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Repair of rat cranial bone defect by using bone morphogenetic protein-2-related peptide

combined with microspheres composed of polylactic acid/polyglycolic acid copolymer and

chitosan

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2015 Biomed. Mater. 10 045004

(http://iopscience.iop.org/1748-605X/10/4/045004)

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1. Introduction

Bone-tissue engineering mainly consists of three

aspects, namely bone biomaterial, seed cells, and

active factors [1]. Polylactic acid (PLA) material is

currently the most frequently investigated and utilized

synthetic material because of its biodegradability

and biocompatibility [2]. Synthetic macromolecule

materials, such as PLA, polyhydroxybutyrate (PHB),

poly(lactide-co-glycolide) (PLGA), and polylactic

acid polyethylene glycol (PLA-PEG), can release the

drug loaded on the surface and inner layers at a slowrate [3]. Furthermore, these materials have almost

no immunogenicity. Thus, they are frequently used

as drug carriers in tissue engineering [4, 5]. The

PLA/polyglycolic acid copolymer is more frequently

applied because of its nonimmunogenicity and

biocompatibility. In addition, the degradation rate

of this copolymer can be adjusted by changing the

mixing ratio of lactic acid and glycolic acid. After the

degradation of the PLA/polyglycolic acid copolymer,

the resulting acidic oligomers or lactic acid and glycolic

acid monomers will form an acidic environment.

This environment can further lead to catalytic

effects that accelerate the degradation. Furthermore,

inflammatory reactions may be induced inside the

body, which limits applications [6]. Chitosan (CS) is

a natural macromolecule polysaccharide that carries

Repair of rat cranial bone defect by using bone morphogeneticprotein-2-related peptide combined with microspheres composed

of polylactic acid/polyglycolic acid copolymer and chitosan

Jingfeng Li1,5, Lin Jin1,2,5, Mingbo Wang3, Shaobo Zhu1and Shuyun Xu4

1 Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Peoples Republic of China2 Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, 430060, Peoples Republic of China3 Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057,

Peoples Republic of China4 Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Peoples Republic of China

E-mail: [email protected] [email protected]

Keywords:BMP-2-related peptide, polylactic acid, chitosan, double microspheres, tissue engineering

Abstract

The effects of the transplanted bone morphogenetic protein-2 (BMP2) -related peptide P24 and

rhBMP2combined with poly(lactic-co-glycolic acid) (PLGA)/chitosan (CS) microspheres were

investigated in promoting the repair of rat cranial bone defect. Forty white rats were selected and

equally divided into four groups (group A: 1g of rhBMP2/PLGA/CS composite; group B: 3 mg

of P24/PLGA/CS composite; group C: 0.5g of rhBMP2+ 1.5 mg of P24/PLGA/CS composite;

group D: blank PLGA/CS material), and rat cranial bone defect models with a diameter of 5 mm

were established. The materials were transplanted to the cranial bone defects. The animals were

sacrificed on weeks 6 and 12 post-operation. Radiographic examinations (x-ray imaging and 3DCT scanning) and histological evaluations were performed. The repaired areas of cranial bone

defects were measured, and the osteogenetic abilities of various materials were compared. Cranial

histology, imaging, and repaired area measurements showed that the osteogenetic effects at two time

points (weeks 6 and 12) in group C were better than those in groups A and B. The effects in groups

A and B were similar. Group D achieved the worst repair effect of cranial bone defects, where a large

number of fibrous connective tissues were observed. The PLGA/CS composite microspheres loaded

with rhBMP2and P24 had optimal concrescence and could mutually increase their osteogenesis

capability. rhBMP2+ P24/PLGA/CS composite is a novel material for bone defect repair with stable

activity to induce bone formation.

PAPER

5 The two authors contributed equally to this work.

RECEIVED

13 January2015

REVISED

16 May2015

ACC EP TED F OR PU BLI CAT ION

21 May2015

PUBLISHED8 July 2015

doi:10.1088/1748-6041/10/4/045004Biomed. Mater. 10 (2015) 045004
mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1088/1748-6041/10/4/045004http://dx.doi.org/10.1088/1748-6041/10/4/045004mailto:[email protected]:[email protected]://crossmark.crossref.org/dialog/?doi=10.1088/1748-6041/10/4/045004&domain=pdf&date_stamp=2015-07-08


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J Li et al

a positive charge. It is biocompatible and degradable

and therefore can be used to produce tissue-engineered

bone microspheres [7]. However, CS is prone to

rapid degradation inside the body because of its high

degradation rate; thus, the period of drug release is

too short to meet the requirement for long-term drug

release. Previous studies used a double emulsification

method: a large number of copolymer microspheres ofPLA and polyglycolic acid are embedded into the CS

microspheres functioning as the substrate to form the

composite microsphere carrier [811]. Experiments

verified that composite microspheres significantly

improve the burst release of copolymer microspheres

of PLA and polyglycolic acid and prolong the period of

drug release [11, 12].

Recently, the osteogenetic activity of bone morpho-

genetic proteins (BMPs) has mainly been utilized in

bone repair and reconstruction. Its role in bone defect

repair has attracted increasing attention. Among all

the members of the BMP family, BMP-2 exhibits thestrongest osteogenetic activity [13]. Natural BMP-2 is

composed of 114 amino-acid residues, of which only

a little over 20 amino acid residues are involved in

the core domain related to osteogenetic activity [14].

The osteogenetic effect of BMP-2 mainly depends on

these primary amino acids. Based on these findings,

the BMP-2-related peptide P24 was designed and suc-

cessfully prepared. This micromolecule polypeptide

consists of 24 amino acids in the BMP-2 functional

domain. In previous studies, P24 significantly promotes

the differentiation of marrow mesenchymal stem cells

into osteoblasts, thereby improving fracture healingand bone repair [1517]. It can also induce in situand

ectopic osteogenesis [18]. P24 is easier to synthesize in

large quantities at a lower cost compared with conven-

tional rhBMP2. P24 not only has the osteogenetic effects

of rhBMP2, but also induces fewer side effects and is

safer [19].

In the present experiment, an emulsification

crosslinking technique was used to prepare the com-

posite microspheres composed of PLGA/CS as bone-

repair bioscaffold with high drug-loading capacity

and good sustained-release effect [12]. Using this

technique, 3 mg of P24, 1g of rhBMP2, and 1.5 mg

of P24 and 0.5g of rhBMP2were loaded with PLGA/

CS composite microspheres to establish three types

of bionic bone materials, which were respectively

rhBMP2/PLGA/CS, P24/PLGA/CS, and rhBMP2+

P24/PLGA/CS composite microspheres w ith the

required activity to induce bone formation. The

three types of scaffold materials were subsequently

used for rat cranial bone defect repair. On weeks 6

and 12 after osteogenesis induction, general observa-

tion, radiographic examination (x-ray imaging and

3D CT imaging), and histological evaluation were

conducted to assess the status of cranial bone-defect

repair. The osteogenetic performance of the materials

was evaluated, and the osteogenetic activities of P24

and rhBMP2were compared.

2. Materials and methods

2.1. Preparation of PLGA microspheres

As detailed previously [20], 0.5 gm of 50 kDa PLGA

(Shandong Medical Appliance Factory, China) was

dissolved in 5 mL of CH2Cl2, and then 50 mg mL1

PBS P24 solution by solving 1.5 mg P24 in 0.03 mL PBS

was added. The solution was treated ultrasonically for30 s under 200 W three times with an interval of 10 s

between the treatments. The water-in-oil emulsion

obtained was added into the mixture of 60 mL of water

and 0.6 mL of sorbitan oleate. The mixture was treated

ultrasonically for 30 s under 600 W three times with

an interval of 30 s between the treatments. The water-

in-oil-in-water emulsion was mechanically agitated

at a moderate rate for 2 h to remove the CH2Cl2and

then allowed to stand for 2 h. The treated emulsion

was washed and centrifuged at 5000 rev min1for

5 min and then freeze-dried at 45 C and 10 Pa to

prepare PLGA microspheres with a molecular weightof 50 kDa.

2.2. Preparation of PLGA/CS microspheres

The prepared PLGA microspheres were used as raw

materials for the second emulsion crosslinking [11,

12]. Dried PLGA microspheres (30 mg) were added

to 9 mL of 3%(w/v) CS (Beijing Chemical Reagents

Company, China) solution and mechanically agitated

to achieve uniform dispersion. The above mixture was

added to 0.03 mL of 50 mg mL1PBS P24 solution

and dispersed uniformly, and then added into the oil

phase composed of 70 mL of liquid paraffin and 2 mL

of sorbitan oleate. The mixture was agitated at high

speed and room temperature for 50 min to obtain

the emulsion. Subsequently, the mixture was slowly

added to 30 mL of 5%(w/v) sodium tripolyphosphate

(TPP), agitated for 2 h, and allowed to stand overnight.

Microspheres synthesized according to the above

procedures were rinsed with petroleum ether and

isopropanol five times and freeze-dried to obtain

dry PLGA/CS composite microspheres, namely the

P24/PLGA/CS microspheres. The rhBMP2/PLGA/CS

composite microspheres were fabricated in a similar

way, adding rhBMP2instead of P24. The synthetic

microspheres in desiccant were stored at 4 C prior to

use.

2.3. Preparation of rhBMP2+ P24/PLGA/CS

microspheres

According to the above method, 0.5g of rhBMP2

and then 1.5 mg of P24 were separately loaded in the

inner PLGA microspheres and chitosan crusts of

PLGA/CS microspheres. The rhBMP2+ P24/PLGA/CS

microspheres were in desiccant at 4C before they were

used in the experiments.

2.4. Morphology analysis

Small amounts of PLGA microspheres and PLGA/CS

composite microspheres were dispersed in adequate

Biomed. Mater. 10 (2015) 045004


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alcohol, added dropwise to a conductive glue, and

dried at room temperature. The PLGA microspheres

and PLGA/CS composite microspheres were quenched

in liquid nitrogen, ground, and then stuck onto theconductive glue. A scanning electron microscope

(Quanta200, FEI Company, Holland) was used to

observe the cross-section morphology after metal

spraying in a vacuum.

2.5. Animal

Forty 46 week-old Sprague-Dawley (SD) rats weighing

between 180 and 220 g were obtained from the Laboratory

Animal Center of Wuhan University, Wuhan, Peoples

Republic of China. All experimental rats were bred at the

Laboratory Animal Center of Wuhan University, with a

standard laboratory diet and in a standard laboratoryenvironment. All animal experiments were approved

and performed according to the regulations of the animal

ethics committee of our university.

2.6. In vivoanimal model and surgical procedures

SD rats were randomly divided into four groups.

Groups A, B, C, and D were transplanted with rhBMP2/

PLGA/CS, P24/PLGA/CS, rhBMP2+ P24/PLGA/CS,

and PLGA/CS composite microspheres, respectively.

Forty SD rats were randomly selected and anesthetized

by intraperitoneal injection of 10% chloral hydrate

at 0.250.3 mL per 100 g. After successful anesthesia,the operative field was disinfected and draped.

A longitudinal incision of about 3 cm was performed

with cranial vault as the center point. Various layers

were exposed successively to the sagittal suture.

A quasicircular cranial bone defect with a diameter

of 5 mm was made by drilling at 4 mm away from the

sagittal suture using a 5 mm drill bit. The corresponding

composite microspheres were then transplanted. Full-

thickness suturation was performed using a thread

after complete hemostasis. After the rats completely

recovered, they were placed back to the labeled cages.

Intraperitoneal injection of 400 000 units of penicillinwas performed once daily for five consecutive days to

prevent infection. The general conditions of the rats

were observed postoperatively.

2.7. General observation, radiographic

examination and histological evaluation

Five SD rats were sacrificed by cervical dislocation

after general anesthesia by intraperitoneal injection of10% chloral hydrate on weeks 6 and 12 post-operation,

respectively. The cranial bone defects of the rats were

exposed for the following tests. (1) General observation

by photography: the inner and outer appearances of the

skulls of all rats in each group were photographed using

a Canon 700D camera. (2) X-ray imaging: the samples

of each group were placed under an x-ray scanner for

imaging. The gray values of the high-density shadows

at the bone cavity interfaces on the x-ray images were

measured with Image ProPlus 6.0 software (Jetta

801, Nanjing, China). (3) 3D CT (GE Lightspeed

Ultra 16, Milwaukee, WI, USA) imaging: the samplesof each group were placed under a CT scanner for

imaging. (4) The 3D images were analyzed using Image

ProPlus 6.0 software by measuring the percentage of

the area of high-density shadows in the area of bone

cavity. The samples were labeled and fixed in 4%

paraformaldehyde, and then stained with hematoxylin

and eosin (H&E) for microscopic observation.

2.8. Statistical analysis

SPSS 20.0 statistical software (SPSS Inc., Chicago, IL,

USA) was employed for data analysis. The differences

between the materials were analyzed using a pairedt-test. The score data were expressed as mean SD

(xs), and one-way ANOVA analysis was performed.

p


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PLGA microspheres. The microstructure of enveloped

microspheres was formed (figure 1(b)).

3.2. General conditions of animals

All animals survived until sampling without evident

abnormalities in physical mobility. All animals also

had a normal diet. Ulceration, infection, nonunion,

swelling, and exudation were not observed on the skins

of transplantation sites of all animals in each group.

At retrieval, the implants were surrounded by a thin

reaction-free fibrous capsule (figure 2).

3.3. X-ray scanning

X-ray observations on week 6 in groups A and B

revealed small pieces of high-density shadows at

the center and on the border of bone defects (figures

3(a) and (b)). In group C, high-density shadows were

present on the obscure borders of the bone defects

(figure 3(c)). In group D, the boundary between the

transplantation sites and the borders of the cranial

bone defects was clear without osteogenesis (figure3(d)). On week 12, the borders of the cranial bone

defects in groups A and B were almost united, but the

high-density shadows on the borders of cranial bone

defect were still obscure (figures3(e) and (f)). In group

C, large pieces of high-density shadows were present

in the bone defects, almost filling the entire defects.

However, the density was lower than that of the normal

bone. The high-density shadows on the borders of the

defect were enhanced, but the defects were not repaired

(figure 3(g)). In group D, bone defect repair was not

observed. The borders of the defects showed very few

high-density shadows, and the cranial bone defects

were not repaired (figure 3(h)). On weeks 6 and 12 after

the operation, the gray values of x-ray images in groups

A, B, and C were statistically significantly higher than

those in group D (p0.05). The gray

values of group C were significantly higher than those

of groups A and B (p


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shadows in bone defects was increased, and several

newly formed bones appeared on the borders (figures

5(e) and (f )). In group C, the majority of the defects

were repaired, and the densities were similar to the

normal bone mineral density (figure 5(g)). In group

D, evident osteogenesis was not observed in the defects

(figure 5(h)). Data obtained through image analysis

software demonstrated that on week 6, the percentages

Figure 3. Radiographs of the implants in four groups at each time-point: (a) group A (rhBMP2/PLGA/CS), (b) group B (P24/PLGA/CS), (c) group C (rhBMP2+ P24/PLGA/CS), (d) group D (PLGA/CS) at 6 weeks post-surgery; (e) group A, (f) group B, (g) group C,(h) group D at 12 weeks post-surgery. The white arrows indicated the areas of the implants.

Figure 4. The gray values of x-ray images in groups A, B and C were statistically significantly higher than those in group D(p0.05). The gray values of group C were significantlyhigher than those of groups A and B (p


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of the area of high-density shadows in the total area of

defect cavities in groups A, B, and C were significantly

higher than those in group D (p0.05). On week 12 after the operation, the

percentages of the area of high-density shadows in the

total area of defect cavities in groups A, B, and C were

significantly higher than those in group D (p0.05). However,

the percentage of the area in group C was significantly

higher those those in groups A and B (p


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and the material started to degrade. A small numberof newly formed bone tissues and osteoblasts were

distributed in a scattered pattern in the cavities of the

materials (figure 7(c)). In group D, the inflammatory

response was observed around the composite

microspheres, but osteoblasts and newly formed

bones were not observed (figure 7(d)). In groups A

and B on week 12, newly formed bones were observed

in the composite microspheres, and the materials

were not completely enveloped. A large number of

fibrillar connective tissues were present in the defects.

A small number of new bones were formed around

the materials, and some composite materials werenot yet degraded (figures 7(e) and (f)). In group C,

the defects were repaired, and the newly formed

bones were observed in the composite microspheres,

which were almost enveloped by the new bones. The

composite material was almost completely degraded(figure7(g)). In group D, newly formed bone was still

not observed, and scars and connective tissues were

observed around the defects (figure 7(h)). On week 6,

the percentages of the newly formed bone in the bone

defect cavities were higher in groups A, B, and C than

that in group D (p0.05). On week 12, the percentages

of the newly formed bones in groups A and B were

similar without significant differences (pa>0.05).

The percentage in group C was higher than those in

groups A and B with significant differences (p


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4. Discussion

Scaffold materials play important roles in bone

tissue engineering. They provide a favorable

microenvironment for cell growth, functioning as

sustained-releasing carriers to increase the release

time of growth factors [21]. Moreover, they are capable

of cell recognition. The BMP-2-related peptide P24independently designed by our research group can

be released slowly to induce bone formation using

an effective scaffold carrier [9, 17, 19, 22]. Recently,

microsphere functioning as a scaffold carrier in bone

tissue engineering has attracted considerable attention

[23]. A microsphere in this context is a spherical drug-

carrying particle composed of polymer materials. This

carrier is degradable; thus, the loaded drug is slowly

released during microsphere degradation [24, 25].

Currently, scaffold-containing microspheres can be

divided into two types, namely artificially synthetic

polymer and ceramics [23]. In terms of applicationtypes, microspheres can be divided into simplex and

composite microspheres, which are composed of

two types of materials. The main raw materials for

preparing synthetic polymeric material include PLA,

lactide, and glycolide copolymers. Because PLA has

lower degradation rate compared with PGA, these

two materials were mixed in several experiments to

prepare a material with controllable degradation rate.

Borden et al[26] speculated that when the ratio of PLA

and polyglycolic acid was 75:25, the scaffold material

exhibited an optimal degradability. Another type of

degradable microspheres originated from natural

materials, such as CS, alginate, and gelatin [27]. Active

groups in the CS can combine with scaffold-containing

microspheres, which is convenient for property control

and application [28]. Previous studies showed that

loaded drug in the microspheres is released in two

ways [12]: burst release after the drug is dissolved in

the solution and has become dispersed, and release of

the loaded drug after polymeric material degradation.

In our experiment, the microsphere drug controlled-

release system was used in bone repair. P24 and

rhBMP2with the ability of inducing bone formation

was synthesized with microspheres with a sustained

releasing function. The synthesis was performed using

a chemical method to achieve the sustained release

of P24 and rhBMP2in the cells. Thus, osteogenesis

was promoted in local tissues. In this experiment,

rhBMP2and P24 were separately loaded in the inner

PLGA microspheres and chitosan crusts of PLGA/CS

microspheres to prepare tissue-engineered bone for

bone-defect repair. Previous studies indicated that

PLGA/CS microspheres (PC10, PC20, and PC50) from

three molecular weights of PLGA could be prepared

by the double-emulsion method [11]. The PC50

microspheres were better than the other two types of

microspheres in terms of drug loading capacity and the

time of controlled release. The PLGA/CS composite

PC50 microsphere adopted in this experiment had a

prolonged release period compared with simplex PLGA

microspheres. In addition, the acidic environment

created by the degradation of simplex PLGA

microspheres could accelerate the degradation rate

of microspheres, which was unfavorable for sustained

release of the loaded drug. PLGA/CS composite

microspheres served as the buffer against the acidic

substances produced by microsphere degradationowing to the CS envelope [28, 29]. As a result, the

degradation rate of the microspheres was reduced and

sustained release was achieved.

In this experiment, rat cranial bone defect models

were established to assess the capacity of rhBMP2+

P24/PLGA/CS composite material in promoting bone

regeneration. Rat cranial bone defect models have been

widely used in bone-tissue engineering tests because of

the advantages of convenience, feasibility, and econ-

omy. Standard bone defect is defined as the critical-

sized bone defect at a specific site of a certain animal

which is incurable by self-repair. Takagi et al[30] ini-tially believed that the critical-sized rat skull defect is

8 mm in diameter. However, during modeling, the sag-

ittal sinus is prone to injury by defect with a diameter

of 8 mm, resulting in hemorrhea which affected the test

results. Subsequently, Mulliken et al[31] prepared cra-

nial bone defects with diameters of 2 and 4 mm. The

cranial bone defects with smaller diameter were not

repaired because of the removal of the periosteum in

the operated areas. In later studies, the cranial bone

defects with diameters of 6 and 7 mm were successively

reported [32, 33]. The standard bone defect with a

diameter of 5 mm is optimal for modeling of rat cranialbone defect [3437]. Given this result, the cranial bone

defects with a diameter of 5 mm were established in this

experiment, and the results are reliable.

The optimal doses of rhBMP2and P24 in bone for-

mation induction were determined in previous studies

[38, 39]. In the present experiment, 0.5g of rhBMP2

and 1.5 mg of P24 were separately loaded in the inner

PLGA microspheres and chitosan crusts of PLGA/CS

microspheres. A comparison of osteogenetic activity

was carried out with 1 g of rhBMP2or 3 mg of P24.

General observation, radiographic examination (x-ray

imaging and 3 D CT imaging), and histological evalua-

tion were performed on weeks 6 and 12 postoperatively,

and the osteogenetic ability of each group was assessed.

General observation indicated that skin ulceration,

infection, nonunion, swelling, and exudation were

not observed on the skins of transplantation sites of all

animals. Meanwhile, the extents of bone defect repair

and degradation of the transplanted composite micro-

spheres in groups A, B, and C on week 12 were higher

than those on week 6. However, the extents of bone-

defect repair and degradation in group A were similar to

those in group B during the entire process, while group

C achieved better results. In group D, a small number

of soft tissues covering the bone defects were observed,

and the PLGA/CS composite microspheres were almost

completely degraded at weeks 6 and 12. The effects of

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J Li et al

induction of bone formation using P24 and rhBMP2

were similar, which was consistent with previous results

[39]. PLGA/CS composite microspheres loaded with

rhBMP2and P24 had optimal concrescence and could

increase their mutual osteogenesis capability. In this

study, general observation, radiographic examination,

and histological evaluation achieved essentially con-

sistent results in evaluating the osteogenetic ability ineach group on weeks 6 and 12 after the transplantation

of composite materials. Specifically, the osteogenetic

effects at two time points (weeks 6 and 12) in group

C were better than those in groups A and B, the latter

two groups obtaining similar effects. Group D exhib-

ited the worst bone-defect repair results, and a large

number of fibrillar connective tissues were observed in

the defects. The composite microspheres induced mild

local inflammatory responses and could achieve effec-

tive controlled drug release. P24 and rhBMP2presented

similar osteogenetic effects. The PLGA/CS composite

microspheres loaded with rhBMP2and P24 were moreeffective than either P24 or rhBMP2used alone.

5. Conclusions

PLGA/CS composite microspheres loaded with

rhBMP2and P24 had optimal concrescence and could

increase their mutual osteogenesis capability. rhBMP2

+ P24/PLGA/CS microspheres are a novel material for

bone defect repair with a stable activity to induce bone

formation.

Acknowledgments

This work was financially supported by the National

Natural Science Foundation of China (Grant Nos:

81301538, 81171684 and 51303094), the International

Science and Technology Cooperation Program of

China (Grant No: 2013DFG32690), and the Youth

Science and Technology Morning Program of Wuhan

(Grant No: 2014072704011256).

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