Biomaterials 33 (2012) 7057e7063

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Osteogenesis induced by a bone forming peptide from the prodomain region

of BMP-7
Hyung Keun Kim a, b, Ji Hyun Kim a, b, Dae Sung Park a, b, Kyung Soon Park b, Seong Soo Kang c, Jun Sik Lee d,
Myung Ho Jeong a, Taek Rim Yoon a, b, *
a
Heart Research Center and Cardiovascular Research Institute of Chonnam National University, Gwangju 501 757, Republic of Korea
b
Department of Orthopaedics Surgery, Chonnam National University Hwasun Hospital, Jeonnam 519 809, Republic of Korea
c
College of Veterinary Medicine, Chonnam National University, Gwangju 500 757, Republic of Korea
d
Department of Biology, College of Natural Science, Chosun University, Gwangju 501 759, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone
Received 23 April 2012 resorption. Many researchers have tried to develop adjuvants as specific suppressors of bone resorption
Accepted 22 June 2012 and stimulators of bone formation for therapeutic purposes in patients with osteoporosis. Therefore,
Available online 15 July 2012
specific stimulators on bone formation are one of therapeutic significance in the treatment of osteo-
porosis. Until now, the regulation of bone generation has been the focus of bone morphogenetic protein-
Keywords:
7 (BMP-7) investigation from mature form. However, new peptides from immature form which has
Bone regeneration
osteogenic activity has not been reported and developments of these proteins are still remained. In this
Bone tissue engineering
Osteogenesis
study, we found a new peptide sequence, called bone forming peptide-1 (BFP-1) and have more high
BMP (bone morphogenetic protein) activities of osteogenic differentiation compared with BMP-7. BFP-1-treated multipotent bone marrow
stromal stem cells (MBSCs) induced the expression levels and activity of alkaline phosphatase (ALP).
Moreover, BFP-1 enhanced the levels of CD44, CD47 and CD51 expression as well as increased Ca2þ
content in MBSCs. In current study, radiography at 8 weeks revealed that BFP-1 pretreated-MBSC
transplanted animals had strongly increased bone formation compared to that in the BMP-7 pretreated
MBSC transplanted animals. Our finding indicates a new insight into peptides from the immature region
of BMP-7 can also be useful in the development of adjuvant therapies for bone-related diseases.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Bone-related disease such as osteoporosis refers to medical
conditions that affect the bone and bone mass. The bone mass
involves a complex process characterized by balance between
osteoblast formation and osteoclast resorption [1]. Many
researchers have tried to develop adjuvants as specific suppressors
of bone resorption and stimulators of bone formation for thera-
peutic purposes in patients with osteoporosis.
Bone morphogenetic proteins (BMPs), the most potent
osteoinductive growth factors, are members of the transforming
growth factor (TGF)-b superfamily, which includes the TGF-
b families, as well as myostatin, growth, and differentiation factors
* Corresponding author. Department of Orthopaedics Surgery, Center for Joint
Disease, Chonnam National University Hwasun Hospital, Jeonnam 519 809,
Republic of Korea. Tel.: þ82 61 379 7677; fax: þ82 61 379 7819.
E-mail addresses: tryoon@jnu.ac.kr, chemokines@naver.com (T.R. Yoon).
[2]. BMPs are expressed in a variety of regions and were originally
investigated due to their ability to regulate new bone formation
[3e5]. Current studies have indicated that BMP-2 and BMP-7 are
also involved in various biological processes, including cell differ-
entiation, proliferation, migration, and bone regeneration [6e8].
BMP-7 plays a role reducing cold ischemic injury and is expressed
in many types of cancers. However, BMP-7 also plays a crucial role
in osteogenic differentiation and proliferation during new bone
generation [9,10]. In addition, the TGF-b superfamily of factors are
secreted as precursors that are approximately four times larger
than the mature form and the sequences of the precursor portions
show limited sequence homology [11e13]. The mature protein is
represented in the terminal domain and appears to be cleaved by
a trypsin-like protease. The pro region appears to be essential for
proper in vivo folding of the mature region. The mature domains of
TGF-b-like proteins are highly conserved across animal species and
are involved in diverse activities in different cell types. Most
described BMP genes are closed in the Vgr-1 gene in their mature
forms such as BMP-2, BMP-3 and BMP-7. Moreover, most BMP

0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2012.06.036
7058 H.K. Kim et al. / Biomaterials 33 (2012) 7057e7063
peptides with biological activity are focused on the mature region
of BMP-7. Until now, the regulation of bone generation has been the
focus of BMP-7 investigations in its mature form and new peptides
from the immature form, which have osteogenic activity, have not
been reported.
In this study, we developed new peptide sequences with oste-
ogenic activity from the immature region of the BMP-7. Interest-
ingly, we found that one of peptide sequences from the immature
region has osteogenic activity more than that of the mature BMP-7
region and induces osteogenesis. This peptide is called bone
forming peptide-1 (BFP-1). This finding suggests new insight into
the osteogenic activity of BFP-1 and its effect on osteoblasts, and
that peptides from the immature region of BMP-7 can also be useful
in the development of adjuvant therapies for bone-related diseases.
2. Materials and methods
2.1. Osteogenic differentiation
Multipotent bone marrow stromal stem cells (MBSCs or D1 cells) were cultured
as previously described [14]. D1 cells were purchased from the American Type
Culture Collection (ATCC, Manassas, VA) and maintained in DMEM containing 10%
fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY) and antibiotics (Gibco). Cells
were seeded at 1  104 cells/well and maintained in culture for 3 days in a humid-
ified 5% CO2 atmosphere at 37  C. Experiments were performed after the cells had
reached about 80% confluence. To induce osteogenic differentiation, the culture
medium was changed at 3 days to osteogenic differentiation medium (ODM; DMEM
supplemented with 50 mg/ml ascorbic acid, 108 M dexamethasone, and 10 mM b-
glycerol phosphate; all from SigmaeAldrich, St. Louis, MO). After culture for another
3 days, one group was cultured only in ODM, whereas another group was cultured in
ODM plus BFP-1 (0.01, 0.1 or 1 mg/ml). Cells were then analyzed 24 and 48 h later.
2.2. Synthesis and purification of the BFP-1
Peptides were synthesized by Fmoc solid-phase peptide synthesis using ASP48S
(Peptron Inc. Daejon, Korea) and purified by reverse-phase high performance liquid
chromatography using a Vydac Everest C18 column (250 mm  22 mm, 10 mm).
Elution was carried out with a watereacetonitrile linear gradient (3e40% [v/v] of
acetonitrile) containing 0.1% (v/v) trifluoroacetic acid. The molecular weight of the
purified peptide was confirmed using liquid chromatography/mass spectroscopy
(Agilent HP 1100 series, Santa Clara, CA).
2.3. Cell viability
Surviving cells was counted using the MTT method (3-(4,5-dimethlythiazol-
2yl)-2.5-diphenyltetrazolium bromide). MTT (20 ml in 7.2 mM phosphate buffer
solution, pH 6.5) was added to each well, and the plates were incubated for an
additional 3 h. After removing the solution from the wells, dimethyl sulfoxide
(DMSO) was added to dissolve the formazan products, and the plates were shaken
for 5 min. The absorbance of each well was recorded from a microplate spectro-
photometer at 570 nm.
2.4. Alizarin red-S staining
Calcification deposits on cells were quantified as described by Chen [15]. Briefly,
cell cultures were washed twice with distilled water, fixed for 1 h in ice-cold 70%
ethanol, and rinsed twice with deionized water. Cultures were stained for 10 min
with Alizarin red-S solution, and excess dye was removed gently with running water.
Calcification deposits, which appeared bright red, were identified by light micros-
copy and photographed. Osteogenic differentiation was quantified by determining
densities and areas of Alizarin red-S staining using an image analysis program (Multi
Gauge V3.0, Fujifilm, Japan).
2.5. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time-PCR
analysis
To assess the effects of BFP-1 on the transcription of genes encoding alkaline
phosphatase (ALP) (50 -ACA CCT TGA CTG TGG TTA CTG CTG A-30 ; 50 -CCT TGT AGC
CAG GCC CGT TA-30 ), osteocalcin (50 -GAG GGC AAT AAG GTA GTG AAC AGA-30 ; 50 -
AAG CCA TAC TGG TCT GAT AGC TCG-30 ), Runx 2 (50 -ACA AAC AAC CAC AGA ACC ACA
AGT-30 ; 50 -GTC TCG GTG GCT GGT AGT GA-30 ) and the housekeeping enzyme glyc-
eraldehyde-3-phosphate dehydrogenase (50 -AAA TGG TGA AGG TCG GTG TG-30 ; 50 -
TGA AGG GGT CGT TGA TGG-30 ), cells grown to 70% confluence on plates with/
without BFP-1 were homogenized using TRIzol reagent (Life Technologies, Carlsbad,
CA). Total RNA was isolated, and 0.5 mg RNA aliquots were reverse transcribed in
20 ml buffer containing 5 AMV reverse transcriptase; 2.5 mM poly dT; 1 mM each of
dATP, dCTP, dGTP, and dTTP; 20 U of RNAse inhibitor; and 20 U of AMV RT. Reverse
transcription was performed using the following conditions: initial incubation at
room temperature for 10 min and then 42  C for 15 min, 97  C for 5 min, and 5  C for
5 min in a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA). Aliquots
of cDNA were amplified in AccuPowerÒ GreenStar qPCR premix (Bioneer Co.,
Daejeon, Korea) using an ExiCyclerÔ 96 Real-time Quantitative Thermal Block
(Bioneer Co.).
2.6. Western blot analysis
Cell were harvested, washed twice with ice-cold PBS and lysed in RIPA buffer
(SigmaeAldrich) supplemented with protease inhibitor cocktail (SigmaeAldrich) for
1 h on ice with vortexing every 10 min. Lysates were centrifuged at 8000 g for
20 min to remove insoluble material, and the protein concentrations in supernatants
were determined using Bradford assay kits with bovine serum albumin (BSA) as the
standard. Equal amounts of protein were separated on 10 or 12% sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis gels, and blots were subsequently
transferred to nitrocellulose membranes (Hybond C, Amersham Life Science,
Uppsala, Sweden). Membranes were treated with primary antibodies against ALP,
osteocalcin, Runx 2, and b-actin (Santa Cruz Biotechnology Inc., CA), and secondary
antibodies were used at 1:500e1:1000. Proteins were detected by an enhanced
chemiluminescence reagent using a commercial kit (Amersham Life Science).
Protein expression was quantified by determining blot densities with an LAS-3000
system (Fujifilm, Tokyo, Japan).
2.7. Immunofluorescence staining
D1 cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for
15 min, permeabilized with 0.1% Triton X-100 for 15 min and then blocked with 5%
BSA in PBS for 30 min. Coverslips were then incubated with primary antibodies
against mouse CD44, CD45, CD47, CD51, ALP, osteocalcin or Runx 2 (eBioscience,
San Diego, CA) at a dilution of 1:100, and secondary antibodies at 1:200, both at
room temperature for 1 h. Cells were then washed with PBS and mounted in 70%
glycerol. Photomicrographs were obtained using an Olympus BX50 fluorescence
microscope (Tokyo, Japan). Nuclei were stained with DAPI solution.
2.8. Calcium assays
The measurement of calcium content followed a previous reported method [16].
Calcium was assayed based on phenolsulfonephthalein dye in a QuantiChrom
calcium assay kit (Gentaur, Voortstraat, Belgium), which forms a very stable blue
colored complex specifically with free calcium. Color intensity, measured at 612 nm
using an Infinite M200 microplate reader, is directly proportional to the calcium
concentration in the sample.
2.9. Flow cytometric analysis
MBSCs (0.5  105) were incubated in staining buffer (PBS containing 0.5% FBS
and 0.1% sodium azide) containing anti-CD44, anti-CD45, anti-CD47 and anti-CD51
(eBioscience) for 30 min in ice. Cells that stained with the appropriate isotype-
matched Ig were used as negative controls. After staining, cells were fixed with
2% paraformaldehyde and analyzed using a FACSCalibur instrument equipped with
CellQuest software (BD Biosciences, San Diego, CA).
2.10. Cell transplantation
BFP-1-treated osteogenically differentiated and BMP-7 treated-MBSCs were
suspended in DMEM at a concentration of 1  106 cells/200 ml. Cells were subcu-
taneously implanted into the right dorsoposterior position region of C57BL/6 mice
and subjected to point projection digital radiography at 26 kV for 3 s using an MX-20
digital microradiography system (Faxitron Bioptics, Lincolnshire, IL). X-ray images
were processed using DicomWorks software.
2.11. Histological evaluation
Tissues were fixed in 10% neutral buffered formalin, followed by decalcification
in 10% EDTA. Paraffin-embedded samples were sectioned to a thickness of 8 mm with
a microtome. The sections were then floated in a water bath at 40  C, positioned on
poly-L-lysine-coated microscope slides, and baked for 2 h at 37  C. The sections were
dewaxed in xylene and rehydrated in various ethanol concentration baths for H&E
staining. Nuclei were stained with hematoxylin for 5 min and with eosin for 1 min.
The sections were then covered with Permount mounting medium and cover-
slipped.
2.12. Statistical analysis
Results are presented as means  standard deviations. Data were analyzed by
one-way analysis of variance followed by Duncan’s post-hoc test using SPSS version
11.0 (Chicago, IL). A p value of less than 0.05 was considered statistically significant.
 H.K. Kim et al. / Biomaterials 33 (2012) 7057e7063 7059

3. Results 3.3. Osteogenic biomarkers activity of BFP-1

3.1. Synthesis of the BFP-1
To investigate the development of new peptide sequences with
osteogenic activity from the immature region of the BMP-7, we
tested the effects of osteogenic differentiation by several peptide
sequences from the immature region of BMP-7 and found that the
GQGFSYPYKAVFSTQ sequence (called BFP-1) had osteogenic effects
in a cell culture system compared with other sequences (data not
shown). As shown in Fig. 1, the complete BFP-1 peptide synthesis
was subsequently cloned (Genbank accession number NM_001719).
3.2. Osteogenic differentiation of BFP-1
To determine whether BFP-1 could be used in osteoporosis
disease therapy as an adjuvant instead of BMP-7, we investigated
the effect of BFP-1 on osteogenic differentiation in MBSCs. First, we
investigated the cytotoxic effect of BFP-1 in MBSCs. As shown in
Fig. 2A, we found that BFP-1 had no cytotoxic effects; thus, the BFP-
1 concentration was increased to >5 mg/ml. MBSCs were treated
with various concentrations of BFP-1 during the initial phase of
osteogenic differentiation. Interestingly, BFP-1 treatment during
the initial phase of differentiation resulted in significantly induced
osteogenic differentiation of MBSCs compared with that of BMP-7
treatment (Fig. 2B).
Next, we investigated whether BFP-1 treatment is sufficient to
enhance osteogenic activity biomarkers such as ALP, Ca2þ content
and osteogenic activity-related gene expression. As shown in
Fig. 3A, BFP-1 increased ALP activity and Ca2þ content in MBSCs in
a dose-dependent manner. To understand the action of BFP-1 on
MBSCs at the molecular level, we determined the effects of BFP-1
on the expression of genes involved in osteogenesis, including
Col-I, ALP and Runx 2. Quantitative real-time PCR analysis showed
that both Col-I, ALP and Runx 2 expression increased during oste-
ogenic differentiation in BFP-1-treated MBSCs compared with that
in ODM-treated MBSCs (Fig. 3B).
3.4. Osteogenic biomarkers expression of BFP-1
To determine whether BFP-1 had more powerful effects on
osteogenic activity compared with those of BMP-7 and whether it
could be used as an adjuvant osteogenesis inducer, we determined
whether BFP-1 treatment induced protein expression of osteogenic
activity biomarkers compared to that of BMP-7 treatment. Protein
expression was assessed by immunofluorescence staining. As
shown in Fig. 4, we found that BFP-1 significantly induced CD44
(Fig. 4A), osteocalcin (Fig. 4B), ALP (Fig. 4C) and Runx 2 (Fig. 4D)
expression. Moreover, osteocalcin and ALP expression levels were

Fig. 1. Synthesis of the BFP-1 peptide. Peptides were synthesized by Fmoc solid-phase peptide synthesis by ASP48S and purified by reverse-phase high performance liquid
chromatography using a Vydac Everst C18 column. The molecular weight of the purified peptides was measured by liquid chromatography/mass spectroscopy (Agilent HP 1100
series).
7060 H.K. Kim et al. / Biomaterials 33 (2012) 7057e7063

strongly enhanced by BFP-1 treatment in MBSCs more than that by

BMP-7. We next investigated whether BFP-1 induces CD44, CD51
and CD47 expression in MBSCs. In Fig. 4E, BFP-1 treatment induced
expression of CD44, CD51 and CD47 in MBSCs. These results suggest
that BFP-1 might induce osteogenesis by enhancing osteocalcin and
ALP expression. They also provide new insight into the possibility
that BFP-1 could be useful as an osteogenesis inducer for bone-
related diseases.

3.5. Bone formation of BFP-1

We investigated BFP-1 bone formation activity and compared it

with that of BMP-7 treatment in vivo. As shown in Fig. 5, radiog-
raphy at 8 weeks revealed that BFP-1 pretreated-MBSC transplanted
animals had strongly increased bone formation compared to that
in the BMP-7 pretreated MBSC transplanted animals (Fig. 5B).
Moreover, we found a larger population of bone cells in BFP-1
pretreated MBSCs than that in BMP-7 pretreated transplanted
animals (Fig. 5C). These data suggest that bone formation increased
significantly in the BFP-1-pretreated MBSC transplanted animals.

4. Discussion

The development of MBSC- and mesenchymal stem cell-based

Fig. 2. BFP-1 induces osteogenic differentiation. MBSCs were treated with various
concentrations (0.01e5 mg/ml) of BFP-1 during the initial phase of osteogenic differ-
entiation. Viable cell numbers were assessed by the MTT assay after 24 h incubation
(A). Cells were treated with various concentrations (0.01e1 mg/ml) of BFP-1 and BMP-7
individually and assessed by Alizarin red-S staining (B). Data are representative of four
independent experiments. Magnification, 20.
therapy for bone regeneration has progressed over the last
decade. However, few studies have investigated the effect of
recombinant BMPs on parameters of distracted long bones [17e19].
Most strategies developed in bone tissue engineering in the past
three decades have been aimed at regenerating or repairing tissue
with forming elements such as BMP and osteoblasts [3,20e23].
These materials are selected as they are known to induce bone
formation. Applying engineering principles to maintain and/or
enable tissue growth is one of the main goals of tissue engineering
[24]. Accordingly, basic requirements to regenerate new tissue
include cells, scaffolds, and growth factors. Utilizing growth factors

Fig. 3. BFP-1 induces ALP activity, Ca2þ production and the expression of Col-I and Runx 2 in MBSCs. Cells were treated with various concentrations (0.01e1 mg/ml) of BFP-1 during
osteogenic differentiation, and ALP activity was assayed by assessing the release of p-nitrophenol from p-nitrophenyl phosphate using a LabAssayÔ ALP assay kit. Ca2þ content was
measured with a Ca2þ detection kit (A). Total RNA was isolated from control, ODM and BFP-1-treated groups and gene expression levels were assayed by quantitative real-time PCR
(B). Data are mean  SD of four independent experiments. **P < 0.01 for the comparison with the ODM control.
 H.K. Kim et al. / Biomaterials 33 (2012) 7057e7063 7061

Fig. 4. BFP-1 enhances the expression levels of ALP, CD44, osteocalcin and Runx 2 in MBSCs. Cells were treated with 1 mg/ml of BFP-1 and BMP-7 individually. After 24 h, the cells
were stained with immunofluorescence-conjugated specific antibodies such as (A) anti-CD44 (red), (B) anti-osteocalcin (green), (C) anti-ALP (green) and (D) anti-Runx 2 (green).
CD44, CD51 and CD47 expression was measured by flow cytometry (E) (Green arrow: ODM only, Pink arrow: ODM with BFP-1 treatment). Data are representative of three
independent experiments. Magnification, 20. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

and proteins such as BMP combined with scaffolds or osteogenic
cells have been studied in a small number of clinical trials [25].
Current approaches to bone repair/healing have focused on incor-
porating bone forming molecules, proteins, and cells into degrad-
able matrices. Until now, the mature region of the BMP-7 was the
only way to enhance osteogenesis, and no information is available
on the differentiation and generation conditions of the immature
region of the BMP-7. In this study, we investigated a new peptide
that has osteogenic activity similar to BMP-7 and focused on
a region other than mature BMP-7. We found that BFP-1, which is in
the immature region of the BMP-7-derived peptide, induced
osteogenesis in MBSCs by enhancing activity of the osteogenic
markers ALP, Runx 2, and the osteogenic phenotype of calcium
deposition (Figs. 3 and 4). At the same time, MBSC osteogenesis was
enhanced by BFP-1 treatment (Fig. 2). These results suggest that
BFP-1-induced osteogenesis may result from controlling the
lineage-specific transcriptional program based on inducing the
Runx 2 gene and protein expression (Figs. 3B and 4D). Runx 2 is the
early and master transcription factor initiating the osteogenic
lineage transcriptional program, which leads to up-regulation of
various bone-related genes and markers such as osteocalcin, a late
bone formation marker involved in constructing the bone matrix.
As shown in Fig. 4B, BFP-1 treatment induced osteocalcin expres-
sion in MBSCs more than that of BMP-7 treatment. Our current data
support the hypothesis that the immature region of the BMP-7 can
also induce osteogenesis mainly by enhancing Runx 2 and osteo-
calcin expression.
CD44, CD51 and CD47 are expressed in a variety of cell types
[26,27]. Several reports have shown that CD44, CD51 and CD47 are
expressed during osteogenic differentiation and can be used as
osteogenesis biomarkers. The surface molecule, CD44, acts as
a receptor for hyaluronic acid, which is involved in adhesion to the
extracellular matrix, cellular proliferation, migration, and differ-
entiation [26,28,29]. CD51 is a ubiquitous receptor that interacts
with several ligands such as osteopontin and metalloproteinase-2.
CD47 is involved in increasing intracellular calcium concentrations
after the cell has adhered to the extracellular matrix and regulates
the TGF-b signaling pathway in osteoblasts [30]. Therefore, we
7062 H.K. Kim et al. / Biomaterials 33 (2012) 7057e7063
Fig. 5. BFP-1 induces effective bone formation activity in vivo. (A) Summary of the experimental protocols. BMP-7 and BFP-1 treated-MBSCs were injected into the left and right
dorsoposterior position region of 6-week-old male mice (day 7). All mice were examined by radiography at 4 and 8 weeks (B). Bone cells were stained with H&E staining (C) and are
shown at various magnifications.
investigated whether BFP-1 induces CD44 expression in MBSCs.
CD44, CD51 and CD47 expression was measured by flow cytometry
and fluorescence microscopy analysis. As shown in Fig. 4E, BFP-1
treatment induced CD44, CD51 and CD47 expression in MBSCs.
Interestingly, BFP-1 had potential bone formation activity in a bone
formation test in vivo (Fig. 5).
5. Conclusions
These study shows that the in vivo and in vitro bone generating
efficacy of BFP-1, which is the immature region of the BMP-7-
derived peptide, can greatly enhance osteogenic differentiation of
MBSCs and up-regulate biological markers during osteogenesis.
These results could have a great impact on osteogenesis and bone
generation in orthopedics and dentistry. However, further studies
are necessary to assess the therapeutic potential of BFP-1. These
results provide new insight into using BFP-1 as an osteogenic
stimulator instead of BMP-7 in further clinical trials of bone
generation engineering.
Acknowledgements
This study was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family
Affairs (A084869 and A100012) and by KOSEF grant funded by the
Korea government (MEST) (No. R01-2008-000-10089-0).
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