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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
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Biomed. Mater. 10 (2015) 045004

doi:10.1088/1748-6041/10/4/045004

Paper

received

13 January 2015
re vised

16 May 2015

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

accep ted for publication

21 May 2015
published

8 July 2015

JingfengLi1,5, LinJin1,2,5, MingboWang3, ShaoboZhu1 and ShuyunXu4

1

Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Peoples Republic of China
Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, 430060, Peoples Republic of China
3
Key Laboratory of Biomedical Materials and Implants, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057,
Peoples Republic of China
4
Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Peoples Republic of China
2

E-mail: zhushaobo2000@163.com and sxu@hust.edu.cn

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
rhBMP2 combined 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: 3mg
of P24/PLGA/CS composite; group C: 0.5g of rhBMP2 + 1.5mg of P24/PLGA/CS composite;
group D: blank PLGA/CS material), and rat cranial bone defect models with a diameter of 5mm
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 3D
CT 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 rhBMP2 and 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.

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 slow
rate [3]. Furthermore, these materials have almost
5

The two authors contributed equally to this work.

2015 IOP Publishing Ltd

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

Biomed. Mater. 10 (2015) 045004

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 of
PLA 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 morphogenetic 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 the
strongest 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 successfully 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 healing
and bone repair [1517]. It can also induce in situ and
ectopic osteogenesis [18]. P24 is easier to synthesize in
large quantities at a lower cost compared with conventional 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 composite microspheres composed of PLGA/CS as bonerepair bioscaffold with high drug-loading capacity
and good sustained-release effect [12]. Using this
technique, 3mg of P24, 1g of rhBMP2, and 1.5mg
of P24 and 0.5g of rhBMP2 were loaded with PLGA/
CS composite microspheres to establish three types
of bionic bone materials, which were respectively
rhBMP 2/PLGA/CS, P24/PLGA/CS, and rhBMP 2 +
P24/PLGA/CS composite microspheres with 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 observation, 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 rhBMP2 were compared.
2

2. Materials and methods

2.1. Preparation of PLGA microspheres
As detailed previously [20], 0.5gm of 50kDa PLGA
(Shandong Medical Appliance Factory, China) was
dissolved in 5mL of CH2Cl2, and then 50mgmL1
PBS P24 solution by solving 1.5mg P24 in 0.03mL PBS
was added. The solution was treated ultrasonically for
30s under 200W three times with an interval of 10s
between the treatments. The water-in-oil emulsion
obtained was added into the mixture of 60mL of water
and 0.6mL of sorbitan oleate. The mixture was treated
ultrasonically for 30s under 600W three times with
an interval of 30s between the treatments. The waterin-oil-in-water emulsion was mechanically agitated
at a moderate rate for 2h to remove the CH2Cl2 and
then allowed to stand for 2h. The treated emulsion
was washed and centrifuged at 5000revmin 1 for
5min and then freeze-dried at45C and 10Pa to
prepare PLGA microspheres with a molecular weight
of 50kDa.
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 (30mg) were added
to 9mL of 3%(w/v) CS (Beijing Chemical Reagents
Company, China) solution and mechanically agitated
to achieve uniform dispersion. The above mixture was
added to 0.03mL of 50mgmL1 PBS P24 solution
and dispersed uniformly, and then added into the oil
phase composed of 70mL of liquid paraffin and 2mL
of sorbitan oleate. The mixture was agitated at high
speed and room temperature for 50min to obtain
the emulsion. Subsequently, the mixture was slowly
added to 30mL of 5%(w/v) sodium tripolyphosphate
(TPP), agitated for 2h, 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 rhBMP 2 instead of P24. The synthetic
microspheres in desiccant were stored at 4C prior to
use.
2.3. Preparation of rhBMP2 + P24/PLGA/CS
microspheres
According to the above method, 0.5g of rhBMP2
and then 1.5mg 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

J Li et al

Figure 1. SEM micrographs of PLGA microspheres (a) and PLGA/CS microspheres (b) (original magnification10000).

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 the
conductive 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 46week-old Sprague-Dawley (SD) rats weighing
between 180 and 220g 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 laboratory
environment. All animal experiments were approved
and performed according to the regulations of the animal
ethics committee of our university.
2.6. In vivo animal 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 0.250.3mL per 100g. After successful anesthesia,
the operative field was disinfected and draped.
A longitudinal incision of about 3cm 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 5mm was made by drilling at 4mm away from the
sagittal suture using a 5mm drill bit. The corresponding
composite microspheres were then transplanted. Fullthickness 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 400000 units of penicillin
was performed once daily for five consecutive days to
prevent infection. The general conditions of the rats
were observed postoperatively.
3

2.7. General observation, radiographic

examination and histological evaluation
Five SD rats were sacrificed by cervical dislocation
after general anesthesia by intraperitoneal injection of
10% 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 samples
of 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 paired
t-test. The score data were expressed as meanSD
(xs), and one-way ANOVA analysis was performed.
p<0.05 was considered statistically significant.

3.Results
3.1. Microsphere morphology
The PLGA microspheres were regular spheres that
exhibited smooth surfaces without cavities (figure
1(a)). The PLGA/CS composite microspheres had
wrinkled surfaces and were dense without cavities. Most
microspheres were spherical, while a small proportion
were spheroidal. The cross-section profiles of PLGA/
CS composite microspheres showed multinucleate
and solid duplex spheres embedded with numerous

Biomed. Mater. 10 (2015) 045004

J Li et al

Figure 2. (a), (e) Photo macrograph of Group A (rhBMP2/PLGA/CS); (b), (f) photo macrograph of Group B (P24/PLGA/CS); (c),
(g) photo macrograph of Group C (rhBMP2 + P24/PLGA/CS); (d), (h) photo macrograph of Group D (PLGA/CS) in the defect sites
at 6weeks and 12weeks post-surgery, respectively. The black arrows indicate the areas of the implants.

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 (figure
3(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 (figures 3(e) and (f)). In group
4

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 (p<0.05). The values in groups A
and B had no significant difference (p>0.05). The gray
values of group C were significantly higher than those
of groups A and B (p<0.05) (figure 4).
3.4. 3D CT scan (3D) images
3D CT scan images indicated that in groups A and B
on week 6, high-density shadows were reduced in the
cranial bone defects and osteogenesis was insignificant
on the borders of the defects (figures 5(a) and (b)).
In group C, several calcification shadows were observed
in the bone defects (figure 5(c)). In group D, the
defect cavities did not evidently change (figure 5(d)).
In groups A and B on week 12, the area of high-density

Biomed. Mater. 10 (2015) 045004

J Li et al

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 6weeks post-surgery; (e) group A, (f) group B, (g) group C,
(h) group D at 12weeks post-surgery. The white arrows indicated the areas of the implants.

Radiographic grey values(%)

160
140
120
rhBMP2/PLGA/CS

100

P24/PLGA/CS

80

rhBMP2+P24/PLGA/CS

60

PLGA/CS

40
20
0
6
12
Implantation time(weeks)

Figure 4. The gray values of x-ray images in groups A, B and C were statistically significantly higher than those in group D
(p<0.05). The values in groups A and B had no significant difference (p>0.05). The gray values of group C were significantly
higher than those of groups A and B (p<0.05) at 6weeks and 12weeks post-surgery.

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
5

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

Biomed. Mater. 10 (2015) 045004

J Li et al

Percentage of the area of highdensity shadows(%)

Figure 5. 3D CT scan photos of the rat cranial bone defect 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 6weeks post-surgery; (e) group A,
(f) group B, (g) group C, (h) group D at 12weeks post-surgery. The black arrows indicate the areas of the implants.

100
90
80
70
60
50
40
30
20
10
0

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

12

Implantation time(weeks)
Figure 6. On week 6 and week 12 post-operation, the percentages of the area of high-density shadows under 3D CT in total area of
defect cavities in groups A, B, and C were significantly higher than those in group D (p<0.05). On week 12 the percentage of area in
group C was significantly higher those that in groups A and B (p<0.05). The values represent meanS.D.(n = 5).

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 (p<0.05). However, no
significant differences were observed between groups A,
B, and C (p>0.05). 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 (p<0.05).
The percentages of the area of high-density shadows in
the total area of defects in groups A and B were similar
without significant difference (p>0.05). However,
the percentage of the area in group C was significantly
6

higher those those in groups A and B (p<0.05) (figure

6). The results were consistent with the gray values of
x-ray images.
3.5. HE staining
HE staining on week 6 indicated that the inflammatory
response did not occur around the composite
microspheres in groups A and B. A small proportion
of transplanted microspheres were degraded, and a
small number of osteoblasts grew on the microspheres
(figures 7(a) and (b)). In group C, more newly formed
bones were observed in the composite microspheres,

Biomed. Mater. 10 (2015) 045004

J Li et al

Percentages of newly formed

bone(%)

Figure 7. The histological images of the bone defect sites 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 6weeks post-surgery; (e) group A, (f) group B,
(g) group C, (h) froup D at 12weeks post-surgery (original magnification200).

100
90
80
70

rhBMP2/PLGA/CS

60
50
40

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

30
20
10
0
6
12
Implantation time(weeks)

Figure 8. On week 6, the percentages of newly formed bone in the bone-defect cavities were significantly higher in groups A, B, and
C than in group D (p<0.05). On week 12, the percentages of newly formed bones in group C was higher than those in groups A and
B with significant differences (p<0.05).

and the material started to degrade. A small number

of 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 were
not 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
7

composite material was almost completely degraded

(figure 7(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 (p<0.05). However, the percentages
in groups A, B, and C were similar without significant
differences (p>0.05). 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<0.05)
(figure 8). The results were consistent with the gray
values of x-ray images and the percentages of the area
of high-density shadows under 3D CT out of total area
of defect cavities.

Biomed. Mater. 10 (2015) 045004

J Li et al

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 P24
independently 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 drugcarrying 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 application
types, 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 controlledrelease system was used in bone repair. P24 and
rhBMP2 with 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 rhBMP2 in the cells. Thus, osteogenesis
was promoted in local tissues. In this experiment,
rhBMP2 and 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
8

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 degradation
owing 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 economy. Standard bone defect is defined as the criticalsized bone defect at a specific site of a certain animal
which is incurable by self-repair. Takagi et al [30] initially believed that the critical-sized rat skull defect is
8mm in diameter. However, during modeling, the sagittal sinus is prone to injury by defect with a diameter
of 8mm, resulting in hemorrhea which affected the test
results. Subsequently, Mulliken et al [31] prepared cranial bone defects with diameters of 2 and 4mm. 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 7mm were successively
reported [32, 33]. The standard bone defect with a
diameter of 5mm is optimal for modeling of rat cranial
bone defect [3437]. Given this result, the cranial bone
defects with a diameter of 5mm were established in this
experiment, and the results are reliable.
The optimal doses of rhBMP2 and P24 in bone formation induction were determined in previous studies
[38, 39]. In the present experiment, 0.5g of rhBMP2
and 1.5mg 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 rhBMP2 or 3mg of P24.
General observation, radiographic examination (x-ray
imaging and 3D CT imaging), and histological evaluation 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 microspheres in groups A, B, and C on week 12 were higher
than those on week 6. However, the extents of bonedefect 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

Biomed. Mater. 10 (2015) 045004

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
rhBMP2 and P24 had optimal concrescence and could
increase their mutual osteogenesis capability. In this
study, general observation, radiographic examination,
and histological evaluation achieved essentially consistent results in evaluating the osteogenetic ability in
each 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 exhibited 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 effective controlled drug release. P24 and rhBMP2 presented
similar osteogenetic effects. The PLGA/CS composite
microspheres loaded with rhBMP2 and P24 were more
effective than either P24 or rhBMP2 used alone.

5.Conclusions
PLGA/CS composite microspheres loaded with
rhBMP2 and 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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