Okubo et al.indd

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Correspondence: Yasunori Okubo, Department of Oral and Maxillofacial Surgery,Graduate School of Medi-
cine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Tel: +81-75-751-3405;
Fax: +81-75-761-9732; Email: okubo@kuhp.kyoto-u.ac.jp
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm

Accelerators of Osteogenesis by Recombinant Human Bone
Morphogenetic Protein-2
Yasunori Okubo1, Kenji Kusumoto2 and Kazuhisa Bessho1
1Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University,
54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. 2Department of Plastic and
Reconstructive Surgery, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka,
570-8507, Japan.

Abstract: Bone morphogenetic protein (BMP) appears to be one of the most promising cytokine and for clinical use in
reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fi broblast growth factor (bFGF),
FK506, elcatonin, and hyperbaric oxygenation (HBO) on osteoinduction by recombinant human bone morphogenetic
protein-2 (rhBMP-2), 2 or 5 µg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation,
the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and
histologically. The amount of new bone in the treatment group was signifi cantly greater than that in the control group. The
alkaline phosphatase activity and calcium content in the treatment group were signifi cantly higher than those in the control
group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by
rhBMP2. These results may be useful when BMP is applied clinically in near future.

Keywords: FK506, Basic fi broblast growth factor (bFGF), elcatonin, hyperbaric oxygenation (HBO), Recombinant human
bone morphogenetic protein.

Introduction
Bone morphogenetic protein (BMP) appears to be one of the most promising biomaterial and for
clinical use in reconstructive surgery for bony defects and augmentation. Therefore, BMP is noted in
the fi eld of bone reconstructive surgery. Since recombinant human bone morphogenetic protein-2
(rhBMP-2) has become available, many animal studies on osteoinduction by rhBMP-2 have been
performed (Fujimura et al.1995; Okubo et al. 1999; Okubo et al. 2000). However for clinical applica-
tion of rhBMP-2 to tissue with low blood supply tissue, e.g. scarred tissue or irradiated tissue, it is
necessary to evaluate the factors that enhance osteoinduction by rhBMP-2.

In the present study, the basic mechanism of osteoinduction by rhBMP-2 and preclinical studies are
discussed and reviewed mainly referring to our previous research regarding accelerators of osteogenesis
and related studies.

Effect of basic Fibroblast Growth Factor (bFGF)
FGF has various effects on cellular proliferation and it has a strong proliferative affected on endothelial
cells, osteocytes and chondrocytes (Connolly et al. 1987; Gospodarowicz et al. 1987; Globus et al.
1988). In addition, FGF and transforming growth factor ß (TGFß) are co-active on proliferating chon-
drocytes and osteoblasts (Iwamoto et al. 1989; Inoue et al. 1989; Nakamura et al. 1995). We evaluated
the effect of FGF on the osteoinductive activity by rhBMP-2. BMP-2 (Genetics Institute, MA) was
provided by Yamanouchi Pharmaceutical Co. Ltd. (Tokyo, Japan). It was dissolved in a buffer (pH 4.5)
containing 5 mM glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80, and stored at −80°C.
FGF-was provided by Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan). Type I collagen solution
(3 mg/ml, pH 3.0) (Cellmatrix LA®; Nitta Gelatin Inc., Osaka, Japan) was used as the carrier for BMP-2
and FGF-2. This collagen was purifi ed from fresh porcine skin, and the telopeptide was removed by
proteolytic digestion. BMP-2 (2 µg) and 0, 16, 80 and 400 ng, and 2, 10 and 50 µg of FGF-2 (n = 10

Drug Target Insights 2007: 2 55–60

Okubo et al

in each group) were mixed with 3 mg of type I
collagen. The mixtures were lyophilized (0.04
Torr) (Eyela® type FDU-830; Tokyo Rika Inc.,
Tokyo, Japan) and shaped into discs (4 mm diam-
eter; 1.5 mm thickness).

Seventy male 10-week-old Wistar rats weighing
240–260 g were used. They were divided into
seven groups. All the rats were anaesthetized with
an intraperitoneal injection of sodium pentobar-
bital (4.0 mg solidus 100 g body weight). After
disinfecting the operative region and incising the
skin, a disc containing BMP-2 (2 µg), bFGF (0,
16, 80 and 400 ng, and 2, 10 and 50 µg in each)
and type I collagen (3 mg) were implanted into
the right calf muscles of the rats. These seven
groups (n = 10 in each group) consisted of group 1
(control), BMP-2 + FGF (0 ng) + type I collagen;
group 2, BMP-2 + FGF (16 ng) + type I collagen;
group 3, BMP-2 + FGF (80 ng) + type I collagen;
group 4, BMP-2 + FGF (400 ng) + type I collagen;
group 5, BMP-2 + FGF (2 µg) + type I collagen;
group 6, BMP-2 + FGF (10 µg) + type I collagen;
and group 7, BMP-2 + FGF (50 µg) + type I
collagen. They were fed rodent chow (Certifi ed
diet MF; Oriental Koubo Inc., Tokyo, Japan) for
the period of the study. Three weeks after the
operation, all the animals were killed with an
intraperitoneal injection of excess sodium pento-
barbital. The specimens with peripheral tissues
were fi xed in 10% formalin neutral buffer solution
(pH 7.4), demineralized in EDTA, and embedded
in paraffi n. They were cut into 4 µm-thick sections
and stained with hematoxylin and eosin. The
samples for quantitative analysis were weighed
and then homogenized in 0.25 M sucrose in a
Polytron homogenizer (Bio-Mixer; type ABM,
Nissei Inc., Osaka, Japan). The sediment was
demineralized in 0.5 N HCl, and the calcium (Ca)

content of the soluble fraction was determined by
the orthocresolphthalein complexone method.
The alkaline phosphatase (ALP) activity and total
protein in the resultant supernatant were deter-
mined by the 4-nitrophenylphosphate method.
The Ca content (µg/mg of tissue) and the ALP
activity (IU/mg of protein) were used as indices
of bone formation. The treatment of each animal
was conducted according to the 1988 guidelines
for animal experiments at Kyoto University.

Three weeks after implantation, ALP was
increased in the 16, 80 and 400 ng FGF-2-treated
groups but decreased in the 50 µg FGF-2-treated
group. Histological examination revealed
increased bone formation in the 16, 80 and 400 ng
FGF-2-treated groups (Table 1). These results
show that combined treatment with FGF-2 and
BMP-2 has a biphasic effect on osteoinductive
activity, i.e. it increases with low doses of FGF-2
and decreases with high doses of FGF-2 (Fujimura
et al. 2002).

Effect of FK506
FK506 has generally been used as an immunosup-
pressant for organ transplantation. We evaluated
the effect of FK506 on osteoinduction by rhBMP-2.
One hundred and twenty male Wistar rats (10
weeks old and weighting 230–250 g) were
randomly divided into the following four groups
of 30 rats each: 1) The short-term FK506 group
(SFG) received a daily intramuscular (i.m.) injec-
tion of 0.1 ml of FK506 (1 mg/kg) from 2 days
before the implantation of lyophilized specimens
until the day of implantation. Then the animals
received a daily injection of 0.1 ml of saline i.m.
from the day of implantation until sacrifi ce. 2) The
medium-term FK506 group (MFG) received a

Table 1.

Dose of FGF Histological fi ndings ALP activity Radiological fi ndings
percentage of bone area (%) IU/mg protein rediopacity area (mm2)

0 15.2 (0.15) 0.88 (0.27) 2.8 (0.6)
16 ng 30.4 (4.6)* 6.1 (2.6)* 5.4 (1.6)*
80 ng 28.2 (4.3)* 4.0 (1.1)* 7.7 (1.8)*
400 ng 32.5 (9.0)* 17.1 (4.3)* 9.0 (2.3)*
2 µg 19.6 (5.9) 0.69 (0.43) 3.5 (1.9)
10 µg 7.6 (6.5) 0.62 (0.34) 1.7 (0.8)
50 µg 0 0.17 (0.1) 0
Data are means (SD).

*Signifi cant difference at p < 0.05, compared with FGF on g group.

Drug Target Insights 2007: 2

Accelerators of Osteogenesis by rhBMP-2

the SFG than in the other groups. Twenty-one days
after implantation, the trabecular bone area was
increased in the CG, but not in the MFG. In the
SFG and LFG, it was decreased at the border of
the implant, and fatty marrow occupied most of
the marrow tissue. Twenty-one days after implan-
tation, the ALP activity was 6.37 ± 0.37 in the CG,
3.34 ± 0.19 in the SFG, 5.40 ± 0.46 in the MFG,
and 1.30 ± 0.24 in the LFG. The values in the SFG,
and LFG were signifi cantly lower than in the CG
and MFG. Twenty-one days after implantation, the
Ca content was 33.81 ± 3.44 in the CG, 18.43 ±
1.94 in the SFG, 24.11 ± 2.61 in the MFG, and
15.24 ± 1.96 in the LFG. Values in the SFG and LFG
were signifi cantly lower than in the CG (Table 2).
These fi ndings demonstrate that short-term admin-
istration of FK506 promotes early osteoinduction.
However, long-term administration accelerates
both bone formation and bone resorption, and
insuffi cient oxygen supply leads to failure of bone
matrix maturation, resulting in poor bone formation
(Kaihara et al. 2002).

Effect of Elcatonin
Elcatonin is a derivative of eel calcitonin synthe-
sized by substituting an ethylene bond for the
disulfi de bond (Morikawa et al. 1976; Otani et al.
1978; Orimo et al. 1990). It has also been reported
from in vivo and in vitro studies that elcatonin
suppresses bone resorption (Orimo et al. 1990;
Yamamoto I et al. 1981). In the clinical fi eld,
elcatonin is used currently for the treatment of
Paget’s disease, and osteoporosis. However, the
role of this hormone in producing an anabolic
effect on osteoblasts is not yet fully understood.
We evaluated the effect of elcatonin on osteoin-
duction by rhBMP-2, especially the anabolic
effect on osteoblasts.

daily injection of 0.1 ml of FK506 (1 mg/kg i.m.)
from 2 days before implantation until 7 days after
implantation. Then a daily injection of 0.1 ml of
saline i.m. was given for the next 7 days until
sacrifi ce. 3) The long-term FK506 group (LFG)
received a daily injection of 0.1 ml of FK506
(1 mg/kg i.m.) from 2 days before implantation
until sacrifi ce. 4) The control group (CG) received
a daily injection of 0.1 ml of saline i.m. from 2
days before implantation until sacrifi ce.

rhBMP-2 was obtained from W. Sebald (Würz-
burg University, Germany, Ruppert et al. 1996).
Atelopeptide type-I collagen (CL) (pH 3.0 was
used as the carrier). rhBMP-2 (5 µg) was mixed
with 3 mg of CL and was lyophilized (EYELA
FDU-830; Tokyo Rikakikai Inc., Tokyo, Japan).
Then the material was compressed in a syringe
to form discs (4 mm in diameter and 1.5 mm
thick).

Rats were anesthetized with intraperitoneal
sodium pentobarbital (5.0 mg per 100 g of body
weight) and lyophilized disc specimens were
implanted into the right calf muscle. After implan-
tation, the fascia and skin were sutured.

FK506 (Tacrolimus; Fujisawa Pharmaceutical
Co., Ltd., Osaka, Japan) was suspended in saline
and injected into the left calf muscle of each rat
(1 mg/kg/day). This dose has already been given
intramuscularly in rat organ transplantation models
(Akahane et al. 1999).

In the radiographic fi ndings, the area of the
shadows at 21days after implantation was in the
order of CG > MFG > SFG > LFG. In the histo-
logical fi ndings, fourteen days after implantation,
new bone surrounded by immature mesenchymal-
type cells was present at border of the implant
around almost the entire circumference in every
group. However, cartilage was still observed in the
LFG. The immature new bone area was larger in

Table 2.

FK506 Ca content ALP activity
µg/mg tissue IU/mg protein
day 7 day 14 day 21 day 7 day 14 day 21
SFG 0.15 (0) 33.5 (1.1)* 18.4 (1.9)* 2.4 (0.4)* 1.9 (0.2) 3.3 (0.2)*
MFG 0.14 (0) 24.4 (3.1) 24.1 (2.6) 2.3 (0.2)* 2.4 (0.3) 5.4 (0.3)
LFG 0.13 (0) 13.3 (1.0)* 15.4 (1.9)* 2.4 (0.3)* 3.1 (0.3) 1.3 (0.4)*
CG 0.1 (0) 23.4 (2.9) 33.8 (3.4) 1.8 (0.4) 2.7 (0.4) 6.3 (0.4)
Data are means (SD).

*Signifi cant difference at p < 0.05, compared with CG.

Drug Target Insights 2007: 2

Okubo et al

Twenty Wistar rats (male; 10 weeks old; weight
240–260 g) were used. Four groups, consisting of
a high elcatonin group (HEG), medium elcatonin
group (MEG), low elcatonin group (LEG) and
control group (CG), were established with 5 rats
in each group.

rhBMP-2 derived from E. coli was obtained
from W. Sebald (Würzburg University, Germany,
Rupport et al. 1996). CL (pH 3.0) was used as a
carrier. Five µg of rhBMP-2 mixed with 3 mg of
CL was lyophilized (EYELA FDU-830; Tokyo
Rikakikai Inc., Tokyo, Japan). The material was
compressed in the injection syringe to discal form
(4 mm in diameter, 1.5 mm in thickness).

As the pharmacological agent, 14-day doses of
elcatonin (Elcitonin®; Asahi Chemical Industry
Co., Ltd., Tokyo, Japan) were prepared, 80 U for
HEG, 8 U for MEG, and 0.8 U for LEG. The total
volume of the elcatonin agent in physiological
saline solution was 0.2 ml for each rhBMP-2
implanted group. The elcatonin solution was placed
into a mini-osmotic pump (alzet® model 2002;
ALZA Co., CA), that would pump out the solution
continuously at a constant rate of 0.5 µl/hour for
14 days. For CG, only 0.2 ml of physiological
saline was placed into the mini-osmotic pump.

All rats were anesthetized with intraperitoneal
administration of sodium pentobarbital. The
lyophilized discal specimens were implanted into
a right calf muscle. After the implantation, the
fascia and skin were sutured. A one-cm-long inci-
sion was made in the paramedian abdominal wall,
including the skin, muscle, and the peritoneum and
the mini-osmotic pump, which had been previously
prepared for each group, was inserted into the
peritoneal space. The abdominal wall was then
closed by suturing layer by layer.

Twenty-one days after the implantation, all rats
were sacrifi ced with an overdose of sodium pento-
barbital. The implanted region was excised with
the surrounding tissue and a radiograph was taken.
Each excised specimen was removed and then cut
into 2 halves, one for histological analysis and the
other for biochemical analysis.

The soft tissue radiographs revealed opaque
shadows morphologically identical to the implanted
specimens. These opaque shadows were observed
in each of the specimens of all groups.

In HEG, there was a relatively vigorous trabec-
ular bone on the outermost edge of the implanted
material. Lining osteoblasts were observed around
the trabecular bone. Bone marrow, including

angioid tissue, was rich at the central side of the
trabecular bone. Fatty marrow occupied a major
part of the marrow tissue. In MEG, there was
trabecular bone on the outermost edge of the
implanted material. The trabecular bone was
thinner than that in HEG. Bone marrow included
fatty tissue. Collagen fi bers remained at the center
of the implanted material. In LEG, less trabecular
bone and marrow were observed compared to the
respective amounts in MEG and HEG. At the
central side of the newly formed trabeculae, a small
area of bone marrow was observed. In CG, espe-
cially, the amount of trabecular bone was clearly
less than in the other groups and few osteoblasts
were observed. The values of ALP activity on day
21 were 5.87 ± 0.43 (mean ± SD IU/mg protein)
in CG, 6.41 ± 0.37 in LEG, 7.10 ± 0.37 in MEG,
and 7.37 ± 0.50 in HEG (Table 3). The value was
highest in HEG and lowest in CG. The values of Ca
content on day 21 were 25.0 ± 1.61 (mean ± SD
µg/mg tissue) in CG, 26.6 ± 0.96 in LEG, 29.0 ±
0.60 in MEG, and 31.3 ± 1.56 in HEG. The ALP
activity and Ca content in HEG were highest and
lowest in CG. In HEG and MEG, the values of
ALP activity and Ca content were signifi cantly
lower than in CG and LEG (p < 0.01). These results
suggested that elcatonin is effective in enhancing
osteoinduction by rhBMP-2, and that elcatonin has
an anabolic effect on osteoblasts in addition to an
anti-resorptive effect (Okubo et al. 2000).

Effect of Hyperbaric Oxygenation
(HBO)
Hyperbaric oxygen (HBO) therapy is an oxygen-
ation method use to treat anoxia by increasing
dissolved oxygen. HBO therapy has been shown
to increase collagen synthesis, capillary ingrowth
(Hunt et al. 1972), neovascularization, and osteo-
genesis (Nilson et al. 1988). Recently, the use of

Table 3.

Elcatonin Ca content ALP activity
µg/mg tissue IU/mg protein
LEG 26.6 (1.0) 6.4 (0.4)
MEG 29.0 (0.6)* 7.1 (0.4)*
HEG 31.3 (1.6)* 7.3 (0.5)*
CG 25.0 (1.6) 5.9 (0.4)
Data are means (SD).

*Signifi cant difference at p < 0.01, compared with CG.

Drug Target Insights 2007: 2

Accelerators of Osteogenesis by rhBMP-2

HBO therapy to improve the rate of bone healing
in conjunction with surgery for dental implant,
osteomyelitis and osteonecrosis has increased. We
compared osteoinduction by rhBMP-2 with and
without HBO therapy. Thirty Wistar rats were
randomly assigned to an HBO group and a control
group of 15 rats each. CL was used as a carrier.
Five µg of rhBMP-2 mixed with 3 mg of CL was
lyophilized (EYELA FDU-830; Tokyo Rikakikai
Inc., Tokyo, Japan). The material was compressed
in an injection syringe to discal form (4 mm in
diameter, 1.5 mm in thickness).

All rats were anaesthetized by intraperitoneal
administration of sodium pentobarbital (5.0 mg per
100 g of body weight). Following disinfection of
the operative region, the lyophilized discal speci-
mens were implanted into a right calf muscle
pouch. The fascia and skin were sutured.

The rats in the HBO group were placed in a
pressure chamber (KHO-100; Kawasaki Engi-
neering Inc., Hyogo, Japan) and exposed to a pres-
sure of 2.0 ATA 100% inspired fl ow oxygen for 60
minutes everyday for 3, 7, and 21 days. During the
fi rst 15 minutes of HBO therapy, the pressure was
increased to 2.0 ATA, and decompression proceeded
for 15 minutes after the treatment.

Three, 7, and 21 days after the implantation, the
rats were sacrifi ced by an overdose of sodium
pentobarbital. Then the implanted region was
excised together with the surrounding tissue and
soft X-rayed. Each excised specimen was removed
and cut into 2 halves, one for histological analysis
and the other for biochemical analysis.

On day 21, soft X-ray revealed opaque shadows
morphologically identical to the implanted speci-
mens in both groups. The oval shadows in the HBO
group were larger with slighter high radio-opacity
than those in the control group. On day 7 after the
implantation, in the HBO group, cartilage tissue was
induced at the outer edge of implanted material. In
the control group, no cartilage or chondrocytes were
detected in these fi ndings. On day 21 after the

implantation, new bone formation was found in both
groups. Around the trabecular bone, lining osteo-
blasts and a few osteoclasts were observed in both
groups. In the control group, trabecular bone tissue
was observed at the outer edge of the implanted
material. In the HBO group, the trabecular area was
greater than that in the control group. The bone
marrow area in the HBO group, including fatty
marrow in part, was wider than that in the control
group. The trabecular area bone in the HBO group
was wider than that in the control group. The results
of the micrograph analysis of the trabecular area and
the percentage of the trabeculum occupying the
overall area are summarized in Table 4.

The ALP activity and Ca content of the HBO
group and the control group are shown in Table 3.
The ALP activity and the Ca content in the HBO
group were signifi cantly higher than those in the
control group on days 7 and 21.

The present results suggest that HBO therapy
accelerates the activity and rate of osteoinduction
by rhBMP-2, since hyperbaric oxygenation may
enhance the effects of rhBMP-2 on the differen-
tiation from immature mesenchymal cells to osteo-
blasts (Okubo et al. 2001).

Conclusions
In skeletal reconstruction using BMPs, lower
amount of BMPs had better induce more bony
tissue. Therefore, some materials have been studied
in vivo for the promotion of osteoinduction. To
date, FGF, FK506, elcatonin, HBO, and prosta-
glandin E1 and other materials have been studied
as the accelerators in our group. These results may
be useful when BMP is applied clinically in near
future.

Acknowledgements
This work was supported in part by Grant-in-Aid for
Young Scientists B(No. 17791453) the Japanese
Ministry of Education, Science, Sports and Culture.

Table 4.

Histological fi ndings Ca content ALP activity
percentage of bone area (%) µg/mg tissue IU/mg protein
HBO group 30.1 (2.2)* 41.0 (3.6)* 7.5 (0.8)*
Control group 16.9 (1.2) 24.0 (2.9) 3.8 (1.2)
Data are means (SD).

*Signifi cant difference at p < 0.05, compared with FGF on g group.

Drug Target Insights 2007: 2

Okubo et al

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/EndPage -1
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/MaxSubsetPct 100
/Optimize true
/OPM 1
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/PreserveEPSInfo true
/PreserveHalftoneInfo false
/PreserveOPIComments false
/PreserveOverprintSettings true
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/TransferFunctionInfo /Apply
/UCRandBGInfo /Preserve
/UsePrologue false
/ColorSettingsFile ()
/AlwaysEmbed [ true
]
/NeverEmbed [ true
]
/AntiAliasColorImages false
/DownsampleColorImages true
/ColorImageDownsampleType /Bicubic
/ColorImageResolution 300
/ColorImageDepth -1
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/EncodeColorImages true
/ColorImageFilter /DCTEncode
/AutoFilterColorImages true
/ColorImageAutoFilterStrategy /JPEG
/ColorACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/ColorImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000ColorACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000ColorImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasGrayImages false
/DownsampleGrayImages true
/GrayImageDownsampleType /Bicubic
/GrayImageResolution 300
/GrayImageDepth -1
/GrayImageDownsampleThreshold 1.50000
/EncodeGrayImages true
/GrayImageFilter /DCTEncode
/AutoFilterGrayImages true
/GrayImageAutoFilterStrategy /JPEG
/GrayACSImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/GrayImageDict <<
/QFactor 0.15
/HSamples [1 1 1 1] /VSamples [1 1 1 1]
>>
/JPEG2000GrayACSImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/JPEG2000GrayImageDict <<
/TileWidth 256
/TileHeight 256
/Quality 30
>>
/AntiAliasMonoImages false
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/MonoImageDownsampleType /Bicubic
/MonoImageResolution 1200
/MonoImageDepth -1
/MonoImageDownsampleThreshold 1.50000
/EncodeMonoImages true
/MonoImageFilter /CCITTFaxEncode
/MonoImageDict <<
/K -1
>>
/AllowPSXObjects false
/PDFX1aCheck false
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/PDFXTrimBoxToMediaBoxOffset [
0.00000
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0.00000
0.00000
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/PDFXSetBleedBoxToMediaBox true
/PDFXBleedBoxToTrimBoxOffset [
0.00000
0.00000
0.00000
0.00000
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/PDFXOutputIntentProfile ()
/PDFXOutputCondition ()
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>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice