effect of compressive force on the expression of inflammatory cytokines and their receptors in...

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 4 8 8 – 4 9 6

avai lable at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journals /arob

Effect of compressive force on the expression ofinflammatory cytokines and their receptors inosteoblastic Saos-2 cells

Yuki Koyama a, Narihiro Mitsui a, Naoto Suzuki b,c, Momoko Yanagisawa a,Rina Sanuki a, Keitaro Isokawa c,d, Noriyoshi Shimizu a,e, Masao Maeno c,f,*aDepartment of Orthodontics, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8310, JapanbDepartment of Biochemistry, Nihon University School of Dentistry, Tokyo 101-8310, JapancDivision of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, JapandDepartment of Anatomy, Nihon University School of Dentistry, Tokyo 101-8310, JapaneDivision of Clinical Research, Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, JapanfDepartment of Oral Health Sciences, Nihon University School of Dentistry, Tokyo 101-8310, Japan

a r t i c l e i n f o

Article history:

Accepted 9 December 2007

Keywords:

Cytokine receptors

Inflammatory cytokines

Mechanical stress

Orthodontic tooth movement

a b s t r a c t

Objective: In orthodontic tooth movement, some cytokines released from periodontal liga-

ment fibroblasts and alveolar bone osteoblasts on the pressure side can alter the normal

processes of bone remodelling, resulting in physiological bone resorption. We examined the

effect of compressive force and interleukin (IL)-1 type I receptor antagonist (IL-1ra) on the

expression of inflammatory cytokines that promote osteoclast formation, as well as on their

receptors, in osteoblastic Saos-2 cells.

Design: The cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal

bovine serum with or without continuous compressive force (0.5–3.0 g/cm2) and/or IL-1ra for

up to 24 h. The gene expression levels of the cytokines and their receptors were estimated by

determining mRNA levels using real-time PCR; the protein levels were determined using

ELISA or immunohistochemical staining.

Results: The expression of IL-1b, IL-1 receptor, IL-6, IL-6 receptor, IL-8 receptor, IL-11 and

tumor necrosis factor-a (TNFa) increased depending on the strength and duration of the

compressive force, whereas the expression of IL-8, IL-11 receptor and TNFa receptor did not

change with the application of compressive force. The expression of cytokines and their

receptors produced by 3.0 g/cm2 of compressive force decreased with the simultaneous

addition of IL-1ra and the decrease was remarkable in IL-8 receptor, IL-11 and TNFa.

Conclusion: These results indicate that mechanical stress induces the production of inflam-

matory cytokines and their receptors in osteoblasts and the phenomenon is enhanced by

the autocrine action of IL-1b, which is increased in amount by mechanical stress.

# 2007 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +81 3 3219 8118; fax: +81 3 3219 8138.E-mail address: [email protected] (M. Maeno).

0003–9969/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2007.12.004

mailto:[email protected]

http://dx.doi.org/10.1016/j.archoralbio.2007.12.004

a r c h i v e s o f o r a l b i o l o g y 5 3 ( 2 0 0 8 ) 4 8 8 – 4 9 6 489

1. Introduction

Tooth movement by the application of orthodontic force is

characterised by remodelling changes in periodontal tissues

such as the periodontal ligament and alveolar bone. These

tissues, when exposed to varying degrees of magnitude,

frequency and duration of mechanical loading, express

extensive macro- and microscopic changes. Mechanical

stimuli exerted on a tooth cause an inflammatory response

in the periodontal tissue. The release of inflammatory

mediators from periodontal tissue triggers the biological

process of alveolar bone resorption.1

Bone remodelling during orthodontic tooth movement is

closely related to the activity of osteogenic cells, including

osteoblasts, osteocytes and osteoclasts. The application of

the optimal force that induces physiological bone remodel-

ling is critical to prevent tooth and periodontal tissue damage.

An important breakthrough in bone biology was the identi-

fication of the role of cytokines in bone remodelling.

Cytokines are released not only by inflammatory cells, but

also by cells that compose the local tissues, such as

fibroblasts, chondrocytes and osteoblasts and are involved

in initiating, amplifying, perpetuating and resolving the

inflammatory response. They play important roles in tooth

movement1 and are also key mediators of tissue damage.

Interleukin (IL)-1, IL-6, IL-8, IL-11 and tumor necrosis factor-a

(TNFa), which are known as typical inflammatory cytokines,

are produced by inflammatory cells and cells that compose

the periodontal tissues upon the application of orthodontic

force.2–6 Of these, the most potent cytokine is IL-1, which

directly stimulates osteoclast function via the IL-1 type I

receptor, which is expressed by osteoclasts.1 However, the

majority of these reports indicate that IL-1 is dominant in

gingival crevicular fluid in vivo.

The cells that most strongly receive a mechanical stress in

periodontal tissues are periodontal ligament cells. We

hypothesized that mechanical stress influences not only

periodontal ligament cells but also osteoblasts in alveolar

bone. We also hypothesized that osteoblasts produce various

cytokines that promotes the osteoclast formation from

mechanical stress and teeth movement according to bone

resorption. However, uncertainty remains regarding the

expression levels of cytokines and their receptors in response

to mechanical stress, especially with respect to the strength of

the mechanical stress to osteoblasts in vitro.

Thus, we examined the effects of different compressive

forces (0.5–3.0 g/cm2) on the expression levels of the inflam-

matory cytokines IL-1b, IL-6, IL-8, IL-11, TNFa and their

receptors IL-1r, IL-6r, IL-8r, IL-11r and TNFr. We also examined

the effects of compressive force and the IL-1 type I receptor

antagonist (IL-1ra) on the expression of these cytokines using

osteoblastic Saos-2 cells.

2. Materials and methods

2.1. Cell culture

Saos-2 cells7,8 from a human osteosarcoma cell line were

obtained from the RIKEN Bioresource Center (Tsukuba, Japan)

and used as osteoblasts. The cells were maintained in growth

medium consisting of Dulbecco’s modified Eagle medium

(Invitrogen, Grand Island, NY, USA) containing 10% (v/v) heat-

inactivated fetal bovine serum (HyClone Laboratories, Logan,

UT, USA) and 1% (v/v) penicillin–streptomycin solution (Sigma

Chemical, St. Louis, MO, USA) at 37 8C in a humidified

atmosphere of 95% air and 5% CO2. The medium was changed

twice weekly.

To examine the effect of IL-1ra on the expression of

cytokines and their receptors, 5.0 ng/mL IL-1ra (Serotec,

Oxford, UK)9 was added to the culture medium, and the cells

were cultured with or without compressive force for up to

24 h.

When gene expression was examined, Saos-2 cells were

cultured with medium containing 10% fetal bovine serum. On

the other hand, when protein expression was examined, the

cells cultured with serum free.

2.2. Application of compressive force

The cells were seeded in the cell culture dishes (inside

diameter: 83 mm) at a density of 2 � 104 cells/cm2. After

overnight incubation, the cells were nearly confluent and

were compressed continuously using a uniform compression

method similar to that described previously.10–15 Briefly, thin

round glass plates were placed over the layer of confluent cells

and the compressive force was adjusted by placing a lead

weight on the glass plates that was washed enough with the

detergent and 70% ethanol and had sterilised by autoclave.

Approximately 90% of the cells in the cell culture dish are

covered with the glass plate (inside diameter: 78 mm). The

weight was positioned so that the force was evenly distributed

across the cell monolayer. Three stainless steel wire bridge

(angle 1208) was placed on the glass plates so that the weight

did not touch the culture medium. The cells were subjected to

0.5, 1.0, 2.0, or 3.0 g/cm2 of compressive force for 1, 3, 6, 9, 12, or

24 h.13–15 Control cells were covered with thin glass plates

without lead weights. Compressive force of this condition was

0.1 g/cm2.

2.3. Real-time polymerase chain reaction (PCR)

Total RNA was isolated from cultured Saos-2 cells using a

commercially available kit (RNeasy Mini kit; Qiagen, Valencia,

CA, USA). Aliquots containing equal amounts of mRNA were

subjected to real-time PCR. First-strand cDNA synthesis was

performed using 1 mg DNase-treated total RNA in 20 mL

solution containing first-strand buffer, 50 ng random pri-

mers, 10 mM dNTP mixture, 1 mM DTT and 0.5 U reverse

transcriptase at 42 8C for 60 min. The cDNA mixtures were

diluted fivefold in sterile distilled water and 2-mL aliquots

were subjected to real-time PCR using SYBR Green I dye. Real-

time PCR was performed in 25 mL solution containing

1 � SYBR1 Premix Ex TaqTM (TaKaRa, Tokyo, Japan) and

0.2 M specific primers (sense and antisense; Table 1). Primers

for IL-8 were designed using Primer3 software (Whitehead

Institute for Biomedical Research, Cambridge, MA, USA).

Other primers were obtained from TaKaRa. PCR was per-

formed in a thermal cycler (Smart Cycler II System, Cepheid,

Sunnyvale, CA, USA) and the data were analysed using Smart

Table 1 – PCR primers used in the experiments

Target Forward primer Reverse primer GenBank accession no.

IL-1b 50-CCAGGGACAGGATATGGAGCA-30 50-TTCAACACGCAGGACAGGTACAG-30 NM000576

IL-1r 50-TTTAAGCAGAAACTACCCGTTGCAG-30 50-TCACGATGAGCCTATCTTTGACTCC-30 NM000877

IL-6 50-AAGCCAGAGCTGTGCAGATGAGTA-30 50-TGTCCTGCAGCCACTGGTTC-30 NM000600

IL-6r 50-GAGGGCTTCTGCCATTTCTGAG-30 50-CCAGGTTCAGCTGACAACAAACA-30 NM000565

IL-8 50-GACTTCCAAGCTGGCCGTG-30 50-CTCCTTGGCAAAACTGCACC-30 NM000584

IL-8r 50-GAAGCACCATCATTCCCGTTG-30 50-GACACAGCTTCAGCCCTGTTCTC-30 NM000634

IL-11 50-CATCTAGGCCTGGGCAGGAA-30 50-TCAGACAAATCGCCCTCAAGTG-30 NM000641

IL-11r 50-AGACCCTGGATGGTGCACTTG-30 50-AGAAGTTCTCATAGTCGGCTGCTTG-30 NM004512

TNFa 50-AGTATCCATGCTCTTGACCTTGTAG-30 50-CCCGTAATTGCTCCAATCTG-30 NM003701

TNFr 50-GCCTGGAGTGCACGAAGTTG-30 50-TCCACCGTTGGTAGCGATACATTA-30 NM001065

GAPDH 50-GCACCGTCAAGGCTGAGAAC-30 50-ATGGTGGTGAAGACGCCAGT-30 NM002046


Cycler software (Version 2.0). The real-time PCR conditions

were 95 8C for 3 s and 60 8C for 20 s, for 35 cycles. The

specificity of the PCR products was verified using a melting

curve analysis between 60 and 95 8C. Each mRNA sample was

tested three times. Each real-time PCR was performed three

times as stated in figure legends and the levels of mRNA

expression were calculated and normalised to the level of

glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA

at each time.

2.4. Enzyme-linked immunosorbent assay (ELISA)

The cells were cultured under each compressive force for

24 h, after which the culture medium was collected for

ELISA. The protein amounts of cytokines in the culture

medium were determined from each standard curve of IL-1b,

IL-6, IL-8, IL-11 and TNFa using commercially available kits

(R&D Systems Inc., Minneapolis, MN, USA). Each sample was

tested three times. Each experiment was performed three

times as stated in figure legends and the absorbance at

492 nm was recorded.

2.5. Immunohistochemistry

Cells grown under a compressive force of 0 (control) and 3.0 g/

cm2 for 24 h were fixed with 70% ethanol for 30 min.

Immunohistochemical staining was conducted as described

previously.16 Briefly, cells were incubated in 2% BSA–PBS

overnight and reacted with antibodies to IL-1r (MAB269; R&D

Systems Inc.), IL-6r (AB227NA; R&D Systems Inc.) and IL-8r

(sc30008; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA)

for 30 min at 4 8C. The bound primary antibodies were

localised using anti-mouse IgG for IL-1r, anti-goat IgG for IL-

6r and anti-rabbit IgG for IL-8r, all of which were conjugated to

rhodamine isothiocyanate (RITC). The primary and secondary

antibodies were used at 1:100 dilution in 1% BSA–PBS. After

nuclear staining with 40,6-diamidino-2-phenylindole (DAPI;

BioGenex, San Ramon, CA, USA), the cells were examined

using an epifluorescence microscope (Eclipse E600, Nikon,

Tokyo, Japan) and photographed digitally.

2.6. Statistical analysis

Each value represents the mean � S.D. Significant differences

were determined using Bonferroni’s modification of Student’s

t-test or ANOVA. Differences at p < 0.05 were considered

significant.

3. Results

3.1. Effect of compressive force on the gene and proteinexpression of cytokine

Saos-2 cells were cultured with or without continuous

compressive force (0.5–3.0 g/cm2) for up to 24 h and the

expression of genes and proteins for IL-1b, IL-6, IL-8, IL-11 and

TNFa in the cells was determined by real-time PCR after 1, 3, 6,

9, 12 and 24 h (Fig. 1a1–a5) and by ELISA of the culture medium

after 24 h (Fig. 1b1–b5), respectively.

The gene expression of IL-1b, IL-6, IL-11 and TNFa

increased with the strength of the compressive force and

increased markedly at 3.0 g/cm2 for 9–24 h (Fig. 1a1, a2, a4 and

a5). In contrast, the gene expression of IL-8 did not differ

significantly with the strength of the compressive force until

24 h of application (Fig. 1a3). The protein expression of each

cytokine showed a tendency similar to the gene expression

(Fig. 1b1–b5).

3.2. Effect of compressive force on the gene and proteinexpression of cytokine receptor


compressive force (0.5–3.0 g/cm2) for up to 24 h and the

expression of genes for IL-1r, IL-6r, IL-8r, IL-11r and TNFr in the

cells was determined by real-time PCR after 1, 3, 6, 9, 12 and

24 h (Fig. 2).

The gene expression of IL-1r, IL-6r and IL-8r increased with

the strength of the compressive force after 12, 12 and 6 h,

respectively, and increased markedly at a force of 3.0 g/cm2

(Fig. 2a–c). In contrast, the gene expression of IL-11r and TNFr

did not change significantly with the strength of the

compressive force, even after 24 h of application (Fig. 2d

and e).


compressive force (3.0 g/cm2) for 24 h and the expression of

proteins for IL-1r, IL-6r and IL-8r in the cells was examined by

immunohistochemical staining (Fig. 3a–f).

Immunohistochemically, increased protein expression of

IL-1r, IL-6 and IL-8r was shown in the cells subjected to a

Fig. 1 – Effect of compressive force (CF) on the gene and protein expression of each cytokine. Saos-2 cells were cultured with

or without continuous CF (0.5–3.0 g/cm2) for up to 24 h and for 24 h, respectively. The gene (a1–a5) and protein (b1–b5)

expression of IL-1b (a1 and b1), IL-6 (a2 and b2), IL-8 (a3 and b3), IL-11 (a4 and b4) and TNFa (a5 and b5) was determined

using real-time PCR and ELISA, respectively, after 1, 3, 6, 9, 12 and 24 h. Each bar indicates the mean W S.D. of three

experiments using ANOVA; *p < 0.05, **p < 0.01, CF treatment vs. control at respective time point.


Fig. 2 – Effect of CF on the gene expression of each cytokine

receptor. Saos-2 cells were cultured with or without

continuous CF (0.5–3.0 g/cm2) for up to 24 h. The gene

expression of IL-1r (a), IL-6r (b), IL-8r (c), IL-11r (d) and TNFr

(e) was determined using real-time PCR after 1, 3, 6, 9, 12

and 24 h. Each bar indicates the mean W S.D. of three

experiments using ANOVA; *p < 0.05, **p < 0.01, CF

treatment vs. control at respective time point.


pressure of 3.0 g/cm2 for 24 h and less intense staining was

observed in their controls.

3.3. Effect of compressive force and IL-1ra on the geneexpression of cytokine


compressive force (3.0 g/cm2) and/or 5.0 ng/mL IL-1ra. The

gene expression of IL-1b, IL-6, IL-11 and TNFa, which increased

significantly with a compressive force of 3.0 g/cm2 (Fig. 1), was

then determined by real-time PCR after 24 h of application

(Fig. 4a–d). The gene expression of each cytokine did not

change significantly with the addition of IL-1ra without the

application of compressive force. In contrast, the gene

expression decreased significantly with the addition of IL-

1ra and the application of compressive force and this decrease

was remarkable in IL-11 and TNFa.

3.4. Effect of compressive force and IL-1ra on the geneexpression of cytokine receptor


compressive force (3.0 g/cm2) and/or 5.0 ng/mL IL-1ra. The

gene expression of IL-1r, IL-6r and IL-8r, which increased

significantly with a compressive force of 3.0 g/cm2 (Fig. 2), was

then determined by real-time PCR after 24 h of application

(Fig. 5a–c). The gene expression of each cytokine receptor did

not change significantly with the addition of IL-1ra without the

application of compressive force. In contrast, the gene

expression decreased significantly with the addition of IL-

1ra and the application of compressive force and this decrease

was remarkable in IL-8r.

4. Discussion

We found that continuous compressive force stimulates the

production of inflammatory cytokines and their receptors by

osteoblasts and that IL-1b is related to the increase in this

expression.

We examined the effect of mechanical stress on the

expression of IL-1b, IL-6, IL-8, IL-11, TNFa and their receptors

in Saos-2 cells for up to 24 h. The strength of the compressive

force used was 0.5–3.0 g/cm2, based on previous experiments

that examined the effect of compressive force on bone

formation in Saos-2 cells13,14 and the expression of matrix

metalloproteinases, plasminogen activators and their inhibi-

tors in Saos-2 cells.15 An experimental period of 24 h was also

used based on previous experiments.13–15 The expression of IL-

1b, IL-6, IL-11 and TNFa increased depending on the strength

and duration of the compressive force applied; however,

the time at which the effect appeared differed among the

cytokines. In contrast, the expression of IL-8 did not change

with the application of compressive force.

IL-1 may be the most important cytokine produced by the

application of orthodontic force. IL-1 directly stimulates

osteoclast function through the expression of IL-1r by

osteoclasts. IL-1 also binds to the IL-1r of osteoblasts and

promotes the formation of osteoclasts through osteoblasts.

Osteoblasts are involved in osteoclast differentiation and

Fig. 3 – Immunohistchemical staining for cytokine receptors in Saos-2 cells grown under a CF of 3.0 g/cm2 (a–c) and without

compression (d–f). Raddish RITC fluorescence represents positive staining for the respective receptors. Staining in the cells

under the compression is more intense than controls. Images in the upper row are in lower magnification and those in the

bottom in higher magnification. Scale bars represent 20 mm.


function via cell-to-cell contact.17 Osteoclast precursors

express the receptor activator of nuclear factor kappa beta

(RANK),18 which is a member of the TNF receptor family,

recognise the RANK ligand (RANKL)10 in cell-to-cell interac-

tions with osteoblasts and differentiate into mononuclear

perfusion osteoclasts (POC) in the presence of macrophage

colony-stimulating factor (M-CSF).19,20 RANKL is also involved

in POC survival and fusion and mature osteoclast activation.

The expression of RANKL in osteoblasts is enhanced by 1a, 25-

dihydroxyvitamin D3, parathormone, prostaglandin E2 (PGE2)

and IL-11.17,21,22 Osteoprotegerin (OPG)23 is a soluble receptor

Fig. 4 – Effect of CF and IL-1ra on the gene expression of each c

continuous CF (3.0 g/cm2) and/or 5.0 ng/mL IL-1ra for 24 h. The

was determined using real-time PCR after 24 h. Each bar indicate

modification of Student’s t-test; *p < 0.05, **p < 0.01, CF + IL-1ra

for RANKL that acts as a decoy receptor in the RANK–RANKL

signalling system.17 Recently, Tanabe et al.24 demonstrated

that IL-1a stimulated the formation of osteoclast-like cells via

an increase in M-CSF and PGE2 production and a decrease in

OPG production by osteoblasts. IL-6 is a multifunctional

cytokine involved in osteoclast recruitment and differentia-

tion into mature osteoclasts.25,26 Osteoblast-derived IL-6, in

particular, is critical to bone remodelling27,28 because excess

IL-6 production may result in an increased numbers of

osteoclasts.29,30 TNFa, another proinflammatory cytokine,

elicits acute or chronic inflammation and stimulates bone

ytokine. Saos-2 cells were cultured with or without

gene expression of IL-1b (a), IL-6 (b), IL-11 (c) and TNFa (d)

s the mean W S.D. of three experiments using Bonferroni’s

treatment vs. CF.

Fig. 5 – Effect of CF and IL-1ra on the gene expression of each cytokine receptor. Saos-2 cells were cultured with or without

continuous CF (3.0 g/cm2) and/or 5.0 ng/mL IL-1ra for 24 h. The gene expression of IL-1r (a), IL-6r (b) and IL-8r (c) was

determined using real-time PCR after 24 h. Each bar indicates the mean W S.D. of three experiments using Bonferroni’s

modification of Student’s t-test; *p < 0.05, **p < 0.01, CF + IL-1ra treatment vs. CF.


resorption. Recent studies8,31–33 have shown that TNFa

directly stimulates the differentiation of osteoclast progeni-

tors to osteoclasts in the presence of M-CSF. Davidovitch et al.8

and Saito et al.31 demonstrated marked increases in the

staining intensity for IL-1 and TNFa in cells of periodontal

ligament and alveolar bone during orthodontic tooth move-

ment in cats.

In light of these findings, our results suggest that

osteoblasts produce various cytokines that promotes the

osteoclast formation from mechanical stress and the produc-

tion of cytokines was dependent on the magnitudes of the

force applied. The amounts of these inflammatory cytokines

may affect the bone resorptive pattern such as a direct or an

undermining bone resorption followed by tooth movement.

For the cytokine receptors, the expression of IL-1r, IL-6r and

IL-8r increased depending on the strength and duration of the

compressive force applied, whereas the expression of IL-11r

and TNFr did not change with the application of compressive

force. These findings suggest that the production of inflam-

matory cytokines that tip the balance of bone remodelling to

bone resorption and of their receptors, increases based on the

strength of the mechanical stress. However, the phenomenon

is not common to all cytokines and their receptors. Two

studies have examined ionic channels in periodontal ligament

fibroblasts to assess the signals that occur in the cells after the

application of mechanical stress and these suggest that the

intercellular Ca2+ concentration is increased by Ca2+ influx

through a Ca2+-permeable ionic channel.34,35 However, we

could not find any studies that examined the signals that occur

in osteoblasts after mechanical stress. In future, we will

examine how the inflammatory cytokines and their receptors

respond differently to the strength of the mechanical stress.

Tanabe et al.24 demonstrated that IL-1 strongly promotes

osteoclast formation by increasing M-CSF and PGE2 produc-

tion and decreasing OPG production by osteoblasts. Therefore,

we evaluated the effect of IL-1ra on the expression of each

cytokine and cytokine receptor to examine the possibility of

autocrine action by IL-1b. The expression of cytokines and

their receptors produced by 3.0 g/cm2 of compressive force

decreased with the addition of IL-1ra and this decrease was

remarkable in TNFa and IL-8r. This suggests that the

production of inflammatory cytokines and their receptors

increased with mechanical stress and also from the increase

in the autocrine action of IL-1b that results from mechanical

stress. On the other hand, the autocrine action with other

cytokines except IL-1b was suggested in the expression of IL-

1b, IL-6, IL-11, IL-1r and IL-6r that did not decrease remarkably

with the addition of IL-1ra and the application of compressive

force.

The application of 3.0 g/cm2 of compressive force sig-

nificantly increased the gene expression of some inflamma-

tory cytokines related to bone resorption and their receptors.

However, the gene expression of some factors related to

bone formation was decreased significantly by the same

force.14 When considering the optimal orthodontic force that

induces physiological bone remodelling, 3.0 g/cm2 will be

excessive; 1.0–2.0 g/cm2 of force, which stimulates factors

related to both bone formation and resorption, is more likely

to be the optimal orthodontic force. We plan to examine the

effect of antagonists other than IL-1ra and signals in the

future.

In conclusion, mechanical stress stimulates the production

of inflammatory cytokines that promote osteoclast formation,

as well as their receptors, in osteoblasts and the phenomenon

depends on the strength of the mechanical stress. In addition,

the production of inflammatory cytokines and their receptors

is enhanced by the autocrine action of IL-1b, which is

increased in amount by mechanical stress.


Acknowledgements

This study was supported by a grant from the Dental Research

Center at Nihon University School of Dentistry and by the Sato

Fund of Nihon University School of Dentistry. This study was

also supported by a special research grant from the Promotion

and Mutual Aid Corporation for Private Schools of Japan.

r e f e r e n c e s

1. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod DentofacialOrthop 2006;129:e1–32.

2. Lee KJ, Park YC, Yu HS, Choi SH, Yoo YJ. Effects ofcontinuous and interrupted orthodontic force oninterleukin-1b and prostaglandin E2 production in gingivalcrevicular fluid. Am J Orthod Dentofacial Orthop 2004;125:168–77.

3. Tuncer BB, Ozmerlc N, Tuncer C, Teoman I, Cakilci B, YucelA, et al. Levels of interleukin-8 during tooth movement.Angle Orthod 2004;75:631–6.

4. Jager A, Zhang D, Kawarizadeh A, Tolba R, Braumann B,Lossdorfer S, et al. Soluble cytokine receptor treatment inexperimental orthodontic tooth movement in the rat. Eur JOrthod 2005;27:1–11.

5. Basaran G, Ozer T, Kaya FA, Hamamci O. Interleukins 2, 6and 8 levels in human gingival sulcus during orthodontictreatment. Am J Orthod Dentofacial Orthop 2006;130:e1–e6.

6. Bletsa A, Berggreen E, Brudvik P. Interleukin-1a and tumornecrosis factor-alpha expression during the early phases oforthodontic tooth movement in rats. Eur J Oral Sci2006;114:423–9.

7. Fogh J, Wright WC, Loveless JD. Absence of HeLa cellcontamination in 169 cell lines derived from human tumors.J Natl Cancer Inst 1977;58:209–14.

8. Davidovitch Z, Nicolay OF, Ngan PW, Shanfeld JL.Neurotransmitters, cytokines, and the control of alveolarbone remodeling in orthodontics. Dent Clin N Am1988;32:411–35.

9. Aida Y, Maeno M, Suzuki N, Namba A, Motohashi M,Matsumoto M, et al. The effect of IL-1b on the expression ofinflammatory cytokines and their receptors in humanchondrocytes. Life Sci 2006;79:764–71.

10. Kanai K, Nohara H, Hanada K. Initial effects of continuouslyapplied compressive stress to human periodontal ligamentfibroblasts. J Jpn Orthod Soc 1992;51:153–63.

11. Watanabe K, Saito I, Hanada K. Effects of conditionedmedium of continuously compressed human periodontalligament fibroblasts on MC3T3-E1. J Jpn Orthod Soc1998;57:173–9.

12. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontalligament cells under mechanical stress induceosteoclastogenesis by receptor activator of nuclear factor kBligand up-regulation via prostaglandin E2 synthesis. J BoneMiner Res 2002;17:210–20.

13. Mitsui N, Suzuki N, Maeno M, Mayahara K, Yanagisawa M,Otsuka K, et al. Optimal compressive force induces boneformation via increasing bone sialoprotein andprostaglandin E2 production appropriately. Life Sci2005;77:3168–82.

14. Mitsui N, Suzuki N, Maeno M, Yanagisawa M, Koyama Y,Otsuka K, et al. Optimal compressive force induces boneformation via increasing bone morphogenetic proteinsproduction and decreasing their antagonists production bySaos-2 cells. Life Sci 2006;78:2697–706.

15. Mitsui N, Suzuki N, Koyama Y, Yanagisawa M, Otsuka K,Shimizu N, et al. Effect of compressive force on theexpression of MMPs, PAs, and their inhibitors in osteoblasticSaos-2 cells. Life Sci 2006;79:575–83.

16. Zohar R, Suzuki N, Suzuki K, Arora P, Glogauer M, McCullochCA, et al. Intracellular osteopontin is an integral componentof the CD44-ERM complex involved in cell migration. J CellPhysiol 2000;184:118–30.

17. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT,Martin J. Modulation of osteoclast differentiation andfunction by the new members of the tumor necrosis factorreceptor and ligand families. Endocr Rev 1999;20:345–57.

18. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC,Tometsko ME, Roux ER, et al. A homologue of the TNFreceptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997;390:175–9.

19. Fuller K, Owens JM, Jagger CJ, Wilson A, Moss R, ChambersTJ. Macrophage colony-stimulating factor stimulatessurvival and chemotactic behavior in isolated osteoclasts. JExp Med 1993;178:1733–44.

20. Jimi E, Shoto T, Koga T. Macrophage colony-stimulatingfactor and interleukin-1a maintain the survival ofosteoclast-like cells. Endocrinology 1995;136:808–11.

21. Feldmann M, Brennan FM, Maini RN. Role of cytokines inrheumatoid arthritis. Annu Rev Immunol 1996;14:397–440.

22. Hofbauer LC, Khosla C, Dunstan CR, Lacey DL, Boyle WJ,Riggs BL. The role of osteoprotegerin and osteoprotegerinligand in the paracrine regulation of bone resorption. J BoneMiner Res 2000;15:2–12.

23. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS,Luthy R, et al. Osteoprotegerin: a novel secreted proteininvolved in the regulation of bone density. Cell 1997;89:309–19.

24. Tanabe N, Maeno M, Suzuki N, Fujisaki K, Tanaka H, OgisoB, et al. IL-1a stimulates the formation of osteoclast-likecells by increasing M-CSF and PGE2 production anddecreasing OPG production by osteoblasts. Life Sci2005;77:615–26.

25. Kurihara N, Bertolini D, Suda T, Akiyama Y, Roodman GD.IL-6 stimulates osteoclast-like multinucleated cellformation in long-term human marrow cultures byinducing IL-1 release. J Immunol 1990;144:4226–30.

26. Okada N, Kobayashi M, Mugikura K, Okamatsu Y, HanazawaS, Kitano S, et al. Interleukin-6 production in humanfibroblasts derived from periodontal tissues is differentiallyregulated by cytokines and a glucocorticoid. J Periodontol Res1997;32:559–69.

27. Chambers TJ, Fuller K. Bone cells predispose bone surfacesto resorption by exposure of mineral to osteoclastic contact.J Cell Sci 1985;76:155–65.

28. Fuller K, Gallagher A, Chambers TJ. Osteoclast resorption-stimulating activity is associated with the osteoblast cellsurface and/or the extracellular matrix. Biochem Biophys ResCommun 1991;181:67–73.

29. Ishimi Y, Miyaura C, Jin CH, Akatsu T, Abe E, Nakamura Y,et al. IL-6 is produced by osteoblasts and induces boneresorption. J Immunol 1990;145:3297–303.

30. Rozen N, Ish-Shalom S, Rachmiel A, Stein H, Lewinson D.Interleukin-6 modulates trabecular and endochondral boneturnover in the nude mouse by stimulating osteoclastdifferentiation. Bone 2000;26:469–74.

31. Saito S, Ngan P, Saito M, Kim K, Lanese R, Shanfeld J, et al.Effects of cytokines on prostaglandin E and cAMP levels inhuman periodontal ligament fibroblasts in vitro. Arch OralBiol 1990;35:387–95.

32. Alhashimi N, Frithiof L, Brudvik P, Bakheit M. Orthodonticmovement induces high numbers of cells expressing IFN-g

at mRNA and protein levels. J Interferon Cytokine Res2000;20:7–12.


33. Alhashimi N, Frithiof L, Brudvik P, Bakheit M. Orthodontictooth movement and de novo synthesis of proinflammatorycytokines. Am J Orthod Dentofacial Orthop 2001;119:307–12.

34. Niisato N, Ogata Y, Furuyama S, Sugiya H. Histamine H1receptor-stimulated Ca2+ signaling pathway in humanperiodontal ligament cells. J Periodontal Res 1996;31:113–9.

35. Nohutcu RM, McCauley LK, Horton JE, Capen CC, Rosol TJ.Effects of hormones and cytokines on stimulation ofadenylate cyclase and intracellular calcium concentrationin human and canine periodontal-ligament fibroblasts. ArchOral Biol 1993;38:871–979.

effect of compressive force on the expression of inflammatory cytokines and their receptors in...

Documents