Biomedicine & Pharmacotherapy 130 (2020) 110516

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Preparation of BMP-2 loaded MPEG-PCL microspheres and evaluation of T

their bone repair properties
Deyin Konga, Yanbin Shia,*, Yan Gaoa, Mengguang Fua, Shengli Konga, Guimei Linb
a
School of Mechanical & Automotive Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
b
School of Pharmaceutical Science, Shandong University, Jinan 250012, China

A R T I C LE I N FO A B S T R A C T

Keywords: Autologous or allogeneic bone grafts are common methods to treat bone defects. Bone tissue engineering
BMP-2 combining carrier material with the active factor can induce a generation of new bone at the bone defect site.
Microsphere However, its clinical application is restricted by the limited donors, the high morbidity at the donor site, the low
Sustained release activity in vivo, and dose-independent adverse effect. To overcome the limitations of traditional therapies, it is
Biocompatibility
urgent to find and develop a repair material that can replace natural bones. Hence, we designed and prepared
Ectopic osteogenesis
suitable MPEG-PCL microspheres loaded bone morphogenetic protein-2 (BMP-2/MPEG-PCL-MS) to effectively
solve the problem mentioned above, prolong its reaction time at the targeted site, and avoid the pain of patients
caused by frequent administration. The physicochemical properties and in vitro release behaviors were good. The
microspheres showed high biocompatibility and strongly induced osteogenesis in vivo. BMP-2/MPEG-PCL-MS
has been proven to exert sustained-release in vivo and maintain the inherent BMP-2 activity. They can be directly
injected into the bone defect site, or implanted to a large bone defect site together with stent material to exert
therapeutic effects. Hence, this smart drug delivery system has promising potential for clinical applications and
provides a well-controlled design for combination of tissue engineering and pharmaceutics for further ex-
ploration.

1. Introduction
There are approximately 26 million patients in the world every year
who need bone defect repair. Bone, as an important organ in the human
body, is incessantly remodeled throughout its life cycle and has a high
self-regeneration ability [1]. Osteoblasts and osteoclasts play an im-
portant role in bone remodeling when damaged. However, bone re-
generation is limited by age, blood, and status of defects. Bone tissue is
often unable to repair itself due to skeletal abnormalities, traumatic
injury, or tumor resection resulting in large defects, which are usually
treated with autologous or allogeneic bone grafting [2–4]. Since 2015,
bone grafting has become the 2nd-ranked transplantation, subsequent
to blood transplantation. However, traditional therapies have many
shortcomings, such as limited donor bone source, lack of high-quality
bone tissue, and disease transmission, resulting in increased treatment
costs [5,6]. Therefore, efforts have been made to develop methods
based on regenerative medicine to accelerate bone healing, using syn-
thetic bone substitutes of biological materials [7]. There have been
several studies devoted to combining biological materials with osteo-
genic stimulation factors to induce massive bone formation [8,9].
The process of bone formation and repair is the process of differ-
entiating bone-forming mesenchymal stem cells into osteoblasts under
the induction of specific growth factors [10,11]. Bone morphogenetic
protein-2 (BMP-2), a member of the TGF-β superfamily, was first dis-
covered for ectopic osteogenesis in 1965 by Marshall R. Urist [12,13]. It
is a key factor in promoting bone formation and has a strong osteogenic
activity. BMP-2 is secreted by osteoblasts and reacts upon osteoblasts. It
can induce osteoblast differentiation and increase alkaline phosphatase
(ALP) activity and mineralization of bone matrix. It is also crucial in
affecting osteoblast differentiation and controls bone differentiation in

Abbreviations: BMP-2, bone morphogenetic protein-2; BMP-2/MPEG-PCL-MS, MPEG-PCL microspheres loaded BMP-2; ALP, alkaline phosphatase; FDA, US National
Drug Administration; EMA, European Medicines Agency; W1, internal aqueous phase; O, oil phase; W2, outer aqueous phase; CCK-8, Cell Counting Kit-8; OD, optical
density; RCV, relative cell viability; IC50, the half maximal inhibitory concentration; BMD, bone density; BV, bone volume; TV, tissue volume; Tb.Th, trabecular
thickness; Tb.N, trabecular number; ROI, region of interest
⁎
Corresponding author at: School of Mechanical & Automotive Engineering, Qilu University of Technology (Shandong Academy of Sciences), No.3501, Daxue Rd,
Jinan 250353, China.
E-mail address: syb@qlu.edu.cn (Y. Shi).

https://doi.org/10.1016/j.biopha.2020.110516
Received 16 May 2020; Received in revised form 27 June 2020; Accepted 7 July 2020
Available online 13 July 2020
0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
D. Kong, et al. Biomedicine & Pharmacotherapy 130 (2020) 110516

various stages of mammalian growth and development [14–16]. It is Table 1

widely used in various clinical treatment trials and has become the most The results of orthogonal experimental design.
commonly used protein bone graft substitute. BMP-2 protein has been Factors A B C D EE (%)
approved by the US National Drug Administration (FDA) and the Eur-
opean Medicines Agency (EMA) for the treatment of spinal fusion, 1 1 1 1 1 76.58
2 1 2 2 2 78.69
which is a unique treatment option that avoids bone grafting [17,18].
3 1 3 3 3 72.36
However, BMP-2 is an unstable protein with a very short half-life 4 2 1 2 3 68.60
and is easily degraded under physiological conditions [19]. It cannot 5 2 2 3 1 62.55
supply a sustained effective concentration on the target site, obviously 6 2 3 1 2 61.54
may not achieve the desired therapeutic effect [20]. Clinical application 7 3 1 3 2 54.31
8 3 2 1 3 67.20
often faces many problems, including poor quality of bone due to
9 3 3 2 1 68.72
continuous administration of large doses, poor patient compliance, in- K1 75.877 66.497 68.440 69.283
flammation, and other side effects [21–26]. Therefore, in order to im- K2 64.230 69.480 72.003 64.847
prove patient compliance, overcome the short half-life of BMP-2 and K3 63.410 67.540 63.073 69.387
R 12.467 2.983 8.930 4.540
off-target problems, and maintain long-term stimulation, choosing a
suitable delivery system is key to controlling the clinical dose and re-
lease [27–30]. Microspheres, to some extent, can control the release of
efficiency). The sediments were washed by deionized water 3 times and
BMP-2 and effectively solve the abovementioned problems.
lyophilized for 48 h to obtain sustained-release BMP-2/MPEG-PCL-MS,
In this study, hydrophilic MPEG was used to modify PCL to reduce
which was also stored at −20 °C.
its hydrophobicity. The obtained block copolymer has both PCL and
MPEG properties, greatly improving the hydrophilicity of PCL.
Adjusting the hydrophilic/hydrophobic balance of the material is ben- 2.4. Characterization of the microspheres
eficial to the compatibility of the material with the protein polypeptide
drug, thereby improving the stability of the drug. MPEG can be directly The particle size, size distributions, and ζ-potentials of microspheres
excluded from the body, further reducing the local acid production were measured on a Zsizer Nano ZS90 instrument (Malvern,
[31,32]. At the same time, the sustained release ability of the micro- Westborough, MA) using the dynamic light scattering method. For the
spheres makes the BMP-2 protein stable and sustainably released in the observation of morphology, the suspension of the microspheres before
target site, which can effectively promote the growth of osteoblasts and and after lyophilization were separately diluted with distilled water,
induce bone formation [33–37]. This design provides a new and pro- dropped onto a glass slide, and observed under an optical microscope.
mising treatment strategy for patients with bone defects. The lyophilized microsphere powder was observed directly under the
super depth microscope (MSD-VHX1000, US). Furthermore, the mi-
2. Material and methods crosphere powder was evenly spread on a double-sided tape, and gold
was splashed with an Eiko IB-5 ion plating apparatus to observe the
2.1. Materials surface morphology under a scanning electron microscope (SEM,
Philips XL30FEG, The Netherlands)
BMP-2 was kindly provided by Yantai Zhenghai Bio-tech Co., Ltd. Due to the unstable physical and chemical properties of BMP-2, we
(China). MPEG-PCL was purchased from Daigang Bio-tech Co., Ltd. determined the concentration of BMP-2 by enzyme-linked im-
(China). munosorbent assay (ELISA). After centrifugation of BMP-2/MPEG-PCL-
MS, the supernatant was collected, and the concentration of BMP-2 in
2.2. Cell line and animals the supernatant was detected by an ELISA kit (Tianjin Anoric Bio-tech
Co., Ltd., China). The entrapment efficiency (EE%) and drug-loading of
The C2C12 cell line (mouse myogenic cells) was purchased from BMP-2 (DL%) of BMP-2/MPEG-PCL-MS were calculated using the fol-
QHI Scientific, Inc. (Jiangyin, China) and cultured in the H-Dulbecco lowing equations:
modified Eagle medium, supplemented 10 % fetal bovine serum, 100
EE% = Wencapsulated BMP-2 / Wtotal BMP-2 × 100 %
U/mL of penicillin, and 100 μg/mL of streptomycin. The cells were
incubated at 37 °C in the presence of 5% CO2. DLDTX% = Wencapsulated BMP-2/Wencapsulated BMP-2 + WMPs × 100 %
All of the in vivo mice experiments were approved by the Shandong
University Animal Care and Use Committee. All Kunming mice were
where Wencapsulated BMP-2 means the weight of BMP-2 encapsulated in
purchased from the Shandong University Animal Center.
microspheres, Wtotal BMP-2 means the total weight of BMP-2 added, and
WMPs means the weight of all the carrier materials in microspheres.
2.3. Preparation of microspheres

The microspheres were prepared using the W1/O/W2 double
emulsion method. A certain concentration of BMP-2 solution (internal
aqueous phase, W1) was added to a dichloromethane solution (oil
phase, O) containing MPEG-PCL, and a high-speed homogenizer was
used at a speed of 5,000–20 000 r/min under ice bath conditions. High-
speed shearing was carried out to form a uniform non-layered colos-
trum (W1/O); the W1/O colostrum obtained after shearing was slowly
and uniformly injected into the PVA solution (outer aqueous phase,
W2), and the high-speed homogenizer was the W1/O/W2 double
emulsion, which was uniformly dispersed at 5,000–20,000 r/min under
ice bath conditions. The emulsion was stirred on a magnetic stirrer at
500 r/min for 4 h to completely evaporate the dichloromethane.
Subsequently, it was centrifuged at 8000 r/min for 6 min, and the su-
pernatant was stored at −20 °C (retained for the encapsulation
2.5. Optimization of preparing technology
To obtain microspheres with better qualities, an orthogonal ex-
periment was used to optimize the preparing technology. Factors af-
fecting the EE% of microspheres were determined by single-factor re-
search, which included MPEG-PCL concentration in the organic phase,
the percentage of PVA, VO/VW1, and VO/VW2, primary shear rate, and
double emulsion shear rate. According to the results of single-factor
experiments, 4 factors that greatly influence EE% were selected to carry
out orthogonal experiments. The key parameters in the preparation
process of microspheres were optimized, including MPEG-PCL con-
centration (A) and the percentage of PVA(B), VO/VW1(C), and VO/VW2
(D), and the best prescription was screened by an L9 (34) orthogonal
design test to get the best condition.

2
D. Kong, et al.
Fig. 1. The morphology photos under optical microscope (A), super depth microscope (B) and SEM (C). The average diameter (E) and zeta potential (F) of mi-
crospheres measured by DLS.
2.6. Stability of microspheres
Influencing factor experiments, including the high temperature test,
high humidity test, and illumination test, were conducted to investigate
the stability of microspheres under different conditions. Changes in
appearance, DL%, EE%, and particle size were recorded to exhibit the
stability of BMP-2/MPEG-PCL microspheres.
High temperature test. Fifty milligrams of dried BMP-2/MPEG-PCL
microsphere powder was sealed and placed at 40℃. The microsphere
powder was taken out on the 5th and 10th days, respectively.
Subsequently, the appearance, DL%, EE%, particle size, and content of
the BMP-2/MPEG-PCL-MS were examined.
High humidity test. Fifty milligrams of dried BMP-2/MPEG-PCL
microsphere powder was sealed and placed at 25℃, RH 75 % (NaCl
saturated solution) and RH 92.5 % (KNO3 saturated solution). The
microsphere powder was taken out on the 5th and 10th days, respec-
tively. Subsequently the appearance, DL%, EE%, particle size, and
content of the BMP-2/MPEG-PCL-MS were examined.
Light test. Fifty milligrams of dried BMP-2/MPEG-PCL microsphere
powder was sealed and placed under light intensity (4500 ± 500) lx,
and the microspheres were taken out on the 5th and 10th days, re-
spectively. Subsequently, the appearance, DL%, EE%, particle size, and
content of the BMP-2/MPEG-PCL-MS were examined.
2.7. Determination of in vitro drug release
In vitro release assays were performed using dynamic membrane
dialysis. The release medium was phosphate-buffered saline (PBS, pH
7.4, containing 0.2 % NaN3). BMP-2 solution (10 μg/mL) or BMP-2/
MPEG-PCL-MS (containing 10 μg BMP-2) were accurately weighed and
placed in a dialysis bag. One milliliter of release medium was then
added. The sterilized thin wires were used to fasten the pockets on both
sides of the dialysis bag. These were placed in centrifuge tubes con-
taining 10 mL of release medium and set to a speed of 100 r/min and a
temperature of 37℃. At the predicted time points (BMP-2 solution
group: 5, 20, 35, 50 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,
7.5 h; BMP-2/MPEG-PCL microsphere group: 0.5, 1, 2, 4, 8, 12, 24, 36,
48, 72, 168, 240, 312, 384, 456, 504, 576, 648, 720, 792, 864, 936,
1008, 1080 h), 1 mL was taken, and an equal amount of fresh release
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Biomedicine & Pharmacotherapy 130 (2020) 110516
medium was added to the centrifuge tube. The BMP-2 concentration of
the sample was measured by ELISA kit. The cumulative release per-
centage Q was calculated according to the following equations]:
Q=[CiV＋(Ci-1Vi-1+Ci-2Vi-2+……+C1V1)]/W × 100 %
where Ci is the concentration of BMP-2 at the ith sampling, V is the total
volume of the release medium, C1 is the concentration of BMP-2 at the
first sampling, and V1 is the total volume of the release medium for the
first sampling, Ci -1, Vi-1, and so on, and W is the amount of BMP-2 in
the dialysis bag.
2.8. Biocompatibility of microspheres
In vitro biocompatibility of microspheres was measured using the
Cell Counting Kit (CCK)-8 method according to the manufacturer’s
protocol (Cell Counting Kit-8, Sigma, US). C2C12 cells were seeded in
96-well plates at 37 °C at a density of 2000 cells/well in 100 μL of
complete medium and incubated overnight. The blank microspheres,
BMP-2/MPEG-PCL-MS, and control group (added an equal volume of
blank complete medium) were then added to each well at the desig-
nated concentration of 0.2 μg/mL and then incubated at 37 °C with 5%
CO2 for 2 d, 4 d, and 6 d. Before analysis, CCK-8 solution (10 μL/well)
was added, followed by further incubation for 1 h. The maximum ab-
sorbance was set at 450 nm, and the optical density (OD) of each well
was scanned on a microplate reader (Denley Dragon Wellscan MK 3,
USA). Relative cell viability (RCV) (%) was calculated as RCV (%)
=ODtest/ODcontrol × 100 %, where ODtest and ODcontrol represent the
OD of cells treated with the test groups and the control group, re-
spectively. The experiments were repeated 6 times independently, and
the half maximal inhibitory concentration (IC50) of the test groups was
calculated using Origin 9.0 software.
2.9. Ectopic osteogenesis study
Kunming mice were intraperitoneally injected with 0.1 mL of 4%
chloral hydrate per 10 g. After anesthesia, the hair of right thigh was
cut, and the surgery site was disinfected. Then, an incision of ap-
proximately 1 cm was made to prepare a quadriceps muscle bag model.
The BMP-2 solution or the BMP-2/MPEG-PCL-MS were mixed with
D. Kong, et al.
Fig. 2. Stability of BMP-2/MPEG-PCL microspheres under different condition after 5d and 10d (n = 3).
C2C12 cells (5 × 106 cells), respectively. After implanting them into the
quadriceps muscle bag gap, the wound was sutured layer by layer. The
control group used C2C12 cells only. The implant sites were marked
and mice were fed normally. Mice were sacrificed at 2, 4, and 8 weeks
after surgery, respectively. The samples were taken out, and the ap-
pearance of the new bone was observed.
To further evaluate the osteogenic activity, ALP assay and calcium
content were measured. After weighing a part of the sample, physio-
logical saline was added to the sample according to the ratio of weight
(g)/volume (mL) = 1/9. The sample was homogenized under ice bath
and centrifuged at 2500 r/min for 10 min. The supernatant was used
determine the related activity according to the manufacturer’s protocol
of the ALP Assay Kit (Beyotime, China) and Calcium Assay Kit (Nanjing
Jiancheng Bioengineering Institute, China). The remaining sample was
fixed in 4% (w/v) paraformaldehyde to maintain the original appear-
ance of the cells.
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Biomedicine & Pharmacotherapy 130 (2020) 110516
2.10. Histological examination
To further examine the osteogenic status of microspheres in vivo, the
fixed samples were embedded in paraffin, sectioned, and stained with
hematoxylin and eosin according to the manufacturer’s instructions.
The stained sections were observed and photographed using a VS120
Virtual Slide Microscope (Olympus Corp., Tokyo, Japan).
2.11. Micro-computed tomography (micro-CT) analysis
The biomechanical strength of bones and the risk of their de-
formation or fracture were determined on the basis of their geometric,
structural, material, and densitometric properties. Studies have shown
that micro-CT can quickly and accurately analyze the microstructure of
mouse bone samples [38–41]. To further evaluate the ectopic osteo-
genic induction ability of microspheres, we analyzed the related in-
dicator of bone using micro-CT. The ectopic osteogenesis sample was
D. Kong, et al.
Fig. 3. Release curve of BMP-2/MPEG-PCL microspheres in vitro (n = 3).
Fig. 4. In vitro cytotoxicity study of C2C12 cells: Cell Viability after cultured
with BMP-2 solution、BMP-2/MPEG-PCL-MS and MPEG-PCL microspheres
after 2 days, 4 days, 6 days (n = 6).
placed in the inspection slot of the micro-CT (SkyScan 1176, Bruker)
system for scanning. The images of different cross-sections were ob-
tained for the same sample. After selecting the region of interest (ROI)
of the images, we reconstructed the model via software NRecon
(NRecon, Skyscan, Belgium) and CTAn (CTAn, Skyscan, Belgium),
subsequently obtaining microstructural parameters, including bone
density (BMD), bone volume (BV), tissue volume (TV), trabecular
thickness (Tb.Th), trabecular number (Tb.N), and total porosity.
2.12. Statistical analysis
Data were presented as mean ± standard deviation. One- and two-
way analysis of variance was used when making multiple comparisons.
All statistical analysis was performed using Origin 9.0 software.
Statistical significance was noted as follows: *, p < 0.05, **, p < 0.01,
***, p < 0.001, ****, p < 0.0001.
3. Results and discussion
3.1. Optimization of preparing technology
The MPEG-PCL concentration in organic phase, the percentage of
PVA, VO/VW1, VO/VW2, primary shear rate, and double emulsion shear
rate were investigated by single factor experiment, and the factors with
greater influence were screened for orthogonal experiments. See the
supplementary information for the results of the single factor experi-
ment. The results of single-factor study (Supplementary information
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Biomedicine & Pharmacotherapy 130 (2020) 110516
Figs. S1-S6) showed that the shear rate was determined to be 10,000 r/
min due to low effect on the EE% of the microspheres. To simplify the
screen of optimal prescription, 4 factors and 3 levels were chosen for
the L9 (34) orthogonal test, that is A: BMP-2/MPEG-PCL concentration
(100 mg/mL, 120 mg/mL, 140 mg/mL), B: PVA concentration (1%, 2%,
3%), C:W1/O (1:3, 1:5, 1:7), and D:O/W2 (1:8, 1:10, 1:12). The results
and analysis are shown in Table 1. The range R indicates the influence
of factors on the result. The larger the R, the greater the influence on
the experimental results. The difference R of the 4 factors is: A >
C > D > B. The average analysis result of each factor is A:1 > 2 > 3;
B:2 > 3 > 1; C:2 > 1 > 3; D:3 > 1 > 2, so the best choice is
A1B2C2D3. The best prescription consisted of: MPEG-PCL concentra-
tion of 100 mg/mL, PVA concentration of 2%, W1/O = 1:5, and O:
W2 = 1:12. Three batches of BMP-2/MPEG-PCL microspheres were
prepared according to the optimized prescription and preparation
process. The EE% and DL% results (Supplementary information Table
S1) showed that the optimal formulation and preparation processes
were reasonable and reproducible.
3.2. Characterization of the microspheres
The BMP-2/MPEG-PCL microspheres were prepared by the double
emulsion method. As shown in the photos observed under optical,
super-depth microscope and SEM (Fig. 1A-C), microspheres had a
round-shaped appearance and good dispersion capabilities due to sui-
table preparation method. The prepared microspheres had no adhesion,
and good fluidity, which can meet the requirements of industrial pro-
duction. The average particle size of BMP-2/MPEG-PCL microspheres
was 4.30 ± 0.19 μm, and the Zeta potential was -23.63 ± 2.31 mV,
which indicated good stability of microspheres (Fig. 1E-F). The en-
capsulation efficiency and drug loading of BMP-2/MPEG-PCL micro-
spheres were determined by ELISA. The prepared microspheres de-
monstrated high EE% of 80.96 ± 1.01 % and DL% of 0.089 ± 0.001
%. It is beneficial for microspheres to be directly injected into the bone
defect site, and they can also be implanted into the large bone defect
site together with the stent material to exert its therapeutic effect.
3.3. Stability of microspheres
The microspheres were placed under high temperature, high hu-
midity, and light conditions for 5 days and 10 days, and their appear-
ance, diameter, DL%, and content were observed. The results are shown
in Fig. 2. Under a high temperature of 40 °C, the microsphere powder
showed slight adhesion, and the diameter increased slightly, which
might be due to the softened MPEG-PCL at high temperatures. The
changed BMP-2 content indicated that high temperature had a certain
effect on the activity of BMP-2. Therefore, the BMP-2/MPEG-PCL mi-
crospheres should be stored at a low temperature. The appearance,
diameter, and BMP-2 content of BMP-2/MPEG-PCL microspheres did
not change much when the humidity was 75 %. However, the micro-
spheres adhered under the humidity of 92.5 %; they became larger, and
the BMP-2 content was relatively lowered. We deduced that a high-
humidity environment could increase the weight of microspheres due to
moisture absorption. Therefore, BMP-2/MPEG-PCL microspheres
should be stored in a relatively dry environment. Light had little effect
on the appearance and diameter of the microspheres. However, the
content of BMP-2 was lower than the initial status, approximately 97 %
of the marked amount, indicating that light also had a certain effect on
microspheres. Hence, BMP-2/MPEG-PCL microspheres should be stored
in a dark environment.
3.4. Determination of in vitro drug release
The cumulative release percentage of BMP-2/MPEG-PCL micro-
spheres (BMP-2/MPEG-PCL-MS) was determined via the dynamic dia-
lysis method. The cumulative release curve of the microspheres was
D. Kong, et al.
Fig. 5. Ectopic bone formation tissue (A) of BMP-2-Solution, BMP-2/ MPEG-PCL-MS group after 2 weeks, 4 weeks and 8 weeks (n = 3). The ALP (B) and Ca2+ (C)
contents of each group (n = 4).
shown in Fig. 3. We observed that the release rate of BMP-2 solution
was faster, reaching 97.33 ± 1.97 % at 7.5 h, whereas the BMP-2/
MPEG-PCL reached 20.76 ± 1.43 % at 24 h, much slower than BMP-2
solution. The results indicated that the microspheres had a slightly
burst release in the first 24 h, and subsequently, the drug release be-
came stable. The main reason for the burst release of the microspheres
was that parts of BMP-2 were adsorbed on the surface of the micro-
spheres instead of encapsulated inside the microspheres. After placed in
the release medium, this part of BMP-2 could quickly enter the release
medium. The cumulative release rate reached 96.61 ± 2.73 % on the
45th day. The results indicated that the BMP-2/MPEG-PCL micro-
spheres showed a good sustainable release effect. The late sustainable
release effect was due to the gradual degradation of the MPEG-PCL, so
the BMP-2 inside would be slowly released. Mathematical model fitting
of BMP-2 solution and BMP-2/MPEG-PCL-MS were performed using
zero-order kinetics, first-order kinetics, Higuchi and Ritger-Peppas
equation, respectively. The results (Supplementary Information Table
S7) showed that the in vitro release property of BMP-2 solution was
consistent with the Ritger-Peppas model with a regression coefficient of
R = 0.9994, while BMP-2/MPEG-PCL microspheres were in accordance
with the Higuchi equation with a regression coefficient R = 0.9875.
3.5. Biocompatibility of microspheres
Medical biomaterial should first meet the requirements of low
toxicity and good biocompatibility. Although BMP-2 protein and bio-
degradable MPEG-PCL polymer have been proven to be biocompatible,
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Biomedicine & Pharmacotherapy 130 (2020) 110516
the safety of BMP-2/MPEG-PCL-MS still needs to be verified. The cy-
totoxicity of different formulations was determined by the CCK-8
method. C2C12 myogenic cells were incubated and were blank micro-
spheres, BMP-2 solution and BMP-2/MPEG-PCL-MS at predetermined
time (2 d, 4 d, and 6 d). After adding CCK-8 solution (10 μL/well) for
1 h, the OD was read via a microplate reader at 450 nm. The significant
difference (P > 0.05) indicates that BMP-2, blank microspheres and
BMP-2/MPEG-PCL-MS have no significant effect on cell growth and
proliferation. The above results fully indicated that BMP-2, BMP-2/
MPEG-PCL-MS, and blank microspheres had no obvious side effects on
C2C12 cells and good biocompatibility, which was in accordance with
the requirement of pharmaceutics (Fig. 4).
3.6. Ectopic osteogenesis study
After 2 weeks, 4 weeks, and 8 weeks of the surgery, 4 mice in each
group were sacrificed by cervical dislocation. We observed that the
BMP-2 group and the BMP-2/MPEG-PCL microsphere group had irre-
gularly shaped new bone-like tissue in the muscle bag, while the blank
group was free of new tissue. After taking out the new tissue and taking
a picture, we compared the appearance of different groups. As dis-
played in Fig. 5A, the new bone-like tissue of the BMP-2/MPEG-PCL-MS
group gradually increased over time, while the BMP-2 solution group
did not differ significantly after several weeks.
ALP is an early indicator of osteoblast differentiation. Elevated ALP
activity can simultaneously initiate calcification and promote the
synthesis of type I collagen. Then, calcium salts can be deposited on
D. Kong, et al. Biomedicine & Pharmacotherapy 130 (2020) 110516

Fig. 6. The H&E microscope photos of each group (×200).

Fig. 7. The analysis of BV, TV, Total porosity, Tb.Th and BV/TV in different groups.

collagen fibers to form calcium nodules. Therefore, the activity of ALP
can be used to recognize osteoblasts and determine the degree of dif-
ferentiation and status of osteoblasts. The level of calcium can also
reflect the degree of osteoblast differentiation, maturation, collagen
fiber formation and new bone calcification. Hence, we examined the
ALP activity and calcium content of all groups (Fig. 5B&C), and the
results showed time-dependence. Only slight differences in ALP activity
and calcium content were found between the 2 groups at the second
week. However, the increase of ALP activity and calcium content in the
BMP-2 group slowed down, whereas the BMP-2/MPEG-PCL-MS group

7
D. Kong, et al.
continued to increase in the later stage. There was a statistically sig-
nificant difference (P < 0.05) both in ALP activity and calcium content
assay between 2 groups at the same time point, which indicates that the
microspheres could maintain the biological activity of BMP-2 in vivo for
a longer time and better achieve its slow release effects.
3.7. Histological examination
Studies have found that both morphological and metabolic char-
acteristics of heterotopic ossification are indistinguishable from normal
ossification. Once damaged, the periosteum and bone marrow of the
damaged parts play a role in the healing process. The ectopic osteo-
genesis can eliminate the false positive result caused by the bone for-
mation of the recipient bone, periosteum, and bone marrow. Therefore,
this paper further observed the ectopic osteogenic induction ability of
each group via H&E stain. In the BMP-2 solution group, relatively ma-
tured chondrocytes were observed at 2 weeks, and a small amount of
woven bone was observed in some parts, which is the histological
transition from cartilage to bone. The bone maturation of BMP-2/
MPEG-PCL-MS was similar during this period. At 4 weeks, a thin layer
of new bone was seen around the edge of the ectopic bone in the BMP-2
solution group, and fibrous tissue was visible in the interstitial bone. In
the BMP-2/MPEG-PCL-MS group, a large amount of matured bone
tissue was observed. The number of trabecular bone and new bones is
significantly higher than that of the BMP-2 solution group. At 8 weeks,
the BMP-2 solution group was similar to that at 4 weeks. However, the
BMP-2/MPEG-PCL-MS group showed a large amount of mature new
bone tissue. Its continuous new bone thickness increased more than that
of the BMP-2 solution and microsphere groups at 4 weeks. Hence, we
deduced that the longer the time, the more matured the new bone.
Furthermore, the degree of bone maturity and the size of the new bone
in the BMP-2/MPEG-PCL-MS group were much larger than those in the
BMP-2 solution group (Fig. 6).
3.8. Micro-CT analysis
To further evaluate the ectopic osteogenic induction ability of mi-
crospheres, we analyzed the related indicator of bone by using micro-
CT, The micro-CT image of the ectopic bone was shown in supple-
mentary information Fig. S7. As vitally intuitive indicators, BV and TV
results showed in Fig. 7, the BMP-2/MPEG-PCL-MS group was much
larger than the BMP-2 solution group, which indicated that micro-
spheres could better promote the growth and differentiation of osteo-
blasts in a period than BMP-2 solution. The total porosity of the ectopic
bone could affect the gene expression and osteogenic differentiation of
osteoblasts, which in turn affect the osteogenesis quality of the ectopic
bone. Generally, the total porosity of ideal osteogenic scaffold material
should exceed 80 %. In the results, both groups were above 80 %, which
might be ideal bone repair material [42]. Low bone density is currently
recognized as a major risk factor for fractures [43]. The trabecular bone
could reflect the quality of the osteogenesis to a certain extent [40]. If
the osteogenesis of the ectopic bone is poor, the osteoclasts would lead
to the resorption of ectopic bone and eventually to the reduction of
Tb.N and Tb.Th. BV/TV also played an important role in the mechanical
properties of bones, which could directly reflect yield strength and
stiffness [44,45]. The data revealed that the BMD, Tb.N, Tb.Th, and BV/
TV in the BMP-2 solution group at the same period were all lower than
that of the BMP-2/MPEG-PCL-MS group, which demonstrated that the
bone quality and mechanical properties in the microsphere group were
better than the control group. All the results showed that both micro-
spheres and BMP-2 solution had an effect on bone growth. However,
the bone quality of BMP-2 solution was much weaker than that of the
microsphere group. Combined with the results in drug release, we de-
duced that microspheres might make BMP-2 more stable and sustain-
able in the targeted site and further improve the quality of osteogenesis.
8
Biomedicine & Pharmacotherapy 130 (2020) 110516
4. Conclusions
In this study, BMP-2/MPEG-PCL sustainable microspheres were
prepared by the double emulsion method. MPEG-PCL was used as
carrier material to overcome the short half-life of BMP-2 and the large
doses in the clinic. The physicochemical properties, in vitro release, and
biocompatibility of the drug-loading system was evaluated, and the
effect of induced osteogenesis in vivo was examined. The results showed
that both the EE% and DL% of the microspheres were high, the particle
size is uniform, the appearance is smooth and round, and the fluidity is
good. The influencing factors test showed that BMP-2/MPEG-PCL-MS
has good stability and can meet the requirements of industrial pro-
duction. BMP-2, BMP-2/MPEG-PCL microspheres, and blank micro-
spheres had no obvious side effects on C2C12 cells and good bio-
compatibility. Ectopic osteogenesis experiments showed that BMP-2/
MPEG-PCL-MS had higher bone size, bone density, ALP activity and
calcium content than BMP-2 solution group. It is proved that BMP-2/
MPEG-PCL-MS can not only exert sustained-release in vivo, but also
maintain BMP-2 activity. The prepared BMP-2/MPEG-PCL-MS can be
directly injected into the bone defect site, or implanted to a large bone
defect site together with stent material to exert therapeutic effects.
Ethical conduct of research
The authors state that they have obtained appropriate institutional
review board approval or have followed the principles outlined in the
Declaration of Helsinki for all human or animal experimental in-
vestigations.
Declaration of Competing Interest
This work was supported by the Major Research & Development
Program of Shandong Province (No. 2015GGX103022). The authors
confirm that there are no known conflicts of interest associated with
this publication and there has been no significant financial support for
this work that could have influenced its outcome.
Acknowledgments
This work was supported by National Natural Science Foundation of
China (No. 21873057), the Major Research & Development Program of
Shandong Province (No. 2015GGX103022) and the Major Research &
Development Program of Shandong Province (No. 2017GGX202007).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2020.110516.
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