al., 1996). Thus, HERS is thought to play an important role in cementum development through the production of enamel matrix proteins. There is a stong possibility that cells of ERM produce enamel matrix protein during cementum repair. In conclusion, bone-related proteins show different mRNA expression patterns in E cells and F cells. Furthermore, this culture model should be useful for elucidating the role of ERM during cementum repair.
Acknowledgements This work was supported in part by the Satake Foundation for Hiroshima University, plus Grant-inAid for Scientific Research (A) (No. 12307051) and Grant-in-Aid for Scientific Research (C) (No. 12897020) from the Japan Society for the Promotion of Science, Japan. References Birek C, Heersche JNM, Jez D, Brunette DM. Secretion of a bone resorbing factor by epithelial cells cultured from porcine rests of Malassez. J Periodont Res 1983;18:75–81.
523
Bosshardt DD, Nanci A. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells. J Hist & Cytochem 1998;46:135–42. Bronckers AL, Farach-Carson MC, Van Waveren EV, Butler WT. Immunolocalization of osteopontin, osteonectin, and dentin sialoprotein during dental root formation and early cemento-genesis in the rat. J Bone Miner Res 1994;9:833–41. Cho MI, Garant PR. Expression and role of epidermal growth factor receptors during differentiation of cementoblasts, osteoblasts, and periodontal ligament fibroblasts in the rat. Anat Rec 1996; 245:342–60. Fong CD, Slaby I, Hammarstro¨m L. Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath. J Bone Miner Res 1996;11:892–8. Gao Z, Mackenzie IC, Williams DM, Cruchley AT, Leigh I, Lane EB. Patterns of keratin-expression in rests of Malassez and periapical lesions. J Oral Pathol 1988;17:178–85. Grant DA, Bernick S. A possible continuity between the epithelial rests and epithelial attachment in miniature swine. J Periodont 1969;40:87–95. Hamamoto Y, Nakajima T, Ozawa H, Uchida T. Production of amelogenin by enamel epithelium of Hertwig’s root sheath. Oral Surg Oral Med Oral pathol Oral Radiol Endod 1996;81:703–9. Hammarstro¨m L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997;24:658–68. Kawamoto T, Pan H, Yan W, Ishida H, Usui E, Oda R et al. Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes. Eur J Biochem 1998;256:503–9. Kiefer MC, Bauer DM, Barr PJ. The cDNA and derived amino acid sequence for human osteopontin. Nucleic Acids Res 1989;17:3306. Kishi F, Matsuura S, Kajii T. Nucleotide sequence of the human liver-type alkaline-phosphatase cDNA. Nucleic Acids Res 1989; 17:2129. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970;227:680–5. Lekic P, Sodek J, McCulloch CAG. Osteopontin and bone sialoprotein expression in regenerating rat periodontal ligament and alveolar bone. Anatom Rec 1996;244:50–8. Lindskog S, Blomlo¨f L, Hammarstro¨m L. Evidence for a role of odontogenic epithelium in maintaining the periodontal space. J Clin Periodont 1998;15:371–3. Loe¨ H, Waerhaug J. Experimental replantation of teeth in dogs and monkeys. Archs Oral Biol 1961;3:176–84. MacNeil RL, Thomas HF. Development of murine periodontium. II. Role of the epithelial root sheath in formation of the periodontal attachment. J Periodontol 1993;64:285–91. McCulloch CAG, Bordin S. Role of fibroblast subpopulations in periodontal physiology and pathology. J Periodontal Res 1991; 26:144–54. Merrilees MJ, Sodek J, Aubin JE. Effects of cells of epithelial rests of Malassez and endothelial cells on synthesis of glycosaminoglycans by periodontal ligament fibroblasts in vitro. Dev Biol 1983; 97:146–53. Myoken Y, Myoken Y, Okamoto T, Sato JD, Takada K. Effect of fibroblast growth factor-1 on the three-dimensional growth and morphogenesis of human salivary gland epithelial cells embedded in collagen gels. In Vitro Cell Dev Biol 1995;31:84–6. Nohutcu RM, McCauley LK, Koh AJ, Somerman MJ. Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro. J Periodontol 1997;68:320–7. Ramakrishnan PR, Lin WL, Sodek J, Cho MI. Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro. Calcif Tissue Int 1995;57:52–9. Sato JD, Kawamoto T, Okamoto T. Cholesterol requirement of P3-X63-Ag8 and X63-Ag8.653 mouse myeloma cells for growth in vitro. J Exp Med 1987;165:1761–6.
524
Y. Mouri et al. / Cell Biology International 27 (2003) 519–524
Sodek J, Chen J, Kasugai S, Nagata T, Zhang Q, Shapiro HS, Wrana JL, Goldberg HA. Sialoproteins in bone remodeling. In: Davidovitch Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation, pp. 127–136. Somerman MJ, Young MF, Foster RA, Moehring JM, Imm G, Sauk JJ. Characteristics of human periodontal ligament cells in vitro. Archs Oral Biol 1990a;35:241–7. Somerman MJ, Shroff B, Argraves WS, Morrison G, Craig AM, Denhardt DT et al. Expression of attachment proteins during cementogenesis. J Biol Buccale 1990b;18:207–14. Somerman MJ, Shroff B, Foster RA, Butler WT, Sauk JJ. Mineralassociated adhesion proteins are linked to root formation. Proc Finn Dent Soc 1992;88(suppl I):451–61. Ten Cate AR. The epithelial cell rests of Malassez and the genesis of the dental cyst. Oral Surg 1972;34:956–64. Ten Cate AR. The role of epithelium in the development, structure and function of the tissues of tooth support. Oral Dis 1996;2:55–62. ´ berg T. EpithelialThesleff I, Vaahtokari A, Kettunen P, A mesenchymal signaling during tooth development. Connect Tissue Res 1995;32:9–15.
Uitto VJ, Larjava H, Peltonen J, Brunette DM. Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells. J Dent Res 1992;71:1203–11. Vainio S, Karavanova I, Jowett A, Thesleff I. Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 1993; 75:45–58. Waerhaug J. Effect of C-avitaminosis on the supporting structure of teeth. J Periodont 1958;29:87–97. Whyte MP, Rettinger SD, Vrabel LA. Infantile hypophosphatasia: Enzymatic detect explored with alkaline phosphatase-deficient skin fibroblasts in culture. Calcif Tissue Int 1987;40:244–52. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW et al. Novel regulators bone formation: molecular clones and activities. Science 1988;242:1528–34. Yamanaka T, Sakamoto A, Tanaka Y, Zhang Y, Hayashido Y, Toratani S et al. Isolation and culture of epithelial cells derived from ERM in human periodontal ligament. In Vitro Cell Dev Biol 2000;36:548–53.