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Int Chin J Dent 2007; 7: 7-13.

Regulation of osteoprotegerin and receptor activator of nuclear
factor-kappa B ligand by interleukin-10 in human dental follicle
cells
Hong Qian, DDS, PhD,* Xuepeng Chen, DDS, PhD,* Yingchun Bi, DDS, PhD,
Yan Wang, DDS, PhD, Yinzhong Duan, DDS, PhD, and Zuolin Jin, DDS, PhD*Equally contribute to the manuscript.

Department of Orthodontics, College of Stomatology, The Fourth Military Medical University, Xi’an,
P. R. China
Purpose: This study was undertaken to determine the expression of interleukin-10 at protein level in humandental follicle cells and the effect of interleukin-10 on gene expression of osteoprotegerin and receptor activatorof nuclear factor-kappa B ligand.
Materials and Methods: Primary human dental follicle cells were cultured. The expression of interleukin-10 at
protein level was detected by immunocytochemistry. The gene expression of osteoprotegerin and receptor
activator of nuclear factor-kappa B ligand treated with 0, 5, 10, 25, 50, and 100 ng/mL interleukin-10 for 1 hour(for osteoprotegerin) or for 6 hours (for receptor activator of nuclear factor-kappa B ligand) was examined byreverse transcription-polymerase chain reaction. Osteoprotegerin secretion treated with 25 ng/mL interleukin-10
at 0, 2, 4, 6, and 8 hours was examined by enzyme-linked immunosorbent assay.
Results: Interleukin-10 was expressed in human dental follicle cells. Interleukin-10 enhanced osteoprotegerin
gene expression and secretion. In contrast, interleukin-10 suppressed receptor activator of nuclear factor-kappa
B ligand gene expresson. Protein kinase C inhibitors, myristoylated protein kinase C peptide inhibitor and
Go6983, decreased interleukin-10-induced osteoprotegerin, whereas protein kinase A inhibitors, cAMPdependent protein kinase peptide inhibitor and KT5720, did not decrease it.
Conclusion: The molecular basis for interleukin-10 on the inhibition of osteoclastogenesis is by enhancement of
osteoprotegerin gene expression and suppression of receptor activator of nuclear factor-kappa B ligand gene
expression in human dental follicle cells. Interleukin-10-induced osteoprotegerin secretion was dependent onprotein kinase C pathway. Interleukin-10 signaling may play an important role in the tooth eruption process.
(Int Chin J Dent 2007; 7: 7-13.)
Key Words: dental follicle cell, interleukin-10, osteoprotegerin, protein kinase A, protein kinese C.

Introduction
Dental follicle, a loose connective tissue sac surrounding the unerupted teeth, is necessary for tooth eruption to
occur.1 Alveolar bone resorption is also required for tooth eruption to form an eruption pathway. The dental
follicle regulates the cellular events of tooth eruption, including recruitment of mononuclear cells to the follicle
and directing the subsequent osteoclastogenesis needed for alveolar bone resorption.2 Receptor activator of
nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) are two essential factors acting as positive
and negative regulators in osteoclast formation. RANKL, a member of tumor necrosis factor (TNF) ligand
family, is expressed on osteoblast/stromal cell membranes. This ligand binds to the receptor activator of nuclear
factor-kappa B (RANK), which is a receptor on the membrane of osteoclastic precursors and on mature
osteoclasts, and induces osteoclast differentiation and activity.3-5 In contrast, OPG, a member of the TNF
receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its
ligand RANKL, thereby preventing RANKL from engaging its receptor RANK.3,6-9 It has been reported that rat
dental follicle cells express both OPG10 and RANKL gene11 and secret OPG.12 In earlier experiments, we also
found the gene expression of OPG and RANKL and OPG secretion in human dental follicle cells (HDFCs),13
suggesting that HDFCs is critical for osteoclastogenesis to occur during tooth eruption.
Interleukin-10 (IL-10), initially described as cytokine synthesis inhibitory factor, is synthesized by T cells,
macrophages, monocytes and B cells and dampens many inflammatory responses,14 e.g. inhibiting TNF-α

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production from LPS-activated monocytes, macrophages.15 IL-10 is expressed in human periodontal ligament
cells16 and rat dental follicle cells as well.17 IL-10 not only suppresses inflammation but also inhibits bone
resorption,18-20 so it may be necessary in both periodontitis and tooth eruption.
Liu et al.17 have reported the effect of IL-10 on gene expression of OPG and RANKL in rat dental follicle
cells. However, whether IL-10 is expressed in HDFCs and regulates gene expression of OPG and RANKL in
HDFCs remains unknown. And the signal transduction pathways through which IL-10 regulates OPG secretion
have not been elucidated. In the present study, we examined IL-10 expression at protein level and the effect of
IL-10 on gene expression of OPG and RANKL, as well as its effect on OPG secretion in HDFCs. In addition,
the signal transduction pathways to regulate IL-10-induced OPG secretion treated with specific protein kinase A(PKA) and protein kinase C (PKC) inhibitors were also investigated.

Materials and Methods
Culture of human dental follicle cells
Prior to commencement, the study protocol was approved by Ethics Committee of the Fourth Military Medical
University. The third mandibular molars were surgically removed from adolescents for the need of orthodontic
treatment with informed consent. HDFCs were cultured as previously described.13 Briefly, the dental follicle
tissues were cleaned after isolation and cut into pieces using a sterilized scalpel and were digested. Then theminced and digested tissues were seeded into culture dishes in Dulbecco’s modified Eagle medium (DMEM,
Hyclone, Logan, UT, USA) with 10% fetal bovine serum and 1mmol/L sodium pyruvate, in a humidified
incubator with 5% CO2 at 37˚C. When the cells had reached confluence, they were detached and subcultured.
Immunocytochemistry
Cells of the fifth passage were grown on coverslips at a density of 2x105 cells/mL, maintained for another 2days, and then fixed with 4% polyoxymethylene for 10 minutes. Next, the fixed cells were permeabilized with
0.3% TritonX-100 in phosphate buffered saline (PBS) for 10 minutes. After endogenous peroxidase activity was
suppressed with 3% H2O2 for 10 minutes, and after non-specific binding sites were blocked with 10% normal
goat serum for 30 minutes, the fixed cells were incubated with rabbit anti-human IL-10 polyclonal antibody(primary antibody, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) at 4˚C overnight. Staining was
performed with goat anti-rabbit IgG (secondary antibody) conjugated with horseradish peroxidase (Santa Cruz
Biotechnology Inc.) and developed with 3,3’-diaminobenzidine-HCl (Dako, Carpinteria, CA, USA). For control
experiment, immunostaining with PBS omitting the primary antibody was performed. Cellular nuclei in all
experiments were counterstained with hematoxylin.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Cells of the fifth passage were grown in 25-cm2 T-flasks until confluent. Then, the cells were incubated with
0, 5, 10, 25, 50, and 100 ng/mL IL-10 (Pepro Tech, Rock Hill, NJ, USA) for 1 hour (for OPG gene expression)or for 6 hours (for RANKL gene expression). After incubation, the cells were harvested for RNA extraction.
Total RNA was extracted from HDFCs using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). To remove
contaminating genomic DNA, the RNA was treated with RNase-free DNase I (Qiagen, Chatsworth, CA, USA)at 37˚C for 30 minutes. Then the RNA concentrations were calculated spectrophotometrically. The RNA
samples had an optical density ratio (OD260/OD280) of at least 1.8.
First-strand cDNA was synthesized using 2 µg of total RNA with Superscript II reverse transcriptase

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(Invitrogen) in a volume of 20 µL, according to the manufacturer’s instructions. For each PCR, 2 µL cDNA was
mixed with PCR buffer, dNTP, primers and Taq DNA polymerase (Invitrogen) to make a total reaction volumeof 25 µL. The following primers were used: OPG (GenBank Accession No. NM002546; forward primer:
5’-TCAAGCAGGAGTGCAATCG-3’; reverse primer: 5’-AGAATGCCTCCTCACACAGG-3’; expected
product

size

342

bp);

RANKL

(GenBank

Accession

No.

NM003701;

forwardprimer:5’-GGCTCATGGTTAGATCTGGC-3’; reverse primer: 5’-TGACCAATACTTGGTGCTTCC-3’; expected
product

size

351

bp);

GAPDH

(GenBank

AccessionNo.

XM0033260;

forwardprimer:5’-GCTCTCCAGAACATCTACC-3’; reverse primer: 5’-GTGTCGCTGTTGAAGTCAG-3’; expected productsize 266 bp). PCR conditions were initial denaturation at 94˚C for 2 minutes, then denaturation at 94˚C for 30 s,
annealing at a appropriate temperature for each primer pair (OPG: 55˚C, RANKL: 59˚C, and GAPDH: 60˚C) for
30 s, and extension at 72˚C for 30 s, all for 30 cycles, a final extension at 72˚C for 10 minutes. PCR products
were subjected to electrophoresis on 1.5% agarose gels and photographed under ultraviolet light after ethidium
bromide staining. Band intensities were measured with the use of NIH Image software. The mRNA levels of
OPG and RANKL were estimated semi-quantitatively by comparison with GAPDH mRNA level.
Enzyme-linked immunosorbent assay (ELISA)
Cells of the fifth passage were seeded into 24-well plates until confluent. Then the cells were cultured in theabsence or presence of 25 ng/mL IL-10 as control group and test group. The culture medium was harvested at 0,
2, 4, 6, and 8 hours for both groups.

OPG secretion in the culture supernatants was measured using

enzyme-linked immunosorbent assay. Cells of the fifth passage were seeded into 96-well plates until confluent.
Then the cells were treated with 25 ng/mL IL-10 with or without PKA inhibitors: 5 mmol/L cAMP-dependent
protein kinase peptide inhibitor (c-AMP inh, Promega, Madison, WI, USA) or 112 nmol/L KT5720 (Calbiochem,
San Diego, CA, USA) or PKC inhibitors: 10 µmol/L Myristoylated protein kinase C peptide inhibitor (Myr,
Promega) or 1 µmol/L Go6983 (Calbiochem). After 24 hours of stimulation, OPG secretion in the culture
supernatants was measured using enzyme-linked immunosorbent assay. OPG in the culture supernatants wasmeasured using commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the
manufacturer’s instructions.
Statistical analysis
All experiments were performed at least three times. Data are expressed as means with standard deviations(SD). Data were subjected to t-test and one-way analysis of variance (ANOVA) with SPSS 10.0 software. The
least significant difference (LSD) test was used for the post hoc comparison of specific groups. A p-value lessthan 0.05 was accepted as statistically significant.

Results
Immunocytochemistry for IL-10
IL-10 was positively immunostained in HDFCs (Fig. 1A), which was in contrast to the negative control (Fig.
1B).
Effect of IL-10 on gene expression of OPG and RANKL
IL-10 enhanced OPG gene expression (p<0.05), with maximal expression at 25 ng/mL IL-10 (Figs. 2 and 3A).
Conversely, IL-10 suppressed RANKL gene expression (p<0.05), with maximal reduction at 25 ng/mL IL-10(Figs. 2 and 3B).

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Fig. 1A
Fig. 1B
Fig. 2
Fig. 1. Positive immunostaining of IL-10 in HDFCs (A) in contrast to the negative control (B), x400.
Fig. 2. RT-PCR products for OPG and RANKL in HDFCs treated with IL-10 in a concentration-dependent study.

Fig. 3A
Fig. 3B
Fig. 3. Ratio between OPG and GAPDH in HDFCs treated with IL-10 (A). Ratio between RANKL and GAPDH in HDFCs treatedwith IL-10 (B). An asterisk (*) represents a statistically significant change from the control value treated without IL-10 (p<0.05).

Effect of IL-10 on OPG secretion
IL-10-induced OPG secretion (25 ng/mL) was increased after 4 hours of stimulation (p<0.05, Fig. 4).
Effect of PKA inhibitors on IL-10-induced OPG secretion
PKA inhibitors, c-AMP Inh and KT5720, did not significantly decreased IL-10-induced OPG secretion in
HDFCs (p>0.05, Fig. 5A).
Effect of PKC inhibitors on IL-10-induced OPG secretion
PKC inhibitors, Myr and Go6983, significantly decreased IL-10-induced OPG secretion in HDFCs (p<0.05,
Fig. 5B).

Fig. 4
Fig. 5A
Fig. 5B
Fig. 4. OPG secretion in HDFCs treated with IL-10 in a time-dependent study. An asterisk (*) represents statistically significant
OPG secretion of the test group compared with that of the control group (p<0.05).
Fig. 5. Effect of PKA inhibitors, c-AMP inh and KT5720 on IL-10-induced OPG secretion in HDFCs (A). Effect of PKC inhibitors,
Myr and Go6983 on IL-10-induced OPG secretion in HDFCs (B). An asterisk (*) represents statistical significance (p<0.05).

Discussion
Wise et al.21 found that the eruption time of the rat first mandibular molar was significantly delayed by 1 day
or more as a result of OPG injection. Kong et al.22 have demonstrated that in null mice devoid of RANKL gene,
the teeth do not erupt. Tomotaka et al.23 have reported that osteoclastogenesis during mouse tooth germ

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development is mediated by RANK-RANKL pathway. All of these studies indicate that OPG and RANKL are
key regulators in osteoclastogenesis needed for alveolar bone resorption during tooth eruption.
The present study has confirmed the expression of IL-10 at protein level in HDFCs. Liu et al.17 has shownthat rat dental follicle cells express IL-10 gene in vitro and it is also expressed in vivo in rat dental follicles frompostnatal day 1 to day 9 during tooth eruption, suggesting that IL-10 is involved in the tooth eruption process.
IL-10 knockout mice have accelerated alveolar bone loss.24 IL-10 may inhibit alveolar bone resorption in thetooth eruption process.
The tooth eruption process in rat has been reported as follows: The eruption time of the rat first mandibularmolar is on postnatal day 3 and day 10.10,25-27 Furthermore, RANKL gene expression is significantly increasedon postnatal days 9-11 compared with the earlier postnatal days,27 while OPG gene expression is decreased on
postnatal day 3.10 On postnatal day 3, the major burst of osteoclastogenesis for tooth eruption occurs.25,26 The
absence of up-regulation of RANKL emphasizes the importance of down-regulation of OPG at this time. A
steady-state output of RANKL, coupled with the decline in OPG, increases the RANKL/OPG ratio to favor
osteoclastogenesis at this time.27 The osteoclasts then erode the alveolar bone to form the eruption pathway,28 so
the tooth eruption occurs. After this day, OPG is increased,10 whereas RANKL remains at a low level, resulting
in the decline of RANKL/OPG ratio to inhibit osteoclastogenesis.27 On postnatal day 10, there is a minor burstof osteoclastogenesis.25,26 The RANKL/OPG ratio is again increased to favor osteoclastogenesis. In summary,
the RANKL/OPG ratio promoting osteoclastogenesis may be greater on postnatal day 3 than day 10.27 We
conclude that the normal tooth eruption in rat occurs at the specific time (postnatal day 3 and day 10) when
osteoclastogenesis arises, resulting in tooth eruption, and there also exists specific period (days except postnatal
day 3 and day 10) in the whole tooth eruption process when the osteoclastogenesis is inhibited and the teeth
remain unerupted. This may be critical to keep the normal morphology of the alveolar bone. It is postulated thatthe phenomenon may be similar in the normal tooth eruption process in humans. Thus, IL-10 may play animportant role in the inhibition of osteoclastogenesis needed for alveolar bone resorption by enhancement of
OPG gene expression and suppression of RANKL gene expression in the tooth eruption process.
Human periodontal ligament cells secrete IL-1019 and OPG29 and express both OPG and RANKL gene.30
After the dental follicle has developed into the periodontal ligament of the adult, it would be necessary to have
limited osteoclastic activity so that the attachment of the periodontal ligament to the alveolar bone would not be
disrupted.12 So it is possible that IL-10 may inhibit osteoclastogenisis by up-regulating OPG gene expression
and down-regulating RANKL gene expression in human periodontal ligament cells after tooth eruption.
The regulation of OPG by cytokines and hormones has been studied in different cell lines. Kondo et al.31 havereported that OPG gene expression in murine marrow stromal cells stimulated with PTH is regulated by PKA
and PKC activation. Villa et al.32 have demanstrated that CGRP inhibits OPG secretion in human osteoblast-likecells via cAMP/PKA-dependent pathway. These reports suggest that IL-10-induced OPG secretion might beregulated by PKA or PKC activity in HDFCs. So we used specific PKA and PKC inhibitors in order to find out
whether PKA or PKC pathway is involved in IL-10-induced OPG secretion.
In the present study, we found that the PKA inhibitors, c-AMP Inh and KT5720, did not decrease
IL-10-induced OPG secretion, indicating that PKA pathway is not involved in IL-10-induced OPG secretion in
HDFCs. Wise et al.33 have indicated that PKA pathway is not involved in OPG gene expression in rat dental
follicle cells. On the contrary, the PKC inhibitors, Myr and Go6983, decreased IL-10-induced OPG secretion,

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suggesting that IL-10-induced OPG secretion is dependent on PKC pathway. The PKC-alpha isoform may be
critical in OPG regulation.33 Other studies also used PKC activators to demonstrate the involvement of PKC
activity in OPG gene expression or secretion. Wise et al.33 have demonstrated that PMA, an activator of PKC
activity, enhances OPG gene expression in rat dental follicle cells. Takami et al.34 have reported that PMA
stimulates OPG gene expression in mouse primary osteoblasts, and PKC inhibitors, such as calphostin andstaurosporin, suppress OPG gene expression. Brandstrom et al.35 have shown that in human bone marrowstromal cells, PdBU, an activator of PKC activity, increases OPG secretion. Given the above, PKC activity is avery important signal transduction pathway in the regulation of OPG gene expression or secretion in some celllines.
In conclusion, the molecular basis for interleukin-10 on the inhibition of osteoclastogenesis is by enhancementof OPG gene expression and suppression of RANKL gene expression in HDFCs. Furthermore, IL-10-induced
OPG secretion is dependent on PKC pathway. IL-10 signaling may play an important role in the tooth eruption
process. Our research suggests further studies on other regulatory molecules and their pathways to elucidate themechanisms of tooth eruption.
Acknowledgment
This research was supported by the Nature Science Foundation of China (No.30400510).

References
1. Cahill DR, Marks SC Jr. Tooth eruption: evidence for the central role of the dental follicle. J Oral Pathol 1980; 9: 189-200.
2. Wise GE, Frazier-Bowers S, D'Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol
Med 2002; 13: 323-34.
3. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis
inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998; 95: 3597-602.
4. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.
Cell 1998; 93: 165-76.
5. Nakagawa N, Kinosaki M, Yamaguchi K, et al. RANK is the essential signaling receptor for osteoclast differentiation factor in
osteoclastogenesis. Biochem Biophys Res Commun 1998; 253: 395-400.
6. Tsuda E, Goto M, Mochizuki S, et al. Isolation of a novel cytokine from human fibroblasts that specifically inhibits
osteoclastogenesis. Biochem Biophys Res Commun 1997; 234: 137-42.
7. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bonedensity. Cell 1997; 89: 309-19.
8. Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): amechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998; 139: 1329-37.
9. Hakeda Y, Kobayashi Y, Yamaguchi K, et al. Osteoclastogenesis inhibitory factor (OCIF) directly inhibits bone-resorbing
activity of isolated mature osteoclasts. Biochem Biophys Res Commun 1998; 251: 796-801.
10. Wise GE, Lumpkin SJ, Huang H, Zhang Q. Osteoprotegerin and osteoclast differentiation factor in tooth eruption. J Dent Res
2000; 79: 1937-42.
11. Nakchbandi IA, Weir EE, Insogna KL, Philbrick WM, Broadus, AE. Parathyroid hormone-related protein induces spontaneous
osteoclast formation via a paracrine cascade. Proc Natl Acad Sci USA 2000; 97, 7296-300.
12. Wise GE, Yao S, Zhang Q, Ren Y. Inhibition of osteoclastogenesis by the secretion of osteoprotegerin in vitro by rat dental
follicle cells and its implications for tooth eruption. Arch Oral Biol 2002; 47: 247-54.
13. Sun H, Zhang Y, Jin Z, Li Z. Regulation of osteoprotegerin and receptor activator of nuclear factor-k B ligand in human dental
follicle cells. Int Chin J Dent 2006; 6: 97-104.
14. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol
2001; 19: 683-765.
15. Bogdan C, Vodovotz Y, Nathan C. Macrophage deactivation by interleukin 10. J Exp Med 1991; 174: 1549-55.
16. Deschner J, Arnold B, Kage A, Zimmermann B, Kanitz V, Bernimoulin JP. Suppression of interleukin-10 release from humanperiodontal ligament cells by interleukin-1beta in vitro. Arch Oral Biol 2000; 45: 179-83.
17. Liu D, Yao S, Wise GE. Effect of interleukin-10 on gene expression of osteoclastogenic regulatory molecules in the rat dental
follicle. Eur J Oral Sci 2006; 114: 42-9.
18. Hong MH, Williams H, Jin CH, Pike JW. The inhibitory effect of interleukin-10 on mouse osteoclast formation involves novel
tyrosine-phosphorylated proteins. J Bone Miner Res 2000; 15: 911-8.
19. Sasaki H, Hou L, Belani A, et al. IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo. J Immunol 2000;
165: 3626-30.
20. Lappin DF, MacLeod CP, Kerr A, Mitchell T, Kinane DF. Anti-inflammatory cytokine IL-10 and T cell cytokine profile inperiodontitis granulation tissue. Clin Exp Immunol 2001; 123: 294-300.
21. Wise GE, Yao S, Liu D. Injections of Osteoprotegerin and PMA delay tooth eruption. Clin Anat 2006; 19: 19-24.
22. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development andlymph-node organogenesis. Nature 1999; 397: 315-23.
23. Suzuki T, Suda N, Ohyama K. Osteoclastogenesis during mouse tooth germ development is mediated by receptor activatorof NFKappa-B ligand (RANKL). J Bone Miner Metab 2004; 22: 185-91.

12Qian et al.

Int Chin J Dent 2007; 7: 7-13.

24. Al-Rasheed A, Scheerens H, Rennick DM, Fletcher HM, Tatakis DN. Accelerated alveolar bone loss in mice lacking
interleukin-10. J Dent Res 2003; 82: 632-5.
25. Cielinski MJ, Jolie M, Wise GE, Ando DG, Marks Sc Jr. Colony-stimulating factor-1 (CSF-1) is a potent stimulator of tooth
eruption in the rat. In: The biological mechanisms of tooth eruption, resorption and replacement by implants. Davidovitch Z,
Ed. Birmingham, AL: Ebsco Media; 1994. p. 429-36.
26. Wise GE, Fan W. Changes in the tartrate-resistant acid phosphatase cell population in dental follicles and bony crypts of rat
molars during tooth eruption. J Dent Res 1989; 68: 150-6.
27. Liu D, Yao S, Pan F, Wise GE. Chronology and regulation of gene expression of RANKL in the rat dental follicle. Eur J OralSci 2005; 113: 404-9.
28. Cahill DR. Eruption pathway formation in the presence of experimental tooth impaction in puppies. Anat Rec 1969; 164:67-77.
29. Wada N, Maeda H, Tanabe K, et al. Periodontal ligament cells secrete the factor that inhibits osteoclastic differentiation andfunction: the factor is osteoprotegerin/ osteoclastogenesis inhibitory factor. J Periodont Res 2001; 36: 56-63.
30. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Dual regulation of osteoclast differentiation by periodontal ligament cells throughRANKL stimulation and OPG inhibition. J Dent Res 2001; 80: 887-91.
31. Kondo H, Guo J, Bringhurst FR. Cyclic adenosine mono-phosphate/protein kinase A mediates parathyroid hormone/parathyroid hormone-related protein receptor regulation of osteoclastogenesis and expression of RANKL andosteoprotegerin mRNAs by marrow stromal cells. J Bone Miner Res 2002; 17: 1667-79.
32. Villa I, Mrak E, Rubinacci A, Ravasi F, Guidobono F. CGRP inhibits osteoprotegerin production in human osteoblast-like cells
via cAMP/PKA-dependent pathway. Am J Physiol Cell Physiol 2006; 291: C529-37.
33. Wise GE, Ren Y, Yao S. Regulation of osteoprotegerin gene expression in dental follicle cells. J Dent Res 2003; 82: 298-302.
34. Takami M, Takahashi N, Udagawa N, et al. Intracellular calcium and protein kinase C mediate expression of receptor
activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts. Endocrinology 2000; 141: 4711-9.
35. Brandstrom H, Bjorkman T, Ljunggren O. Regulation of osteoprotegerin secretion from primary cultures of human bonemarrow stromal cells. Biochem Biophys Res Commun 2001; 280: 831-5.

Correspondence to:Dr. Zuolin Jin
Department of Orthodontics, College of Stomatology, The Fourth Military Medical University
145 Changle Xi Road, Xi’an, 710032, P. R. China
Fax: +86-29-83223047 E-mail: zuolinj@fmmu.edu.cn
Received January 18, 2007. Accepted February 11, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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