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Amelogenin-mediated Regulation of Osteoclastogenesis, and Periodontal Cell Proliferation and Migration

¹ Functional Genomics Section and
² Cell Biology Section, Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, MSC 4395, Bldg. 30, Room 122, Bethesda, MD 20892, USA;
³ Cartilage Biology and Orthopedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA; and
⁴ Department of Anatomy and Cell Biology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA

^* corresponding author, ak40m{at}nih.gov

	ABSTRACT

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We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

KEY WORDS: amelogenins • LRAP • osteoclastogenesis • knockout mice

	INTRODUCTION

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Amelogenins are highly conserved proteins that constitute 90% of the enamel organic matrix and are mainly produced by the ameloblasts. At least 14 mRNA splice forms of amelogenin are generated in the ameloblasts (Simmer et al., 1994; Yuan et al., 2001; Veis, 2003). Although amelogenins have been implicated in tissue-specific epithelial-mesenchymal signaling during tooth development (Simmer et al., 1994; Veis et al., 2000; Zeichner-David, 2001), their distribution and the precise functions associated with the individual peptides derived from the splice variants are still unclear. Recent reports have indicated that amelogenins, mainly leucine-rich amelogenin peptide (LRAP), have the potential to induce bone formation (Veis et al., 2000; Viswanathan et al., 2003; Boabaid et al., 2004). Additionally, Emdogain (enamel-matrix-derived proteins) and the full-length amelogenin induced mineralization in cementum and in bone (Viswanathan et al., 2003; Venezia et al., 2004). We have previously reported the expression of two amelogenin isoforms, M180 and LRAP, in the periodontal region of WT mice (Hatakeyama et al., 2003). A lack of amelogenin transcripts correlated well with the cementum defects and abnormal osteoclastogenesis around tooth roots of amelogenin knockout mice (Hatakeyama et al., 2003). However, the functions of the amelogenin splice variants, especially M180 and LRAP, in the periodontal region are not clearly understood. The aim of the present study was to identify the potential functions of M180 and LRAP in periodontal cells, especially in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells.

	MATERIALS & METHODS

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Amelogenin-knockout Mice
Wild-type (WT) and amelogenin-KO mice (Gibson et al., 2001) were housed in a specific pathogen-free animal facility and maintained according to standard NIH guidelines for housing and breeding practices. Mice were given a dough diet (Bio-Serv, Holton Industries Co., Frenchtown, NJ, USA) and autoclaved water ad libitum.

Osteoclast Differentiation Assay
Osteoclast differentiation assays were performed as described (Kanzaki et al., 2001, 2002), with minor modifications. Briefly, the cementoblast/periodontal ligament cells were isolated from the root surface of the mandibular molars of either the WT or KO mice (Hatakeyama et al., 2003). Bone marrow (BM) cells from the long bones of WT mice were used as the source for osteoclast progenitor (OP) cells. The WT-BM cells (5 x 10⁵ cells/cm²) were co-cultured with cementoblast/periodontal ligament cells (2 x 10⁴ cells/cm²) from either WT or KO mice for 7 days in DMEM containing 10% fetal calf serum supplemented with 10^–9 M 1,25(OH)₂D₃ in 96-well plates (Miura et al., 2002). The recombinant porcine P172 (89% homology to mouse M180) and recombinant porcine LRAP (85% homology to mouse LRAP) (Simmer et al., 1994; Ryu et al., 1999; Boabaid et al., 2004) were solubilized in 0.5% BSA, and added to the co-cultures at 0, 1, 10, or 100 ng/mL. Culture media were changed every 3 days. After 7 days, the adherent cells were stained for tartrate-resistant acid phosphatase (TRAP) activity, for analysis of the presence of osteoclasts, with the use of a leukocyte acid phosphatase kit (Sigma Diagnostics, St. Louis, MO, USA).

RNA Isolation and RT-PCR
Total RNA from cementoblast/periodontal ligament cells cultured in six-well plates with 100 ng/mL of recombinant P172 or LRAP for 0, 12, 24, 48, or 72 hrs was isolated by means of the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Inc., Valencia, CA, USA). After DNase treatment, 1 µg of each sample of the total RNA was subjected to first-strand cDNA synthesis in the SuperScript II^TM First-strand Synthesis System (Invitrogen Life Technology, Carlsbad, CA, USA). We performed PCR to analyze mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), as previously described (Hatakeyama et al., 2003). GAPDH was used as a control. The data were consistent, as confirmed by three independent experiments.

Cell Proliferation Assay
Twelve-well culture dishes were coated with various concentrations of P172 or LRAP (0, 1, 10, 100 ng/mL) overnight at 4°C and then washed with DMEM medium 3 times. Cementoblast/periodontal ligament cells (4.0 x 10⁴ per well) were seeded on the coated dishes and cultured in DMEM containing 10% FBS. Total cell number was counted after 1, 3, and 5 days of culture, by means of a Beckman Coulter Counter.

Migration Assay
The cell migration assay was performed as described previously (Lemire et al., 2002). The cementoblast/periodontal ligament cells from WT and KO mice, and mouse embryonic fibroblasts from 14-day-old WT mouse embryos, were separately cultured on six-well plates in DMEM medium supplemented with 10% FCS. Mouse embryonic fibroblasts served as negative controls for the assay. Cell monolayers were disrupted (wounded) by being scraped with a 200-µL pipette tip, which resulted in a < 600-µm lane free of cells. Cells were washed twice with DMEM medium, and then cultured with or without P172 or LRAP at the indicated concentrations with 0.2% FCS. In one of the experimental groups, the conditioned medium from three-day cultures of WT-cementoblast/periodontal ligament cells was added to the KO-cementoblast/periodontal ligament cells. Cell migration distance was recorded every 10 min for a period of 12 hrs, by time-lapse video microscopy (Axiovert 25, Carl Zeiss Microimaging, Inc., Thornwood, NY, USA) and a video camera module (XC-ST50/50CE, Sony Electronics, Inc., Park Ridge, NJ, USA).

Statistical Analysis
All data were analyzed by Student’s t test. All values are reported as mean ± standard deviation (SD).

	RESULTS

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Osteoclast Differentiation
Bone marrow (BM) cells differentiate into osteoclast progenitors when co-cultured with cementoblast/periodontal ligament cells in the presence of 1,25(OH)₂D₃ (Kanzaki et al., 2001). In co-cultures of WT-BM cells + KO-cementoblast/periodontal ligament cells, a significant increase was observed in the number of osteoclasts (TRAP-positive multinucleated cells) as compared with those in the co-cultures of WT-BM cells + WT-cementoblast/periodontal ligament cells (p < 0.01) (Fig. 1A). Interestingly, treatment with increasing amounts of LRAP resulted in a proportional decrease in the number of TRAP-positive cells in the co-cultures of WT-BM cells + KO-cementoblast/periodontal ligament cells (Figs. 1B, 1D, 1F), as well as in the co-cultures of WT-BM cells + WT-cementoblast/periodontal ligament cells (Figs. 1G, 1I, 1K). In contrast, treatment with P172 did not affect the number of TRAP-positive cells in either of the co-cultures (Figs. 1C, 1E, 1F, 1H, 1J, 1K).

View larger version (57K): [in this window] [in a new window]   Figure 1. LRAP inhibits osteoclastogenesis. (A) Number of osteoclasts (tartrate-resistant alkaline phosphatase [TRAP])-positive multinucleated cells, as indicated by arrows in the subsequent panels in the co-cultures of wild-type (WT) osteoclast progenitor (bone marrow cells) and amelogenin-KO (KO) or WT-cementoblast/periodontal ligament cells treated with 10–8 M of 1,25(OH)2D3 for 7 days. Increased numbers of osteoclasts were observed in the co-cultures of osteoclast progenitor and KO cementoblast/periodontal ligament cells (compare panels B and G). (B–K) Osteoclast formation in the co-cultures of WT osteoclast progenitor and KO or WT-cementoblast/periodontal ligament cells treated with LRAP or P172 for 7 days in 96-well plates in the presence of 10–8 M of 1,25(OH)2D3. Different amounts of LRAP and P172 (1, 10, and 100 ng/mL) were added in the culture medium. TRAP staining of co-cultures of cementoblast/periodontal ligament cells from KO mice (B–E) and WT mice (G–J) treated with either LRAP or P172 as indicated. Data are expressed as the mean ± SD (n = 18). **P < 0.01. Scale bar: 100 µm.

View larger version (37K): [in this window] [in a new window]   Figure 2. LRAP inhibits the osteoclastogenic pathway. (A,B) RANKL and osteoprotegerin (OPG) mRNA levels as measured by RT-PCR analysis of total RNA extracted from cementoblast/periodontal ligament cells treated either with 100 ng/mL of LRAP (A) or P172 (B) for 0, 12, 24, 48, or 72 hrs. GAPDH was used as the internal control. WT, wild-type mice; KO, amelogenin-KO mice.

View larger version (39K): [in this window] [in a new window]   Figure 3. Both LRAP and P172 induce the proliferation of cementoblast/periodontal ligament cells. (A) Amelogenin-KO (KO) cementoblast/periodontal ligament cells showed reduced proliferation. **P < 0.01. (B,C) Increased proliferation rate of KO cementoblast/periodontal ligament cells treated with either LRAP or P172. Amelogenin-KO cementoblast/periodontal ligament cells were cultured with various concentrations of LRAP (B) or P172 (C) for 1, 3, and 5 days. *P < 0.05 vs. day 0, **P < 0.01 vs. day 0, #P < 0.05 vs. 0 ng/mL group, and ##P < 0.01 vs. 0 ng/mL group. (D,E) Wild-type (WT) cementoblast/periodontal ligament cells were cultured with various concentrations of LRAP (D) or P172 (E) for 1, 3, or 5 days. Data are expressed as the mean ± SD (n = 3). *P < 0.05 vs. day 0, **P < 0.01 vs. day 0, #P < 0.05 vs. 0 ng/mL group, and ## P < 0.01 vs. 0 ng/mL group.

View larger version (32K): [in this window] [in a new window]   Figure 4. Both LRAP and P172 promote migration of cementoblast/periodontal ligament cells. (A) Amelogenin-KO (KO) cementoblast/periodontal ligament cells exhibited a reduced cell migration rate. (B) Addition of the conditioned medium from the three-day cultures of wild-type cementoblast/periodontal ligament cells increased the migration rate of amelogenin KO-cementoblast/periodontal ligament cells. (C,D) Increased cell migration rates in the WT and amelogenin KO-cementoblast/periodontal ligament cells cultured with 100 ng/mL LRAP (C) and P172 (D) for 12 hrs. Data are expressed as the mean ± SD (n = 3) of the µm distance covered by cementoblast/periodontal ligament cells. *P < 0.05 vs. day 0 and **P < 0.01 vs. day 0.

	DISCUSSION

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M180, the major amelogenin in mouse enamel, is believed to play a key role in crystal formation through supermolecular aggregations (Fincham and Moradian-Oldak, 1995; Zeichner-David, 2001). Additionally, M180 interacts with other proteins, such as cytokeratin and N-acetyl glucosamine (Ravindranath et al., 2001, 2003). LRAP lacks 121 amino acids that are present in M180. Recently, it has been demonstrated that LRAP could not rescue the hypoplastic enamel phenotype in amelogenin-KO LRAP transgenic mice (Chen et al., 2003). Although several reports indicate that LRAP may function as a signaling molecule (Veis et al., 2000; Veis, 2003; Viswanathan et al., 2003; Bobaid et al., 2004), specific functions of LRAP in the periodontal regions have not been identified.

Previously, we reported that the presence of increased cementum resorption and increased localization of osteoclasts near the cementum correlated with defects in the amelogenin-KO mice (Hatakeyama et al., 2003). The presence of an increased number of osteoclasts near the cementum of mice lacking amelogenin splice variants also suggests a potential role for these proteins in protecting cementum by controlling active osteoclastogenesis. Because the amelogenin splice variants are secreted molecules, porcine recombinant P172 (89% homology to mice) and LRAP (P59- 85% homology to mice) were added to bone marrow and cementoblast/periodontal ligament cells in co-culture systems, so that their function in cell-to-cell communication could be examined. The number of osteoclasts decreased significantly following treatment with LRAP, but not with P172.

In the periodontal region, alveolar bone remodeling requires both bone resorption and bone induction. Active remodeling of alveolar bone takes place in clinical conditions, such as orthodontic tooth movement, in which bone resorption occurs near the pressure side and bone formation at the tension side of the tooth root (Proffit and Fields, 2000). Bone resorption is mediated by active osteoclastogenesis and requires the expression of several regulatory molecules, such as RANKL, RANK, osteoprotegerin, and TRAF 6 (Takahashi et al., 1999). RANKL interacts with its receptor RANK on osteoclast progenitor cells during active osteoclastogenesis. Osteoprotegerin, a soluble decoy receptor of RANKL, competes with RANK for RANKL binding and serves as an inhibitor of osteoclastogenesis. The periodontal ligament cells express both RANKL and OPG (Kanzaki et al., 2001; Hasegawa et al., 2002), and RANKL expression can also be induced by mechanical stress in periodontal ligament cell culture systems (Kanzaki et al., 2002). Unlike the periodontal ligament cells used in our study, the immortalized cementoblasts, OCCM-30, cultured with ascorbic acid and a high level of LRAP (2 µg/mL) for 72 hrs, displayed higher expression of osteoprotegerin expression and slightly reduced expression of RANKL (Boabaid et al., 2004). Consistent with the increased RANKL expression near the cementum of amelogenin-KO mice (Hatakeyama et al., 2003), the amelogenin-KO cementoblast/periodontal ligament cells also showed increased RANKL expression without stimulation by mechanical stress. However, reduced expression of RANKL in the amelogenin-KO cementoblast/periodontal ligament culture system in the presence of LRAP (0.1 µg/mL) indicates that LRAP plays a key role in the regulation of the osteoclastogenic pathway. The epithelial cell rests of Malassez are the cell aggregates of Hertwig’s epithelial root sheath that are located in the periodontal region between the alveolar bone and tooth root, and express amelogenins (Hammarström et al., 1997). Our findings suggest that LRAP secreted by epithelial cell rests of Malassez may confer protection to the cementum by controlling osteoclastogenesis through the RANKL pathway.

We also found that both LRAP and P172 induce cementoblast/periodontal ligament cell proliferation and migration. During periodontal regeneration, periodontal ligament cells proliferate and migrate to form ligament attachments between alveolar bone and cementum. Emdogain, a mixture of proteins extracted from developing porcine enamel matrix, has been used to treat periodontal diseases. Amelogenins are the major components in Emdogain preparations. Emdogain treatment restores bone growth and periodontal regeneration in experimentally induced periodontitis in monkeys (Hammarström et al., 1997). The addition of Emdogain to cementoblasts has been demonstrated to induce cell proliferation (Viswanathan et al., 2003). Emdogain treatment in in vitro wound-healing models has a dramatic effect on wound closure in periodontal ligament fibroblasts and gingival fibroblasts (Rincon et al., 2003). In our experiments, both P172 and LRAP stimulated proliferation of cementoblast/periodontal ligament cells. In addition, in vitro cell migration results indicate that LRAP and P172 enhanced migration of cementoblast/periodontal ligament cells. However, mouse embryo fibroblasts did not show any change in cell migration in the presence of either P172 or LRAP, suggesting that activity of these peptides may be specific to periodontal cells. The enhanced cell proliferation and migration by these variants imply their potential role in periodontal regeneration, an essential process in maintaining tooth roots anchored to alveolar bone by establishing new attachments.

Our findings demonstrate that the external signals that control osteoclastogenesis are mediated mainly by LRAP. Additionally, LRAP also mediates the induction of migration and proliferation of cementoblast/periodontal ligament cells. Although migration and proliferation are induced by P172, it appears less significant than LRAP. It is not clear whether this difference in periodontal cell function is due to the presence of an additional 121 amino acids in the P172 protein, which could alter the structure and conformation of the amelogenin protein, changing the functional specificity.

	ACKNOWLEDGMENTS

 
We thank Drs. Mary Jo Danton, Marian Young, and Yoshihiko Yamada for critical reading of the manuscript, and Drs. Satoru Takahashi, Masako Miura, Tomokazu Hasegawa, and Takashi Nakamura for helpful discussions. These studies were supported by grants from the Division of Intramural Research of the National Institute of Dental and Craniofacial Research (NIDCR) (HK and ABK), National Institutes of Health, and NIDCR grant DE 011089 (CWG).

Received March 23, 2005; Last revision October 28, 2005; Accepted November 3, 2005

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Boabaid F, Gibson CW, Kuehl MA, Berry JE, Snead ML, Nociti FH Jr, et al. (2004). Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis. J Periodontol 75:1126–1136.[ISI][Medline]

Chen E, Yuan ZA, Wright JT, Hong SP, Li Y, Collier PM, et al. (2003). The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype. Calcif Tissue Int 73:487–495.[ISI][Medline]

Fincham AG, Moradian-Oldak J (1995). Recent advances in amelogenin biochemistry. Connect Tissue Res 32:119–124.[ISI][Medline]

Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, et al. (2001). Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem 276:31871–31875.[Abstract/Free Full Text]

Hammarström L, Heijl L, Gestrelius S (1997). Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 24:669–677.[ISI][Medline]

Hasegawa T, Yoshimura Y, Kikuiri T, Yawaka Y, Takeyama S, Matsumoto A, et al. (2002). Expression of receptor activator of NF-kappa B ligand and osteoprotegerin in culture of human periodontal ligament cells. J Periodontal Res 37:405–411.[ISI][Medline]

Hatakeyama J, Sreenath T, Hatakeyama Y, Thyagarajan T, Shum L, Gibson CW, et al. (2003). The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice. J Biol Chem 278:35743–35748.[Abstract/Free Full Text]

Kanzaki H, Chiba M, Shimizu Y, Mitani H (2001). Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition. J Dent Res 80:887–891.[Abstract/Free Full Text]

Kanzaki H, Chiba M, Shimizu Y, Mitani H (2002). Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 17:210–220.[ISI][Medline]

Lemire JM, Merrilees MJ, Braun KR, Wight TN (2002). Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro. J Cell Physiol 190:38–45.[ISI][Medline]

Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, et al. (2002). A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation. Biochem Biophys Res Commun 291:987–994.[ISI][Medline]

Proffit WR, Fields HW (2000). Contemporary orthodontics. 3rd ed. St. Louis, MO: Mosby.

Ravindranath RM, Tam WY, Bringas P Jr, Santos V, Fincham AG (2001). Amelogenin-cytokeratin 14 interaction in ameloblasts during enamel formation. J Biol Chem 276:36586–36597.[Abstract/Free Full Text]

Ravindranath RM, Basilrose RM Sr, Ravindranath NH, Vaitheesvaran B (2003). Amelogenin interacts with cytokeratin-5 in ameloblasts during enamel growth. J Biol Chem 278:20293–20302.[Abstract/Free Full Text]

Rincon JC, Haase HR, Bartold PM (2003). Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model. J Periodontal Res 38:290–295.[ISI][Medline]

Ryu OH, Fincham AG, Hu CC, Zhang C, Qian Q, Bartlett JD, et al. (1999). Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins. J Dent Res 78:743–750.[Abstract/Free Full Text]

Simmer JP, Lau EC, Hu CC, Aoba T, Lacey M, Nelson D, et al. (1994). Isolation and characterization of a mouse amelogenin expressed in Escherichia coli. Calcif Tissue Int 54:312–319.[ISI][Medline]

Takahashi N, Udagawa N, Suda T (1999). A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 256:449–455.[ISI][Medline]

Veis A (2003). Amelogenin gene splice products: potential signaling molecules. Cell Mol Life Sci 60:38–55.[ISI][Medline]

Veis A, Tompkins K, Alvares K, Wei K, Wang L, Wang XS, et al. (2000). Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J Biol Chem 275:41263–41272.[Abstract/Free Full Text]

Venezia E, Goldstein M, Boyan BD, Schwartz Z (2004). The use of enamel matrix derivative in the treatment of periodontal defects: a literature review and meta-analysis. Crit Rev Oral Biol Med 15:382–402.[Abstract/Free Full Text]

Viswanathan HL, Berry JE, Foster BL, Gibson CW, Li Y, Kulkarni AB, et al. (2003). Amelogenin: a potential regulator of cementum-associated genes. J Periodontol 74:1423–1431.[ISI][Medline]

Yuan ZA, Chen E, Gibson CW (2001). Model system for evaluation of alternative splicing: exon skipping. DNA Cell Biol 20:807–813.[ISI][Medline]

Zeichner-David M (2001). Is there more to enamel matrix proteins than biomineralization? Matrix Biol 20:307–316.[ISI][Medline]

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