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Effects of compressive stress on the expression of M-CSF, IL1β, RANKL and OPG mRNA in periodontal ligament cells

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Objective: The aim of this study was to determine if human PDL cells can produce osteoclastogenic mRNA and examine how compressive stress affects the expression of osteoclastogenic mRNA in human PDL cells. Methods: Human PDL cells were obtained from biscupids extracted for orthodontic treatment. The compressive force was adjusted by increasing the number of cover glasses. PDL cells were subjected to a compressive force of 0.5, 1.0, 2.0, 3.0 or 4.0 g/cm2 for 0.5, 1.5, 6, 24 or 48 hours. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to examine levels of M-CSF, IL-1β, RANKL, OPG mRNA expression. Results: Human PDL cells could produce M-CSF mRNA. Human PDL cells under compressive stress showed increased M-CSF, IL-1β and RANKL mRNAs expression in a force (up to 2 g/cm2) and time-dependent manner. However, OPG mRNA expression was constant regardless of the level and duration of stress. Conclusions: Continuous compressive stress induced the mRNA expression of osteoclastogenic cytokines including M-CSF, RANKL, IL-1β in PDL cells. Together with an unchanged OPG mRNA level, these results suggest that compressive stress-induced osteoclastogenesis in vivo is partly controlled by M-CSF, RANKL and IL-1β expression in PDL cells. (Korean J Orthod 2009;39(4):248-256).
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ORIGINAL ARTICLE
248
a
Former Resident,
b
Professor,
c
Assistant Professor,
d
Full-time
Lecturer, Department of Orthodontics, Dental Hospital, East-West
Neo Medical Center.
Corresponding author:
Jong-Hyun Nahm.
Department of Orthodontics, Dental Hospital, East-West Neo
Medical Center, 149, Sangil-dong, Gangdong-gu, Seoul 134-
727, Korea.
+82 2 440 6205; e-mail, orthopia@unitel.co.kr.
Received January 30, 2008; Last Revision June 8, 2009;
Accepted June 13, 2009.
DOI:10.4041/kjod.2009.39.4.248
Effects of compressive stress on the expression of M-CSF,
IL-1
β
, RANKL and OPG mRNA in periodontal ligament cells
Ji-Woong Kim, DMD, MSD,
a
Ki-Soo Lee, DMD, MSD, PhD,
b
Jong-Hyun Nahm, DMD, MSD, PhD,
c
Yoon-Goo Kang, DMD, MSD, PhD
d
Objective: The aim of this study was to determine if human PDL cells can produce osteoclastogenic mRNA
and examine how compressive stress affects the expression of osteoclastogenic mRNA in human PDL
cells. Methods: Human PDL cells were obtained from biscupids extracted for orthodontic treatment. The
compressive force was adjusted by increasing the number of cover glasses. PDL cells were subjected to
a compressive force of 0.5, 1.0, 2.0, 3.0 or 4.0 g/cm
2
for 0.5, 1.5, 6, 24 or 48 hours. Reverse transcription
polymerase chain reaction (RT-PCR) analysis was performed to examine levels of M-CSF, IL-1β, RANKL,
OPG mRNA expression. Results: Human PDL cells could produce M-CSF mRNA. Human PDL cells under
compressive stress showed increased M-CSF, IL-1β and RANKL mRNAs expression in a force (up to 2
g/cm
2
) and time-dependent manner. However, OPG mRNA expression was constant regardless of the level
and duration of stress. Conclusions: Continuous compressive stress induced the mRNA expression of os-
teoclastogenic cytokines including M-CSF, RANKL, IL-1β in PDL cells. Together with an unchanged OPG
mRNA level, these results suggest that compressive stress-induced osteoclastogenesis in vivo is partly
controlled by M-CSF, RANKL and IL-1β expression in PDL cells.
(Korean J Orthod 2009;39(4):248-256)
Key words: Human PDL cell, Mechanical stress, Osteoclastogenesis
INTRODUCTION
Orthodontic tooth movement occurs during the se-
quential periodontal tissue remodeling, especially al-
veolar bone, induced by therapeutic mechanical stress.
1
It is a generally accepted that the periodontal ligament
tissue plays a key role in tooth movement as a re-
sponse to an applied mechanical stress, due to parad-
ental tissue remodeling including bone resorption and
formation. “Ankylosed teeth”, in which the cementum
of the tooth root is connected directly to the alveolar
bone, cannot be moved by therapeutic mechanical
stress due to the lack of a periodontal ligament.
2
Osteoclastogenesis has been an important subject in
the field of bone cell biology for a long time. Recent-
ly, the molecular determinants of osteoclastogenesis
were identified. Membrane-bound proteins, receptor ac-
tivator of nuclear factor
κ
B ligand (RANKL), and
soluble macrophage colony-stimulating factor (M-CSF)
are considered essential factors for osteoclastogenesis
produced by osteoblasts and bone marrow stromal
cells.
3-6
In contrast, osteoprotegerin (OPG), a soluble
tumor necrosis factor (TNF) receptor homolog, was
found to inhibit osteclastogenesis by competing with
the binding of RANKL to the RANK (receptor of
RANKL).
7
Vol. 39, No. 4, 2009. Korean J Orthod
Effects of compressive force on the periodontal ligament cells
249
Fig 1. Method used to apply a compressive stress.
Pre-cultured PDL cells were compressed continuously
using a different number of round-shaped cover glas-
ses. Round-shaped cover glasses were placed over a
confluent cell layer in each well of a 6-well plate. The
amount of compressive force was adjusted by increas-
ing or decreasing the number of cover glasses placed.
Cultured cells derived from the PDL, mostly fibro-
blasts, can express and produce the cytokines asso-
ciated with osteoclastogenesis. Hasegawa et al
8
re-
ported that PDL cells derived from deciduous and per-
manent teeth synthesized both RANKL and OPG, and
could regulate the differentiation of osteoclasts. It was
also reported that PDL cells secrete M-CSF in re-
sponse to TNF-
α
stimulation.
9
Wada et al
10
showed
that human PDL fibroblastic cells have the capacity to
produce and secrete OPG. Furthermore, it was reported
that inflammatory cytokines, such as PGE2, IL-1
α
,
IL-1
β
, IL-6 and TNF-
α
, are produced by mechan-
ically stimulated PDL cells.
11-13
Previous reports showed that PDL cells can partic-
ipate in osteoclastogenesis but the production of the re-
lated cytokines in PDL cells has not been fully charac-
terized. In particular, for M-CSF and IL-1
β
, their pro-
duction in mechanically stimulated PDL cells has not
been documented. This study examined the mode of
osteoclastogenetic cytokine production including MCSF,
RANKL, IL-1
β
and OPG in PDL cells in response to
compressive mechanical stimulation.
MATERIAL AND METHODS
Primary human PDL cells
Biscupids extracted from 5 patients for orthodontic
reasons were used in this study. Informed consent was
obtained from all volunteers. Immediately after ex-
traction, the teeth were placed in
α
-MEM containing
15% FBS (Sigma-aldrich, St. Louis, MO, USA) and
3-fold-reinforced antibiotics (Antibiotics and Antimy-
cotics, Gibco BRL, Grand Island, NY, USA) in a 50
ml conical tube (Corning, NY, USA). Using a No. 15
surgical blade, a piece of PDL was obtained exclu-
sively from the middle of the tooth roots in order to
exclude the intermixture of gingivae and dental
pulp.
9,10
The PDL tissue obtained was treated with 1.10
unit/ml dispase (Gibco BRL, Grand Island, NY, USA)
and 264 unit/ml collagenase (Collagenase Type II;
Gibco BRL, Grand Island, NY, USA) for 1 hour at
37
o
C. After washing with
α
-MEM, ligament samples
were cultured on a 100 mm primary culture dish
(Corning, NY, USA) in
α
-MEM containing 15% FBS
and antibiotics. The cells proliferating from the extracts
were passaged. For all experiments, PDL cells from
the 4
th
to 8
th
passages were used.
Cell culture
All PDL cells were cultured in
α
-MEM containing
15% FBS and antibiotics at 37
o
C in a 5% CO
2
incubator. The culture medium was changed twice a
week throughout the experiment.
Application of compressive stress
After sufficient cultivation, PDL cells from each pa-
tient were transferred to 6-well plates. Each well of a
6-well plate contained 1 × 10
6
PDL cells. Two days
later, the PDL cells were allowed to adhere to the well
plate base, and PDL cells were compressed con-
tinuously using the uniform compression method illus-
trated in Fig 1.
Briefly, round-shaped cover glasses (30 mm diame-
ter, Marienfeld, Louda-Könlgshofen, Germany) were
placed over a confluent cell layer in each well of a
6-well plate. Each cover glass weighed 0.245 gm. The
compressive force was adjusted by increasing or de-
Kim JW, Lee KS, Nahm JH, Kang YG
대치교정지
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250
Table 1. Primers used for RT-PCR
Annealing
Sp e c ific it y Olig onucle otide sequ ence (5'-3') Accession no. Product (BP )
temperatu re (
o
C)
M-CSF DSO
TM
prim er NM _00 0757 N.A 63
IL- 1
β
DSO
TM
prim er NM _00 0576 N.A 63
RANK L AGC AGA GAA AGC GAT GG T AF0 19047 341 58
GGG TAT GAG AAC TTG GGA TT
OPG TCA AGC AGG AGT GCA ATC G U943 32 342 57
AGA ATG CCT CCT CAC ACA GG
GAPDH CGG AGT CAA CGG ATT TGG TCG TA T N M_002046 306 56
AGC CTT CTC CAT GGT GGT GAA GAC
Sequence of DSO
TM
primer is not shown according to patent protection law.
creasing the number of cover glasses. Because the di-
ameter of a well in a 6-well culture plate is approx-
imately 35 mm, the periphery of each well was not
fully covered by the cover glass. However, the un-
covered area was minor and did not appear to affect
the results of the study. The PDL cells were subjected
to a compressive force of 0.5, 1, 2, 3 or 4 g/cm
2
(total
given force was 3.43, 7.015, 14.21, 21.315, 28.175 gm,
which was 0.49, 1.01, 2.01, 3.01, 3.99 g/cm
2
) for 0.5,
1.5, 6, 24 or 48 hours. The PDL cells without com-
pressive stimulation served as the control group.
RNA extraction and first-strand comple-
mentary DNA synthesis
After each culture period, the total RNA was ex-
tracted from each culture. After removing the culture
medium, the PDL cells from each well were homogen-
ized using Trizol reagent (Invitrogen Co., Carlsbad,
CA, USA). After homogenization, 0.2 ml of chloro-
form (Sigma-aldrich, St. Louis, MO, USA) per 1 ml of
Trizol reagent was added. The samples were centri-
fuged at 4
o
C, 12,000 rpm for 15 minutes. An aqueous
phase was transferred to a fresh tube, and 0.5 ml of
isopropyl alcohol (Sigma-aldrich, St. Louis, MO, USA)
per 1 ml of Trizol reagent was added. The samples
were centrifuged again under the same conditions.
Removing the supernatant, the RNA pellets were wash-
ed with 75% alcohol (Sigma-aldrich, St. Louis, MO,
USA), and dried for 5 to 10 minutes. The RNA was
dissolved in 0.1% diethyl pyrocarbonate (DEPC) water
(Fermentas, Glen Burnie, MD, USA).
For complementary DNA synthesis, a mixture of 500
ng mRNA, 2
μ
l of 10
μ
M Oligo dT (Fermentas, Glen
Burnie, MD, USA) and 3
μ
l DEPC water was in-
cubated at 80
o
C for 3 minutes, and chilled on ice for
2 minutes. Subsequently, 4
μ
l of 5X RT buffer
(Fermentas, Glen Burnie, MD, USA), 20 units of
RNase inhibitor (Fermentas, Glen Burnie, MD, USA),
200 units of RevertAid
TM
M-MuLV RT (Fermentas
Inc., Glen Burnie, MD, USA) and 4
μ
l of 2.5 mM
dNTP Mix (Fermentas, Glen Burnie, MD, USA) were
added to the mixture and incubated at 42
o
C for 90
minutes.
Reverse transcription polymerase chain re-
action assays
First-stranded complementary DNA was subjected to
polymerase chain reaction (PCR) amplification using
gene specific PCR primers. PCR for M-CSF and IL-1
β
was carried out using a GeneXP
TM
kit (Seegene, Seoul,
Korea). Each 10
μ
l reaction mixture contained 2
μ
l of
5X Human CYTO-X DSO
TM
primer, 5
μ
l of 2X mas-
ter mix and 20 ng of cDNA. Each cycle consisted of
the following: heat denaturation at 94
o
C for 30s, an-
nealing at 63
o
C for 90s and extension at 72
o
C for 90s.
PCR amplification can only start if all two parts of the
DSO
TM
(Dual Specificity Oligonucleotide; Seegene,
Seoul, Korea) primer bind to cDNA, which result in
Vol. 39, No. 4, 2009. Korean J Orthod
Effects of compressive force on the periodontal ligament cells
251
Fig 2. Compressive stress up-regulated M-CSF and
IL-1β mRNAs expression in PDL cells. A, RT-PCR
analysis of PDL cells. The PDL cells were loaded at
different compressive stress (0, 0.5, 1, 2, 3 or 4 g/cm
2
)
for 48 hours; B, Densitometry analysis. The results are
expressed as the mean ratio to GAPDH expression o
f
five independent experiments. M-CSF and IL-1β
mRNAs expression at 2 g/cm
2
was significantly differ-
ent compared to those of the control.
Fig 3. Compressive stress up-regulated M-CSF and
IL-1β mRNAs expression in a time dependent
manner. A, RT-PCR analysis of PDL cells. The PDL
cells were loaded at a constant compressive stress (2
g/cm
2
) for 0, 0.5, 1.5, 6, 24 or 48 hours; B, Densi-
tometry analysis. The results are expressed as the
mean ratio to GAPDH expression of five independent
experiments. M-CSF and IL-1β mRNAs expression
was significantly different from the control after 6
hours and 24 hours, respectively.
higher specificity and sensitivity than when using the
usual primer.
In PCR for RANKL and OPG, 2X PCR Master Mix
(Fermentas, Glen Burnie, MD, USA) was used for the
reaction. Each 50
μ
l reaction mixture contained 20
pmol of the sense and antisense PCR primers, 200 ng
of cDNA and 25
μ
l of 2X PCR Master Mix. Each cy-
cle consisted of the following: denaturation at 94
o
C for
30s, annealing at a temperature optimized for each pri-
mer pair (Table 1) for 90s and extension at 74
o
C for
90s.
The PCR products were electrophoresed and vi-
sualized on a 2% agarose gel containing ethidium bro-
mide with UV light illumination. The relative intensity
of the gel bands was measured using Scion Image
(Scion, Frederick, MD, USA) for Windows XP.
Statistical treatment
Statistical significance was evaluated by analysis of
variance (ANOVA) and a multiple-comparison test
(Scheffé's test) using SPSS V.13 (SPSS inc., Chicago,
IL, USA). A p value
0.05 was considered signi-
ficant. The values are expressed as the mean ± SD.
RESULTS
Effects of various compressive stress and
duration on the expression of M-CSF and
IL-1β mRNAs
The expression of M-CSF and IL-1
β
mRNAs in hu-
man PDL cells was assessed after applying various
mechanical compressive stresses for various durations.
Kim JW, Lee KS, Nahm JH, Kang YG
대치교정지
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, 2009
252
Fig 4. Compressive stress up-regulated RANKL mRNA
expression in PDL cells. In contrast, OPG mRNA ex-
pression did not change. A, RT-PCR analysis of PDL
cells. The PDL cells were loaded at different com-
pressive stresses (0, 0.5, 1, 2, 3 or 4 g/cm
2
) for 48
hours; B, Densitometry analysis. The results are ex-
pressed as the mean ratio to GAPDH expression o
f
five independent experiments. RANKL mRNA ex-
pression was significantly different from the control at
2 g/cm
2
. In contrast, OPG mRNA expression was con-
stant throughout the experiment.
Fig 5. Compressive stress up-regulated RANKL mRN
A
expression in a time dependent manner. In contrast,
OPG mRNA expression did not change. A, RT-PCR
analysis of PDL cells. The PDL cells were loaded at a
constant compressive stress (2 g/cm
2
) for 0, 0.5, 1.5,
6, 24 or 48 hours; B, Densitometry analysis. The re-
sults are expressed as the mean ratio to GAPDH ex-
pression of five independent experiments. RANKL
mRNA expression was significantly different from the
control after 6 hours. In contrast, OPG mRNA ex-
pression was constant throughout the experiment.
PDL cells under compression showed an increase in
M-CSF and IL-1
β
mRNAs expression in a force-de-
pendent manner up to 2 g/cm
2
, but the expression de-
creased after applying 3 g/cm
2
. The expression of M-
CSF and IL-1
β
mRNAs in the experimental groups
reached a maximum of 1.7- and 1.5-fold, respectively,
at a compressive load of 2 g/cm
2
, and were signifi-
cantly different from those of the control group which
was without compressive stress (0 g/cm
2
) (Fig 2).
M-CSF and IL-1
β
mRNAs expression was similar to
that of the control group at 0.5 hour of 2 g/cm
2
com-
pression but a time-dependent increase was evident af-
ter 6 and 24 hours, respectively, for up to 48 hours
(Fig 3).
Effects of various compressive forces and
duration on the expression of RANKL and
OPG mRNAs
Expression of RANKL and OPG mRNA in human
PDL cells was assessed after applying various com-
pressive mechanical stresses for various durations. PDL
cells under compression showed increased RANKL
mRNA expression in a force-dependent manner up to
2 g/cm
2
, but expression was decreased after 3 g/cm
2
.
The expression of RANKL mRNA in the experimental
groups reached a maximum of 2.1-fold at 2 g/cm
2
, and
was significantly different from those of the control
group (Fig 4). RANKL mRNA expression was similar
to that of the control group after 0.5 hours of 2 g/cm
2
compression. However, an increase was evident after 6
hours, and it increased in a time-dependent manner for
Vol. 39, No. 4, 2009. Korean J Orthod
Effects of compressive force on the periodontal ligament cells
253
up to 48 hours (Fig 5). In contrast, OPG mRNA ex-
pression did not change, regardless of the amount of
compressive force applied and the duration of com-
pression (Figs 4 and 5).
DISCUSSION
In this study, human PDL cells were cultured under
various levels of compressive stress for different peri-
ods to determine the effects of mechanical stress on
the expression of M-CSF mRNA in PDL cells. A RT-
PCR assay was used to analyze target mRNA
expression.
Generally, PDL includes multipopulation cells con-
sisting mainly of fibroblasts with high alkaline phos-
phatase activity,
14
and fibroblasts from PDL share
many physiological characteristics with osteoblasts.
15,16
Some studies already described methods to isolate PDL
cells from PDL, and an almost identical method was
used in this study. It is possible that osteoblastic cells
in the PDL subpopulation transduce mechanical stress.
However, the effects of other types of PDL cells were
negligible in this experiment because most cells in the
population in this culture system were spindle-shaped
fibroblastic cells.
Many researchers have already examined the effects
of mechanical stress on PDL cells. Various methods
have been used to impart mechanical stress to cultured
cells in the manner of tension, compression and fluid
shear stress, etc.
17-26
Among the manner of mechanical
stress, compressive stress was used to confirm the role
of PDL cell in the tissue remodeling process of the
compressive side during orthodontic tooth movement
with particular focus on osteoclastogenesis. Hydrostatic
compression,
22,23
reverse-tension compression,
24
and di-
rect contact compression methods
19,25,26
were used to
provide compressive stress on cultured cells. The direct
contact compression method appeared to mimic most
in vivo systems in orthodontic treatment because a stat-
ic compressive force is applied to the tooth in daily or-
thodontic practice and that PDL tissue is compressed
directly between two hard structures, bone and cemen-
tum. Therefore, this study adopted the direct contact
compression method, which provides a compression
force by squeezing the cells between two hard surfa-
ces, the culture dish surface and cover glass.
Kanzaki et al.
19
used glass cylinder and lead gran-
ules to provide compression stimulation. They placed a
glass cylinder over a confluent cultured PDL cell layer
and adjusted the compressive force by adding lead
granules to the cylinder. Similarly, Yamaguchi et al.
25
applied compressive stimulation by placing a 30 mm
cell disk over a confluent cell layer followed by a
glass cylinder on top of it. They also used lead gran-
ules to control the amount of compressive force by
placing them into the glass cylinder. The present meth-
od used only cover glasses in order to simplify the
equipment. In addition, by stacking same diameter
glasses, a more uniform force could be delivered to the
underlying cells than the lead granules, which can roll
around in the glass cylinder. In addition, the cover
glass weight was small enough (0.245 g) to control the
force in a more accurate manner.
The M-CSF and RANKL evaluated in this study are
essential factors for osteoclastogenesis produced by os-
teoblasts and bone marrow stromal cells.
3-6
M-CSF is
essential for not only the proliferation of osteoclast
progenitors, but also for differentiation into mature os-
teoclasts and survival in vitro.
27
In contrast, OPG was
reported to inhibit osteclastogenesis competing with the
binding of RANKL to the RANK. IL-1
β
is a proin-
flammatory cytokine that stimulates M-CSF and PGE
2
production,
28
and up-regulated PGE
2
mediates RANKL
expression in PDL cells under a static compressive
stress.
19
However, there is some controversy regarding the
effects of M-CSF in osteoclastogenesis. Although M-
CSF is an essential factor for the formation of osteo-
clasts, it also has been reported that the addition of
M-CSF to a culture inhibits osteoclast formation.
Udagawa et al.
3
reported that M-CSF, while stimulat-
ing murine osteoclast survival, did not induce pit-form-
ing activity. Perkins et al.
29
showed that the addition of
exogenous M-CSF to a co-culture of mouse spleen
cells and ST2 cells caused a dose-dependent decrease
in the number of osteoclasts formed, and was accom-
panied by an increase in the number of macrophages.
Recently, Yasuda et al.
30
reported that a high concen-
tration of M-CSF (
40 ng/ml) suppressed osteoclast
formation in murine spleen cell cultures stimulated
Kim JW, Lee KS, Nahm JH, Kang YG
대치교정지
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254
with soluble RANKL. Considering that these studies
examined osteoclast formation in animal models, spe-
cies differences and different requirements for M-CSF
between the different culture systems can account for
these divergent findings.
Through RT-PCR assays, PDL cells under a com-
pressive stress showed increased M-CSF, IL-1
β
and
RANKL mRNAs expression in a force- (up to 2 g/
cm
2
) and time-dependent manner, but OPG mRNA ex-
pression was constant regardless of the amount of
compressive stress or duration of compression. This
can be explained by the fact that PDL cells under
compressive stress regulate bone remodeling through
an increase in the production of osteoclastogenetic cy-
tokines but not OPG. In addition, compressive stress-
induced intracellular changes are not involved in OPG
mRNA expression. However, it was reported that ten-
sion type stimulation up-regulates OPG mRNA ex-
pression.
31,32
Therefore compression and tension stim-
ulation appears to have separate signal transduction
pathways.
These results partly coincide with previous reports.
Kanzaki et al.
19
reported that compressive stress up to
2 g/cm
2
up-regulated RANKL mRNA in a time-de-
pendent manner. They also measured the level of OPG
and reported no change. Nishijima et al.
26
examined
levels of RANKL and OPG in gingival crevicular fluid
during orthodontic tooth movement and found that the
RANKL level was elevated while the OPG level was
decreased. They also performed an in vitro study and
reported that compression stressed PDL cells increased
the secretion of RANKL in a time-dependent manner
but decreased the secretion of OPG in a time-depend-
ent manner. Nakajima et al.
33
carried out a similar
compressive stimulation study on PDL cells. They also
reported that RANKL production was elevated in a
time and force-dependent manner up to 4 g/cm
2
, the
maximal force that they used. On the other hand, they
reported an increase in OPG production in com-
pression-stressed PDL cells. Overall, it appears that
compressive stress up-regulates RANKL mRNA and
increases the production of RANKL in PDL cells.
However, for OPG, there are discrepancies between
studies, which will need more investigation to establish
the mode of OPG production in PDL cells.
With the exception of Nakajima et al.'s
33
study, oth-
er studies examining compressive stress effects on
PDL cells reported that a 2 g/cm
2
force is the optimal
level of force that can elicit a maximal PDL cell
response.
19,23,26
These results also support the optimal
force level of 2 g/cm
2
. At an applied force magnitude
of 3 g/cm
2
, the level of M-CSF, IL-1
β
and RANKL
mRNA expression decreased. This is probably due to
the 3 g/cm
2
applied force being too heavy making cell
survival difficult. Indeed, a microscopic observation af-
ter applying 4 g/cm
2
showed that the compressive
stress was so heavy that PDL cells were partially dam-
aged and?the number of cells decreased (data not
shown). Nakao et al.
23
suggested that 2 g/cm
2
was suit-
able but 7.0 g/cm
2
was too heavy for cell survival.
This in vitro result can be applied clinically. Although
the applied orthodontic force cannot be distributed uni-
formly on the desired tooth root surface, the area of
root surface that faces a compressive orthodontic force
according to the type of tooth movement can be esti-
mated and the desirable amount of force can be calcu-
lated by multiplying 2 g/cm
2
. The validity of this as-
sumption may be proven by another systemized clin-
ical trial investigation.
After 6 hours of 2 g/cm
2
compressive mechanical
stress, there was a significant increase in the level of
M-CSF and RANKL mRNA expression compared to
the control. However, the level of IL-1
β
mRNA ex-
pression increased significantly after 24 hours. This
means that IL-1
β
mRNA up-regulation is delayed
compared to M-CSF and RANKL. In addition, other
cytokines might mediate signal transduction during a
compressive mechanical stress. To the best of our
knowledge, there are no reports on the effect of com-
pressive stress on the expression of M-CSF and IL-1
β
mRNA. These results suggest that the expression of
M-CSF and IL-1
β
mRNA increases in a time- and
force-dependent manner (up to 2 g/cm
2
). In addition, it
appears that a compressive force directly regulates
M-CSF mRNA expression but indirectly regulates IL-1
β
mRNA expression. More signal transduction studies
will be needed to confirm the mechanism of com-
pressive force induced M-CSF and IL-1
β
mRNA
expression.
Vol. 39, No. 4, 2009. Korean J Orthod
Effects of compressive force on the periodontal ligament cells
255
CONCLUSION
Continuous compressive stress up-regulates M-CSF,
IL-1
β
and RANKL mRNA expression in cultured
hPDL cells in a force- and time-dependent manner.
However, a compressive force does not regulate OPG
mRNA expression in PDL cells. These results suggest
that a compressive stimulated PDL cells regulate osteo-
clastogenesis by up-regulating the stimulatory cytokines
but not down-regulating the OPG production. There-
fore, in orthodontic tooth movement, PDL cells partic-
ipate in bone resorption by up-regulating osteoclastoge-
netic cytokines, including M-CSF, IL-1
β
and RANKL.
- 국문초록 -
압박력이 치주인대 세포의 M-CSF, IL-1
β
, RANKL
OPG mRNA 발현에 미치는 영향
김지웅
a
이기수
b
남종현
c
강윤구
d
연구의 목적은 배양된 사람 치주인대 세포에서 파골세
포의 형성에 관련된 물질을 합성
,
분비할 있는지를 알아
보고 압박력이
M-CSF, IL-1
β
, RANKL
OPG mRNA
발현에 미치는 영향을 알아보고자 하였다
.
교정치료를 목적
으로 발치된 소구치에서 얻은 치주인대세포를 배양한
양한 크기
(0.5, 1.0, 2.0, 3.0, 4.0 g/cm
2
)
기계적 자극을
양한 기간
(0.5, 1.5, 6, 24, 48 hours)
동안 적용하고
, M-CSF,
IL-1
β
, RANKL, OPG mRNA
발현양의 변화를 검사하였다
.
각각의 실험군에서 얻어진
mRNA
대해 역전사 중합효소
연쇄반응검사를 시행하였다
.
검사 결과 압박력은 사람 치주
인대 세포에서
M-CSF mRNA
발현시켰으며
M-CSF, IL-1
β
,
RANKL mRNA
발현양은 자극의 크기와 기간에 따라 증가
하였다
.
그러나 압박력은 사람 치주인대 세포에서
OPG
mRNA
발현양에 영향을 미치지 않는 것으 타났다
.
상의 결과는 기계적 자극이 치주인대 세포에서
M-CSF, IL-1
β
,
RANKL mRNA
발현양을 조절함으로 파골세포의 분화에
영향을 미칠 있음을 시사한다
.
주요 단어
:
주인대세포
,
기계적 자극
,
파골세포형성
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... This method is also attracting considerable attention for its possible use as in vitro research technique, where it could bridge the gap between traditional two-dimensional (2D) cell cultures and in vivo 3D biological reality (Li et al. 2011). Although many studies have tried to mimic orthodontic force using 2D or 3D cultured periodontal ligament cell models (Kim et al. 2009;Mitsui et al. 2006;Nakajima et al. 2008), the characteristics of these systems including gene expression profiles have shown considerable variability. Previous studies have also not reported cell survival in their models (Sheldrake 1974), raising questions about cell sustainability under compressive stress and whether these in vitro models mimic in vivo conditions. ...
... It was previously observed that RANKL mRNA expression was significantly increased at compressive stress loads of 25 g/cm 2 or greater, peaking at 6 h at 25 g/cm 2 (Li et al. 2011). Another report also showed that RANKL mRNA expression under 48 h of 2.0 g/cm 2 compressive stress was significantly higher compared than that at 0 or 4.0 g/ cm 2 , and that RANKL mRNA expression increased with the duration of compressive stress (Kim et al. 2009). RANKL is believed to be an essential osteoclastogenesis inducer; therefore, the peak of RANKL expression deduced from the polyline should provide a more expedient representation of compressive stress for osteoclastogenesis (De Araujo et al. 2007). ...
... Studies using two dimensional (2D) periodontal ligament culture systems also showed similar trends. Kim et al. (2009) showed that under 0-4 g/cm 2 compressive stress, expression of OPG did not significantly change until 48 h, and Nakajima et al. (2008) indicated that under 0-4 g/cm 2 compressive stress, OPG expression increased with longer compressive stress durations. ...
Article
This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-L-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm(2) compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic "double-peak" expression at 15 and 35 g/cm(2) on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm(2) on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.
... Osteocytes are important mechanosensors and transducers of applied mechanical strain Krishnan & Davidovitch, 2009). But studies show that osteoblasts and PDL cells also respond to mechanical strain by production of mediators such as cytokines, growth factors, prostaglandin and nitric oxide after loading in-vitro (Chen et al., 2014;de Araujo, Oba, & Moriyama, 2007;Kang et al., 2013;Kanjanamekanant, Luckprom, & Pavasant, 2013;Kikuta et al., 2013;Kim, Lee, Nahm, & Kang, 2009;Kim, Park, Park, Lee, & Kang, 2013;Kook et al., 2011a;Koyama et al., 2008;Ku et al., 2009;Lee et al., 2007;Li, Yi, Yang, Zhang, et al., 2016;Li et al., 2011Li et al., , 2013Maeda et al., 2007;Nakajima, Yamaguchi, Kojima, Takano, & Kasai, 2008;Nakao et al., 2007;Proff et al., 2014;Ritter et al., 2007;Tripuwabhrut et al., 2012). Therefore we can conclude that besides the osteocytes in the alveolar bone, also the PDL cells and osteoblasts in the PDL mediate cell activation and differentiation of the cells. ...
... Interestingly, although less than the upregulation of RANKL (Taddei et al., 2013), the expression of OPG is simultaneous upregulated but more pronounced at the tension side (Kobayashi et al., 2000;Taddei et al., 2013;Taddei, Andrade, et al., 2012;Taddei, Moura, et al., 2012). In-vitro compressive forces increased the expression of RANKL and decreased the expression of OPG in osteoblasts and PDL cells Kikuta et al., 2013;Kim et al., 2009;Lee et al., 2015;Li, Yi, Yang, Zhang, et al., 2016;Nakajima et al., 2008;Nakao et al., 2007;Nettelhoff et al., 2016;Nishijima et al., 2006;Tripuwabhrut, Mustafa, Gjerde, Brudvik, & Mustafa, 2013;Wongkhantee, Yongchaitrakul, & Pavasant, 2007). This is consistent with the idea that compressive force upregulates the RANKL/OPG ratio and promotes osteoclast differentiation . ...
Article
Objectives: The aim of the present systematic review was to offer a timeline of the events taking place during orthodontic tooth movement(OTM). Materials and methods: Electronic databases PubMed, Web of Science and EMBASE were searched up to November 2017. All studies describing the expression of signaling proteins in the periodontal ligament(PDL) of teeth subjected to OTM or describing the expression of signaling proteins in human cells of the periodontal structures subjected to static mechanical loading were considered eligible for inclusion for respectively the in-vivo or the in-vitro part. Risk of bias assessment was conducted according to the validated SYRCLE's RoB tool for animal studies and guideline for assessing quality of in-vitro studies for in-vitro studies. Results: We retrieved 7583 articles in the initial electronic search, from which 79 and 51 were finally analyzed. From the 139 protein investigated, only the inflammatory proteins interleukin(IL)-1β, cyclooxygenase(COX)-2 and prostaglandin(PG)-E2, osteoblast markers osteocalcin and runt-related transcription factor(RUNX)2, receptor activator of nuclear factor kappa-B ligand(RANKL) and osteoprotegerin(OPG) and extracellular signal-regulated kinases(ERK)1/2 are investigated in 10 or more studies. Conclusion: The investigated proteins were presented in a theoretical model of OTM. We can conclude that the cell activation and differentiation and recruitment of osteoclasts is mediated by osteocytes, osteoblasts and PDL cells, but that the osteogenic differentiation is only seen in stem cell present in the PDL. In addition, the recently discovered Ephrin/Ephs seem to play an role parallel with the thoroughly investigated RANKL/OPG system in mediating bone resorption during OTM.
... We found that cell viability was significantly decreased in groups C and VC, however the cell morphology was unaffected; which was similar to previous reports (27,28). Indeed, compressive force can be increased up to 2 g/cm 2 with no any damage to the cells (29). Our and previous studies indicated that mechanical stimuli affected cell proliferation but did not damage PDL cells. ...
... The effects of compressive force on OPG were still controversial. In our study, compressive force had no significant effect on OPG expression, in agreement with previous studies (12,29). However, one study reported compressive force (0.5-4.0 g/cm 2 ) upregulated OPG (30), while another reported exposure to compressive force downregulated OPG (11). ...
Article
Objective: Vibration can be used to accelerate tooth movement, though the exact mechanisms remain unclear. This study aimed to investigate the effects of low magnitude high frequency (LMHF) vibration combined with compressive force on periodontal ligament (PDL) cells in vitro. Materials and methods: Human PDL cells were isolated from extracted premolar teeth of four individuals. To determine the optimal frequency for later used in combination with compressive force, three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. To investigate the effects of vibration combined with compressive force, PDL cells were subjected to three cycles of optimal vibration frequency (V) or 1.5 g/cm2 compressive force for 48 h (C) or vibration combined with compressive force (VC). Cell viability was assessed using MTT assay. PGE2, soluble RANKL (sRANKL), and OPG production were quantified by ELISA. RANKL, OPG, and Runx2 expression were determined using real-time PCR. Results: Cell viability was decreased in groups C and VC. PGE2 and RANKL, but not OPG, were increased in groups V, C, and VC, thus increasing the RANKL/OPG ratio. The highest level was observed in group VC. sRANKL was increased in groups V, C, and VC; however, no significant different between the experimental groups. Runx2 expression was reduced in groups C and VC. Conclusions: Vibration increased PGE2, RANKL, and sRANKL, but not OPG and Runx2. Vibration had the additive effects on PGE2 and RANKL, but not sRANKL in compressed PDL cells.
... In addition, the tensile and compressive stress response shown in Figure 5 is consistent with the PDL tissue response described in the literature. 25 It has been reported that the optimal time for wearing thermoplastic appliances is 2 weeks; during which time, the teeth are typically under treatment for approximately 21-23 h each day. 26 However, different patients may experience PDL movement for different durations. ...
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The dynamic remodelling processes in the periodontal ligament (PDL) account for the reaction of PDL cells to different orthodontic force simulations. These occur mostly by degradation and synthesis of collagen types I, III, V, VI, XII and XIV. The purpose of this study was to quantify specific collagen types in the PDL from zones of tension and compression of experimental teeth. Such changes could then be correlated with the processes of orthodonticstimulated tissue breakdown. Maxillary and mandibular premolars of three females and one male patient were orthodontically moved with a box loop for a total of 14 days, prior to tooth extraction. Teeth from the contralateral side of either the maxilla or the mandible served as the untreated controls. A total of seven experimental and seven control teeth were used in this investigation. PDL fibroblasts from the cervical third of the roots corresponding to the compression and tension zones of the experimental and control teeth, respectively, were scraped and cultured in vitro at 37°C in a humidified incubator with 5 per cent CO2/95 per cent air. Collagen synthesis of types I, III, V and VI was quantified by using an ELISA. Application of orthodontic forces in the experimental teeth showed a significant increase (P < 0.05) of the synthesis of all collagen types in the compression as opposed to the tension zones. Collagen synthesis on the compression zone of experimental teeth was not significantly different in the mandible when compared with those of the maxilla. In addition, the proportional distribution of different types of collagen was also not significantly different in the PDL fibroblasts from either zone of experimental teeth of either the maxilla or the mandible. Collagen metabolism in response to orthodontic stimulation appears to be higher in the compression zones and lower in the tension zones. Contrary to what is traditionally assumed in the literature, such findings indicate that in addition to bone resorption, tissue remodelling is very active in zones of compression following the disappearance of the hyalinized areas. These findings constitute a model for future studies on collagen metabolism during orthodontic-stimulated tooth movement.
Article
We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cell (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines (NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells-through a mechanism involving cell-to-cell and/or cell-to-matrix contact.
Article
The production of cytokines has been associated with the biology of tooth movement in animal populations. The purpose of this study was to measure tumor necrosis factor-α (TNF) directly in the human gingival sulcus before and after the application of an orthodontic force. To recover TNF from the sulcus, paramagnetic beads, coated with monoclonal antibodies for TNF, were introduced into the gingival sulcus of 50 teeth undergoing orthodontic tooth movement (by two force systems) in 20 patients. Retrieval was performed by a permanent magnetic device designed to fit the periodontal sulcus. The samples were taken before force application (controls), and at a fixed time after force application. The amount of immunoabsorbed TNF was quantified with an immunochemical assay. There was a greater than twofold increase in TNF recoverable from the gingival sulcus after application of orthodontic forces (mean of 12.9 ng vs 30.5 ng). A Student's t test for paired samples demonstrated statistical significance at p < 0.01. We conclude that the quantity of paradental TNF, found in human gingival sulcus, is elevated during tooth movement. The source may be from the adjacent gingiva, but more likely the compressed periodontal ligament and the resorbing bone adjacent to the root surface. (AM J ORTHOD DENTOFAC ORTHOP 1995;108:519-24.)
Article
We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1α,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1α,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.
Article
Neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), may be associated with bone remodeling in response to mechanical stress during orthodontic tooth movement. To investigate this hypothesis, we examined the effects of neuropeptides on the expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (PDL) cells under compression in vitro. PDL cells were subjected to compressive force (2.0 g/cm2) continuously in the presence or absence of SP or CGRP for 2–4 days. The expression of the SP receptor, neurokinin 1-receptor (NK1-R), in PDL cells was confirmed by RT–PCR and immunofluorescent staining. The effects of neuropeptides (SP and CGRP) on the expression of RANKL and OPG mRNA were determined using RT–PCR. PDL cells constitutively expressed NK1-R on both the mRNA and protein levels. Compressive force decreased OPG mRNA expression and increased RANKL mRNA expression. In the presence of neuropeptides, the OPG level decreased synergistically with compression. Neuropeptides stimulated RANKL expression without compression, whereas they decreased RANKL mRNA expression with compression. These results indicate that PDL cell compression induces the up-regulation of RANKL and down-regulation of OPG, whereas neuropeptides suppress the RANKL expression induced by compression. Therefore, the neuropeptides SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement.
Article
We have reported that osteoclast differentiation factor (ODF) expressed on the plasma membrane of osteoblasts/stromal cells is a ligand for osteoclastogenesis inhibitory factor (OCIF). A genetically engineered soluble form of ODF (sODF) induced osteoclast-like multinucleated cells (OCLs) in the presence of M-CSF in mouse spleen cell cultures. Osteoblasts/stromal cells were not required in this process. To elucidate the mechanism of human osteoclastogenesis, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days with sODF and human M-CSF in the presence or absence of dexamethasone. Treatment of human PBMCs with sODF together with M-CSF induced OCLs, which expressed tartrate-resistant acid phosphatase and vitronectin receptors, produced cAMP in response to calcitonin, and formed resorption pits on dentine slices. OCLs were also formed from the adherent cell population of human PBMCs. Dexamethasone was required for human OCL formation in culture of whole PBMCs but not in culture of the adherent cell population. OCL formation was strongly inhibited by OCIF simultaneously added. These results clearly indicate that like in mouse osteoclastogenesis, ODF is a critical factor for human osteoclastogenesis. The present study also indicates that OCIF acts as a naturally occurring decoy receptor for ODF in inhibiting signal transduction in human osteoclast formation.
Article
Cathepsin is a typical and well-characterized lysosomal cysteine protease that, under pathological conditions, is involved in tissue destruction. A recent immunocytochemical study demonstrated that cathepsins B (CAB) and L (CAL) were localized in the periodontal ligament (PDL) of the rat molar, and they were expressed in compressed sites during experimental tooth movement. Further, we demonstrated previously that the levels of CAB and CAL in gingival crevicular fluid increased during orthodontic tooth movement. Therefore, CAB and CAL may play important roles in the process of collagen degradation during orthodontic tooth movement, and our in vitro study examined the secretion of CAB and CAL in PDL cells following mechanical stress. PDL cells were subjected to 0.5, 1.0, 2.0, or 3.0 g/cm2 of compression force or an increase in surface area by tension force of 0.28%, 0.95%, 1.72%, or 2.50% for 24 hr. For detection of CAB and CAL in conditioned medium, commercially available ELISA kits were used. We found compression and tension significantly increased the secretions of both CAB and CAL in PDL cells, which were exhibited in a time- and force magnitude-dependent manner. The compression-stimulated secretion of CAB was increased approximately 3-fold and that of CAL 4-fold, as compared with the control. Further, tension-stimulated secretion of CAB was increased by 1.5-fold and that of CAL 2-fold compared with the control. When analyzed using a semiquantitative polymerase chain reaction assay, CAB and CAL mRNA were increased in response to both compression and tension forces. These findings demonstrated that mechanical stress (compression and tension forces) causes an increase in secretion of CAB and CAL in PDL cells in vitro.
Article
A wide variety of laboratory apparatuses have been devised for mechanical stimulation of cell and tissue cultures. This article reviews the functional attributes of several dozen systems developed for that purpose, including their major advantages and disadvantages. These devices can be categorized in terms of their primary loading modality: compression (hydrostatic pressure or direct platen contact), longitudinal stretch, bending, axisymmetric substrate bulge, in-plane substrate distention, fluid shear stress, or combined substrate distention and fluid shear.
Article
The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.