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Injectable Alginate Hydrogel Cross-Linked by Calcium Gluconate-
Loaded Porous Microspheres for Cartilage Tissue Engineering
JinFeng Liao,
†,‡
BeiYu Wang,
†
YiXing Huang,
∥
Ying Qu,
†
JinRong Peng,
†
and ZhiYong Qian*
,†
†
State Key Laboratory of Biotherapy and Cancer Center, and Collaborative Innovation Center for Biotherapy, West China Hospital
and
‡
State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of
Stomatology, Sichuan University, Chengdu 610041, P. R. China
∥
Department of Orthopaedic Surgery, Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, P. R. China
*
SSupporting Information
ABSTRACT: A great interest has been shown in the
injectable scaffolds for cartilage tissue regeneration because it
can fill irregularly shaped defects easily through minimally
invasive surgical treatments. Herein, we developed a new
injectable three-dimensional (3D) alginate hydrogel loaded
with biodegradable porous poly(ε-caprolactone)−b-poly-
(ethylene glycol)−b-poly(ε-caprolactone) microspheres
(MPs/Alg) as the calcium gluconate container to cross-link
alginate. Suspensions of chondrocytes/alginate and porous
microspheres turned into a gel because of the release of
calcium gluconate; thus, the injectable composite hydrogels
give a 3D scaffold to fit the defects perfectly and integrate the extracellular-matrix-mimicking architecture to efficiently
accommodate cartilage cells in situ. Tissue repair in a full-thickness cartilage defect model was controlled at 6, 12, and 18 weeks
after the implant by micro-CT and immunohistochemistry to evaluate the healing status. The defect in the MPs/Alg+ cells group
achieved an almost complete repair at 18 weeks, and the repaired chondrocytes regained a normal tissue structure. Moreover, the
MPs/Alg+ cells-treated group increased the quality of tissue formed, including the accumulated glycosaminoglycan and the
uniformly deposited type II collagen. The results point out the promising application of the injectable MPs/Alg-chondrocytes
system for cartilage tissue engineering.
1. INTRODUCTION
The self-repairing ability of articular cartilage after damage is
very limited because of the low metabolic and biosynthetic
activities of mature chondrocytes and the nonavailability of
chondrogenic cells.
1−3
Many strategies have been applied to
enhance the cartilage defect repair with the ultimate aim of
filling the defects with the same morphological and functional
repaired cartilage tissue. Osteochondral allografting, periosteal
and perichondral tissue grafting, chondrogenic cell trans-
plantation, and subchondral drilling have been widely employed
in preclinic and clinic trials.
4
The concept of tissue engineering
has been used to develop cell-based repair biomaterials.
5−8
Numerous studies using chondrocytes or cells with chondro-
genic potential (mesenchymal stem cells and adipose stem
cells) have suggested that different biomaterials can support the
proteoglycan-containing tissues and the formation of type II
collagen (COL II).
9,10
Specific extracellular matrix components
of the articular cartilage, such as glycosaminoglycan (GAG) and
collagen, play critical roles in supporting chondrogenesis and
regulating the expression of the chondrocytic phenotype.
11,12
Injectable scaffolds have been paid particular attention for
cartilage tissue engineering because of their ability to fill
irregularly shaped defects by minimally invasive procedures.
The injectable tissue engineering system has applications in
numerous materials. The scaffold of an injectable tissue
engineering system must possess physical properties that
allow it to be injected via a syringe or catheter.
13−15
However,
when implanted in the body, the scaffold materials should
maintain a desired form or shape in defect location without
diffusion or movement, acquiring more significant mechanical
properties.
16
Hydrogels are a class of materials that meet the
requirements for a successful injectable tissue engineering
system. Many methods have been employed to prepare
injectable in situ forming hydrogels, including photopolymeri-
zation,
17,18
enzymatic cross-linking,
19,20
and chemical cross-
linking with cross-linker agents (such as glutaraldehyde,
carbodiimide, genipin, adipic dihydrazide, etc.).
21−24
Unfortu-
nately, photopolymerization usually needs a photosensitizer
and prolonged irradiation, which limit its applications. On the
other hand, toxic chemical cross-linkers are the major obstacles
for chemical cross-linking to the use of injectable in situ
forming scaffolds.
25
It is worth mentioning that alginate is most
commonly used as an instant hydrogel for bone tissue
engineering because of its hydrophilicity, biocompatibility,
Received: December 11, 2016
Accepted: January 30, 2017
Published: February 9, 2017
Article
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© 2017 American Chemical Society 443 DOI: 10.1021/acsomega.6b00495
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This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
and biodegradability.
26,27
Alginate hydrogel with different
shapes can be prepared by cross-linking at normal temperature
in the absence of organic solvents. Chondrocytes encapsulated
within calcium alginate hydrogels have shown a great promise
as engineered scaffolds to repair articular cartilage, with the
chondrocytes secreting cartilage markers, such as GAGs and
COL II.
28
On the other hand, most injectable hydrogels have
limitations such as having sufficient mechanical stability and
durability to support cell proliferation/differentiation before the
formation of new tissue. One of the attempts to overcome this
challenge was to use biodegradable microparticles, which have
also been applied as injectable matrices for tissue regeneration
and cell delivery.
29
Mixtures of hydrogel and microspheres were
widely used in cartilage repair;
30
however, a simple mixing of
these results in ignoring some properties like strength moduli
and environmental responsibility.
Porous biodegradable microparticles are viewed as a useful
tool for the delivery of proteins,
31
antitumor drugs,
32
and
temporal templates for various tissue regeneration applica-
Figure 1. Preparation scheme of calcium gluconate loaded in porous PCEC microspheres/alginate hybrid hydrogel in situ formed in cartilage defect
repair.
Figure 2. (A-1−C-1) Light microscope images of the porous PCEC microspheres made from PCEC copolymer with different molecular weights. A-
1, B-1, and C-1 are the magnifications of A-1, B-1, and C-1, respectively.
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tions.
33,34
Porous microparticles made from block polymers,
such as poly(D,L-lactic acid) (PDLLA) and poly(D,L-lactic-co-
glycolic acid) (PLGA), were generally prepared by a water-in-
oil-in-water (w/o/w) double-emulsion solvent evaporation
technique in tissue engineering.
35−37
However, PDLLA and
PLGA had some disadvantages, including the possibility of
acute or chronic inflammatory responses, potential localized pH
decreases because of the relative-acidity-degraded byproducts,
and a retarded clearance rate.
38,39
To overcome these
limitations, amphiphilic poly(ε-caprolactone)−b-poly(ethylene
glycol)−b-poly(ε-caprolactone) (PCL−PEG−PCL, abbrevi-
ated to PCEC) polymers were synthesized in our study and
applied to prepare porous microspheres, which were loaded
with calcium gluconate crystals as the cross-linker to cross-link
alginate. The microspheres can release calcium gluconate once
combined with an alginate solution. In this respect, we
developed a novel hybrid injectable alginate hydrogel with
porous PCEC microspheres as a container of calcium gluconate
(MPs) and used the respective hydrogels as models for three-
dimensional (3D) scaffolds for in vitro and in vivo experiments.
The suspension of the calcium gluconate/alginate mixture
loaded with porous PCEC microspheres can be injected into a
cartilage defect site and cross-linked in situ under normal
physiological conditions for about 3 min with the release of
calcium ions, thereby eliminating the need for invasive
implantation procedures and retrieval surgeries (the prepara-
tion procedure of the hybrid hydrogel and cartilage defect
repair in rabbit is shown in Figure 1). More specifically, the
mechanical strength and the degradation rate of the scaffold
may be controlled by the incorporation of porous PCEC
microspheres. Furthermore, calcium gluconate may further
enhance the cartilage tissue repair with calcium supply.
40,41
This injectable hybrid hydrogel may yield synergetic effects,
such as good mechanical strength, shape-persistent ability,
stabilization of the microspheres, and biological performance.
We would expect that this hybrid scaffold has a special
advantage in cartilage and bone engineering. Furthermore, this
is an unexplored biomaterial system so far to the best of our
knowledge.
2. RESULTS AND DISCUSSION
2.1. Preparation and Characterization of Porous PCEC
Microspheres. The PCEC copolymer was synthesized by
ring-opening polymerization of ε-CL and PEG 4000. PCEC
was successfully synthesized by characterization with 1H NMR,
as shown in Figure S1. The porous PCEC microspheres were
prepared by a double-emulsification/solvent evaporation
method with ammonium bicarbonate as the porogen. We
tuned the molecular weight of the PCEC polymers to control
their structural and morphological characteristics. As shown in
Figure 2, for 30, 50, and 60 kDa PCEC polymers, the average
diameters of the microspheres were 124 ±23, 203 ±35, and
218 ±40 μm, respectively. The porous structure is
homogeneous, and the pores were interconnected. With
increasing molecular weight, the microspheres became
spherical, and a more porous structure could be obtained. On
the other hand, when the molecular weight of the polymer
increased, the pores become smaller (pore sizes of micro-
spheres made of 30, 50, and 60 kDa were ∼10, ∼8, and ∼7μm,
respectively). Considering the more optimized morphology for
cell growth, we chose the polymer with a molecular weight of
50 kDa to prepare porous microspheres.
Next, scanning electron microscopy (SEM) of the micro-
spheres (50 kDa) was observed, as shown in Figure 3A-2,A-3.
The average diameter of the open pore was about 8 μm in the
dry state. The pores may be larger than 8 μm in the wet state,
Figure 3. Microscope and SEM images of (A-1−A-3) porous PCEC microspheres; (B-1−B-3) porous PCEC microspheres loaded with calcium
gluconate on the surface; and (C-1−C-3) porous PCEC microspheres loaded with calcium gluconate in the holes.
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which was suitable for cell growth in the inner sides of the
microspheres (Figure 3A-1). The wrinkling of the microspheres
in the dry state is due to their hydrophilicity (amphiphilicity the
PCEC polymer). The porosity of the PCEC microspheres was
greater than 90%, indicating a highly interconnected porous
structure.
2.2. Porous PCEC Microspheres as the Container of
Calcium Gluconate. Considering the insolubility of calcium
gluconate in EtOH, we have investigated the preparation
method of calcium gluconate deposited in porous PCEC
microspheres. Porous PCEC microspheres were first soaked in
EtOH, and a 3% calcium gluconate solution was added. This
process was performed three times. Calcium gluconate
deposited on the surface of the porous PCEC microspheres
was observed by upright microscope and SEM (Figure 3B-1−B-
3). Energy-dispersive X-ray spectroscopy (EDX) scans obtained
from the loaded calcium gluconate crystals showed that the
weight percentage of calcium is 10.09% (Figure 4A). In another
situation, porous PCEC microspheres were soaked in a 3%
calcium gluconate solution first. Then, the calcium gluconate
solution was decanted, and EtOH was added to deposit the
calcium gluconate particles in the pores of the microspheres.
This process was also performed three times. The holes of the
porous PCEC microspheres were loaded with calcium
gluconate, thereby decreasing the transmittance of light (Figure
3C-1), and the microspheres became satiated (Figure 3C-2,C-
3). EDX analysis suggested that a trace amount of calcium
element was deposited on the surface of the microspheres
(Figure S2). To demonstrate calcium gluconate in the holes, we
used liquid nitrogen to facilitate cryo-fracturing of the
microspheres. As shown in Figure 4B, the broken microspheres
were rough, and the calcium gluconate crystals were in the
holes of the microspheres. EDX scans demonstrated that
calcium gluconate was successfully loaded in the interior of the
porous microspheres. The percentage of calcium in the holes
was lower than that on the surface because of the interference
of microspheres. Furthermore, to quantify the calcium content,
the microspheres were soaked in water to release the whole
loaded calcium ions. The amounts of calcium gluconate
deposited on the surface or inner holes of porous PCEC
microspheres measured by inductively coupled plasma-atomic
emission spectrometer (ICP-AES) were 96 and 183 μg/mg,
respectively. Considering that the calcium gluconate crystals
loaded on the surface of the microspheres tend to fall offand
the time to form hydrogel when mixed with an alginate solution
was short (∼30 s), we chose the other one (loaded in the inner
pores of the microspheres) for further study.
Figure 4. EDX element analysis of calcium gluconate crystal (A) on the surface and (B) in the holes of porous PCEC microspheres.
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2.3. Characterization of Porous PCEC Microspheres/
Alginate Hybrid Hydrogel. Calcium gluconate crystals
loaded in the pores of the microspheres (50 mg) were mixed
with a 1.5% alginate solution (0.75 mL). After homogenizing
the mixture, these crystals can release calcium ions when
contacting with water to cross-link alginate. The gel formation
time was only ∼3 min, leaving enough time to conduct the in
vitro and in vivo experiments. Figure 5 displays the cross-
sectional morphology of the prepared porous PCEC micro-
spheres/alginate hybrid hydrogel (MPs/Alg). The hybrid
hydrogel had interconnected pores of mean diameter ∼195
μm, and the pores were partly incorporated with the
microspheres. The compressive modulus of the MPs/Alg
hybrid hydrogel scaffold was investigated. The mechanical
properties of the MPs improved significantly when they
blended with the alginate hydrogel. The MPs/Alg hybrid
hydrogel displayed an increased compressive modulus of 123.6
kPa on addition of PCEC microspheres, compared to that of
the alginate hydrogel itself (18.7 kPa).
2.4. Cell Morphology on PCEC Microspheres/Alginate
Hydrogel. Chondrocytes have a good tolerance to the calcium
alginate scaffold because of the inert alginate scaffold
maintaining their morphology and phenotype as reported.
42
To assess the porous PCEC microspheres and the MPs/Alg
hydrogel for the cultivation of chondrocytes, articular
chondrocytes were seeded and cultured on the two scaffolds
for 3 days. Then, SEM was used to observe the chondrocytes
grown on the scaffolds to evaluate cell morphology and
proliferation. Figure 6A, B shows chondrocytes growing on
PCEC microspheres and MPs/Alg hydrogel, respectively. It was
found that the chondrocytes were first anchored on the
superficial area of both scaffolds and kept their spherical
morphology. Furthermore, the production of GAG is a critical
feature of cartilage cells, which plays an important role in the
phenotype regulation of the chondrocytes. As Figure 6C
displays, the GAG amount of chondrocytes culture on the
microspheres/alginate hydrogel increased significantly over
time. This indicates that the microspheres/alginate hydrogel
Figure 5. (A) SEM images and (B, C) the magnification of the cross section of porous PCEC microspheres/alginate hydrogels (scale bars were 100
μm).
Figure 6. SEM photographs of (A) porous PCEC microspheres and (B) porous PCEC microspheres/alginate hydrogel cultured with cartilage cells
for 3 days (scale bars were 10 μm); (C) GAG quantification assays after 1, 3, and 7 days of culture with cartilage cells on the microspheres/alginate
hydrogel.
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was compatible and supplied a microenvironment, which
facilitates the proliferation of chondrocytes.
2.5. In Vivo Formability and Absorption of Hybrid
Scaffold. To examine the application potential of using the
PCEC microspheres/alginate hydrogel for injectable therapy,
the PCEC microspheres, alginate, and hybrid scaffold were
implanted into mice for in vivo formability and degradability.
The PCEC microspheres/alginate hydrogel could be success-
fully administered by injection through a syringe, instead of a
surgical procedure. The scaffolds with diameter of up to 1 cm
were collected into a suspension solution for injection. The
PCEC microspheres/alginate hydrogel could pass through an
18 G needle without eliciting a clogging problem in the needle
during the injection. Tissue mounds were formed immediately
after the injection (Figure 7C-1−C-3). However, the micro-
spheres cannot be concentrated in one place and may diffuse to
other sites after implantation (Figure 7A-1−A-3), which may
cause inflammation and embolization.
43
Also, an 1.5% alginate
adhesive solution is not stable enough to fix a circle under the
subcutaneous tissue (Figure 7B-1−B-3).
After implantation for 1 week, the Alg and MPs/Alg scaffold
had no obvious loss (Figure S3B-1,C-1). Over time, progressive
absorption of the implanted composite occurred. The materials
(Alg and MPs/Alg scaffold) became relatively small at 2 weeks
compared to the state at 1 week (Figure S3B-2,C-2), indicating
that part of the implanted composite has been absorbed.
However, the Alg group needed shorter time to be absorbed by
the body, and the remaining Alg scaffold was significantly
smaller compared to the hybrid scaffold at 3 weeks (Figure
S3B-3,C-3). Thus, the longer time required for the degradation
of the MPs/Alg scaffold was suitable for cartilage tissue
engineering. Furthermore, the shape of the MPs/Alg hydrogel
remained unchanged during the implantation process. How-
ever, it is difficult to observe the formability and absorption of
PCEC microspheres (Figure S3A-1−A-3).
2.6. In Vivo Study of the PCEC Microspheres/Alginate
Hydrogel in Rabbit Cartilage Defects. The ability of the
scaffold to improve cartilage regeneration was evaluated by the
reconstruction of full-thickness cartilage defect on the left knees
of rabbits. The images of different treated groups are presented
in Figure S4. During postoperative period, the rabbits did not
show significant inflammation and rejection response. Their
incised skins healed gradually, and all animals remained in good
health. Then, we assessed the cartilage regenerative capability
by micro-CT reconstruction evaluation, histological score
analysis, and immunohistochemical assessment.
2.6.1. Micro-CT Reconstruction Evaluation. In the animal
experiment, a full-thickness cartilage defect was used as the
Figure 7. Fixed shapes of (A-1−A-3) porous PCEC microspheres, (B-1−B-3) 2% alginate solution, and (C-1−C-3) porous PCEC microspheres/
alginate hydrogels after subcutaneous injection in Wistar rats.
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defect model, which stretched deep beneath the tidemark,
without penetrating the subchondral bone plate.
44
Thus, this
defect model involved the cartilage and part of the subchondral
bone. As shown in the 3D (Figure 8A) and two-dimensional
(2D) (Figure 8B) reconstruction images, the newly regenerated
tissue generally grew from the margin of defects to the central
areas. After 18 postoperative weeks, the defects treated with
MPs/Alg hydrogel and MPs/Alg hydrogel with cells supple-
ment were mainly covered with a new cartilage and a
subchondral bone. On the contrary, the regeneration of defects
in other four groups appeared depressed, and there was still a
blank zone after 18 weeks. Then, we isolated the defect areas as
the region of interest (ROI) (Figure 8C). The regenerated
cartilage and bone were indicated by blue and gray, respectively.
It is worth noting that the defect in the MPs/Alg+ cells-treated
group was almost repaired, and a regenerated matching
cartilage layer was formed.
2.6.2. Semiquantitative Histological Scoring Analysis. As
the repair efficacy is an important feature of defect substitutes,
the regeneration percentage of bone tissue is generally marked.
Because the defect sections were treated as ROI, the
quantitative data about the volume of cartilage and subchondral
bone could be analyzed. The percentage of newly grown bone
volume in the MPs/Alg group progressed from 43.8% at 6
weeks to 89.9% at 18 weeks after implantation (Figure 9A).
Moreover, 96.7% of the osteochondral tissue was regenerated
by MPs/Alg+ cells at 18 weeks, which spread the largest
Figure 8. Micro-CT images acquired from (A) 3D reconstruction (red circles indicated the defect areas), (B) 2D reconstruction in longitudinal view
(red squares indicated the defect areas), and (C) 3D reconstruction of the repaired cartilage defect of different groups (the newly grown cartilage and
bone are indicated by blue and gray, respectively) after operation at different periods.
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volume of the defect compared to that of blank, MPs, Alg, and
cells groups.
The histological score assessment was evaluated from five
aspects, as shown in Table S1. The score of the MPs/Alg+ cells
group was better (i.e., lower) than that of other groups at
determined time points after operation (Figure 9B); however,
there was no significant difference between the MPs/Alg group
and the MPs/Alg+ cells group. The results indicated that the
scaffold alone played an important role in the cartilage
regeneration. The structure of the scaffold had space for the
growth of cartilage cells. Moreover, the characteristics of the
scaffold may enhance the immigrated MSC cells to
chondrogenic differentiation. Along with the cell proliferation,
the scaffold degraded and was absorbed by the body, and a new
cartilage was formed instead of scaffold.
2.6.3. Histological Immunohistochemical Evaluation. By
H&E staining analysis, 6 weeks after treatment with the MPs/
Alg+ cells, the newly formed cartilage cells were uniformly
distributed without orderly aligning in the defects, whereas few
newly formed tissues appeared in the control group (Figure
10A). A group of scaffold-treated defects could be observed in
some chondrocytic cells. It is interesting that the MPs/Alg+
cells group regrew a layer of cartilage, which highly expressed
GAG (Figure 10B) and COL II (Figure 10C). The group of
MPs/Alg also showed the recovered hyaline cartilage and the
subchondral bone. In contrast, defects in control or those
transplanted with Alg, MPs, or cells only showed a limited
repair, resulting in a negligible expression of GAG and COL II.
Over time, a number of fibrocartilage cells appeared in control,
MPs, and cell groups. The Alg-group-induced repair tissues
exhibited an irregular link with the original cartilage compared
to that of the MPs/Alg+ cells and MPs/Alg groups. However,
the Alg-treated group was superior to the MPs and cells groups.
It is worth noting that the MPs/Alg+ cells-treated group
displayed even a normal regenerated subchondral bone and
chondrocytes. Meanwhile, it is difficult to distinguish the
interface between the repaired cartilage and the host cartilage
through the homogeneous staining of COL II and GAG. The
MPs/Alg group had a relatively thinner newly regenerated
cartilage, and the junction of this cartilage and the host cartilage
was irregular. The MPs/Alg hydrogel with cell supply showed a
successful repair in the full-thickness cartilage defect. Thus, the
injectable MPs/Alg hydrogel has been demonstrated to be an
effective carrier for chondrocytes and worthy of further
investigation toward the desired clinical application. The
MPs/Alg hydrogel will also be evaluated as a carrier for other
cells to regenerate other tissues in the future.
3. CONCLUSIONS
In summary, we demonstrated the feasibility of calcium
gluconate cross-linked alginate hydrogel prepared using
biodegradable porous microsphere as the cross-linker carrier
as an injectable hybrid scaffold. This injectable scaffold may be
useful to meet different shape defects and regrow cartilage
layers by a minimally invasive approach. The hybrid hydrogel
has desirable features, such as interconnected pores, enhanced
compressive modulus, good formability, and reasonable
degradability. Chondrocytes seeded on the hydrogel could
proliferate well and maintain their chondrogenic property.
Then, the reparative ability of the porous PCEC microspheres/
alginate hydrogel was assessed in repairing full-thickness
cartilage defects in a rabbit model. The results indicated that
the porous PCEC microspheres/alginate hydrogel is a suitable
substrate for cartilage tissue engineering.
4. EXPERIMENTAL SECTIONS
4.1. Materials. Poly(ethylene glycol) (PEG, Mn= 4000), ε-
caprolactone (ε-CL), stannous octoate (Sn(Oct)2), and
poly(vinyl alcohol) (PVA, average Mn= 30 000−70 000)
were obtained from Sigma-Aldrich Company. Calcium D-
gluconate monohydrate was purchased from Aladdin Industrial
Corp., Shanghai, China. Alginate, ammonium bicarbonate
(NH4HCO3), dichloromethane, phosphate buffer saline, and
sodium hydroxide were acquired from Kelong Chemicals,
Chengdu, China. Deionized water (18.2 MΩcm) obtained
from Milli-Q Gradient System was used in all of the
preparations.
For the animal experiment, we purchased 54 New Zealand
White rabbits (male; initial weight: 2−2.5 kg) from the
Experimental Animals Center of Sichuan Province, China. They
were separated into six groups evenly on the basis of the
different time points. A total of 36 Wistar rats (male; weight:
200 g), purchased from Beijing HFK Bioscience Co., Ltd., were
used to investigate the formability and absorption of the
scaffolds in vivo. All animal studies were approved by the
animal care and use committee of the State Key Laboratory of
Biotherapy, Sichuan University.
4.2. Preparation of PCEC Copolymer. PCEC copolymers
with different designed molecular weights were synthesized by
ring-opening polymerization of poly(ε-caprolactone) (ε-CL)
and poly(ethylene glycol) (PEG, Mn= 4000) catalyzed by
Sn(Oct)2at 130 °C in accordance with our previous reports.
45
The obtained PCEC copolymers were characterized by 1H
NMR spectroscopy (Bruker 400 spectrometer, German) and
GPC (HLC-8320GPC, EcoSEC, TOSOH, Japan).
4.3. Preparation of Porous PCEC Microspheres. Porous
PCEC microspheres were fabricated by a w/o/w double-
emulsion method. In brief, 3.75 mL of 5% NH4HCO3solution
Figure 9. (A) Volume of newly formed bone tissue and (B)
histological scoring for reparative tissues in different groups at 6, 12,
and 18 weeks after operation. (*p< 0.05).
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was injected into 12 mL of 62.5% PCEC methylene chloride
solution using a Powergen 700 homogenizer at 5000 rpm for 3
min. Then, the first w/o emulsion was poured into 450 mL of
0.5% PVA solution immediately and re-emulsified using an
overhead propeller (LR-400A, Fisher Scientific Co.) at 1500
rpm for 8 h. After methylene chloride was evaporated, the
obtained microspheres were separated by a filter net, etched
with NaOH, washed with distilled water, and lyophilized by a
lyophilizer. The morphologies of the microspheres were
observed by a digital image system (Nikon E 600 Microscope
with a Nikon Digital Camera DXM 1200, Nikon Corporation,
Japan) and SEM (JSM-5900LV, JEOL, Japan).
4.4. Calcium Gluconate Deposited in PCEC Micro-
spheres. Porous PCEC microspheres were soaked in a 3%
calcium gluconate solution for 3 h. Then, the entire calcium
gluconate solution was decanted, and EtOH was added to
deposit calcium gluconate in the pores of PCEC microspheres.
The above process was conducted three times. Initially, when
the microspheres were soaked in EtOH with the reverse
sequence, calcium gluconate was loaded on the surface of the
microspheres. The obtained calcium gluconate-loaded micro-
spheres were lyophilized. The morphologies of the calcium
gluconate deposited in the microspheres were observed by
SEM, and elemental analysis was performed by an EDX setup
installed in the SEM. The amounts of calcium gluconate were
measured by ICP-AES (SPECTRO ARCOS, Spectro, Ger-
many).
4.5. Preparation and Characterizations of PCEC
Microspheres/Alginate Hydrogel. First, 10 mL of 1.5%
stock alginate solution was prepared. Then, 50 mg of calcium D-
gluconate monohydrate-loaded microspheres were mixed with
the alginate stock solution (0.75 mL) while homogenizing the
mixture to obtain a homogeneous distribution of microspheres
and calcium ion cross-linking. The cross-sectional morpholo-
gies of the microspheres/alginate hydrogel were analyzed by
SEM after lyophilization. The mechanical properties of the
PCEC microspheres/alginate (MPs/Alg) hydrogel were
measured by an Instron 5500 mechanical tester (Instron Cor.).
Figure 10. (A) H&E staining, (B) Saf-O staining, and (C) COL II staining in six groups at 6, 12, and 18 weeks after operation (magnification 100×).
Note: the arrows represent the host cartilage and repair cartilage boundary; RC and HC represent repaired cartilage and host cartilage, respectively.
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4.6. Cartilage Cells Culture on the PCEC Microspheres
and Hybrid Microspheres/Alg Hydrogel. Primary articular
cartilage cells were obtained from the knee joints of newborn
rabbits. The cartilage layers were isolated and cut into small
pieces, and 0.2% collagenase II was used to digest the cartilage
slices. To obtain the chondrocytes, the digested cell suspension
was filtered and centrifuged. The primary chondrocytes were
cultured in Dulbecco’s Modified Eagle Medium with 100 U/
mL of penicillin, 10% fetal calf serum, and 100 μg/mL of
streptomycin at 37 °C under 5% CO2. When the chondrocytes
were cultured to passage 3, they were digested to grow on
scaffolds; 1.0 ×104chondrocytes were added into the sterilized
microspheres and the hybrid hydrogel in 24-well culture plates.
The cell-seeded plates were kept in an incubator for
predetermined times. Then, the chondrocytes/scaffolds com-
posites were fixed in 10% formalin solution, dehydrated in
graded ethanol series, and observed by SEM. The GAG
contents for different time intervals were also analyzed.
4.7. In Vivo Formability and Absorbability Inves-
tigation. The formability and biocompatibility of the micro-
spheres/alginate hydrogel were in vivo carried out by
implanting the scaffolds into the back subcutaneous tissue of
Wistar rats. Cobalt-60 irradiation of 25 kGy for 2 h (Sichuan
Academy of Agricultural Sciences) was applied to sterilize the
porous microspheres in this study. The alginate solution was
filtrated by a 0.22 μmfilter membrane for sterilization. The
microspheres, alginate solution, and their mixture were
separately injected into the back subcutaneous tissue of the
rats. Then, three rats in each group at each time point were
sacrificed by an overdose of chloral hydrate. The residual
scaffolds in the subcutaneous tissues were photographed to
record the formability and absorption of scaffold and tissue
appearance around the treated site.
4.8. Cartilage Defect Repair in Vivo. The cartilage defect
experiment in vivo was carried out in our previous study.
46
Briefly, we created full-thickness defect (thickness: 3 mm;
diameter: 4 mm) through the articular cartilage and
subchondral bone of the patellar groove using an electric
drill. The cartilage defects in the left leg of New Zealand White
rabbits were treated with nothing, microspheres, alginate
hydrogel, cartilage cells, microsphere/alginate hydrogel, and
microsphere/alginate hydrogel with cell supplement (each
group, n= 9). The animals were given antibiotic for 1 week
postoperatively, housed at a constant temperature (22 °C), and
given food and tap water. Joint samples were gathered at
predetermined time points after operation and processed for
micro-CT reconstruction and histological analysis. A micro-CT
scanner (Y. Cheetah, YXLON International GmbH, Germany)
was used to reconstruct the cartilage defect and analyze the
results. The scan settings were as follows: X-ray voltage = 56
kV, X-ray current = 61 μA, scaling coefficient = 50, and voxel
resolution=0.012mm.Thescanimageswerethen
reconstructed to create a 3D geometry using VGStudioMax
software. For histological examination, the tissues underwent a
series of processes and were stained with Hematoxylin and
Eosin (H&E), Safrannin-O (Saf-O), and Collagen type II
(COL II).
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsomega.6b00495.
Characterization data, degradation ability, cartilage
defect, and standard of histological score assessment
(PDF).
■AUTHOR INFORMATION
Corresponding Author
*E-mail: anderson-qian@163.com. Tel/Fax: +86-28-85501986.
ORCID
ZhiYong Qian: 0000-0003-2992-6424
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was financially supported by the National Natural
Science Foundation of China (31525009 and 31271021),
National High-Tech Project of China (863-Project,
2015AA020316), Sichuan Innovative Research Team Program
for Young Scientists (2016TD0004), the International Science
& Technology Cooperation Program of China
(2013DFG52300), and Distinguished Young Scholars of
Sichuan University (2011SCU04B18). The authors thank Hui
Wang (Analytical & Testing Center, Sichuan University) for
her help in SEM analysis.
■REFERENCES
(1) Rothenfluh, D. A.; Bermudez, H.; O’Neil, C. P.; Hubbell, J. A.
Biofunctional Polymer Nanoparticles for Intra-articular Targeting and
Retention in Cartilage. Nat. Mater. 2008,7, 248−254.
(2) Sridhar, B. V.; Brock, J. L.; Silver, J. S.; Leight, J. L.; Randolph, M.
A.; Anseth, K. S. Development of a Cellularly Degradable PEG
Hydrogel to Promote Articular Cartilage Extracellular Matrix
Deposition. Adv. Healthcare Mater. 2015,4, 702−713.
(3) Naranda, J.; Sušec, M.; Maver, U.; Gradišnik, L.; Gorenjak, M.;
Vukasović, A.; Ivković, A.; Rupnik, M. S.; Vogrin, M.; Krajnc, P.
Polyester Type PolyHIPE Scaffolds with an Interconnected Porous
Structure for Cartilage Regeneration. Sci. Rep. 2016,6, No. 28695.
(4) Fragonas, E.; Valente, M.; Pozzi-Mucell, M.; Toffanin, R.; Rizzo,
R.; Silvestri, F.; Vittur, F. Articular Cartilage Repair in Rabbits by
Using Suspensions of Allogenic Chondrocytes in Alginate. Biomaterials
2000,21, 795−801.
(5) Singh, Y. P.; Bhardwaj, N.; Mandal, B. B. Potential of Agarose/
Silk Fibroin Blended Hydrogel for in Vitro Cartilage Engineering. ACS
Appl. Mater. Interfaces 2016,8, 21236−21249.
(6) Pelttari, K.; Pippenger, B.; Mumme, M.; Feliciano, S.; Scotti, C.;
Mainil-Varlet, P.; Procino, A.; Rechenberg, B.; Schwamborn, T.; Jakob,
M.; Cillo, C.; Barbero, A.; Martin, I. Adult Human Neural Crest-
derived Cells for Articular Cartilage Repair. Sci. Transl. Med. 2014,6,
No. 251ra119.
(7) Chen, W.; Chen, S.; Morsi, Y.; El-Hamshary, H.; El-Newhy, M.;
Fan, C.; Mo, X. Superabsorbent 3D Scaffold Based on Electrospun
Nanofibers for Cartilage Tissue Engineering. ACS Appl. Mater.
Interfaces 2016,8, 24415−24425.
(8) Armstrong, J. P. K.; Shakur, R.; Horne, J. P.; Dickinson, S. C.;
Armstrong, C. T.; Lau, K.; Kadiwala, J.; Lowe, R.; Seddon, A.; Mann,
S.; Anderson, J. L. R.; Perriman, A. W.; Hollande, A. P. Artificial
Membrane-Binding Proteins Stimulate Oxygenation of Stem Cells
During Engineering of Large Cartilage Tissue. Nat. Commun. 2015,6,
No. 7405.
(9) Lee, J. E.; Kim, K. E.; Kwon, I. C.; Ahn, H. J.; Lee, S.-H.; Cho, H.;
Kim, H. J.; Seong, S. C.; Lee, M. C. Effects of the Controlled-Released
TGF-β1 from Chitosan Microspheres on Chondrocytes Cultured in a
Collagen/Chitosan/Glycosaminoglycan Scaffolde. Biomaterials 2004,
25, 4163−4173.
ACS Omega Article
DOI: 10.1021/acsomega.6b00495
ACS Omega 2017, 2, 443−454
452
(10) Zhang, J.; Wang, J. H.-C. Kartogenin Induces Cartilage-like
Tissue Formation in Tendon-bone Junction. Bone Res. 2014,2,
No. 14008.
(11) Giammanco, G. E.; Carrion, B.; Coleman, R. M.; Ostrowski, A.
D. Photoresponsive Polysaccharide-Based Hydrogels with Tunable
Mechanical Properties for Cartilage Tissue Engineering. ACS Appl.
Mater. Interfaces 2016,8, 14423−14429.
(12) Joeng, K. S.; Long, F. Wnt7b can Replace Ihh to Induce
Hypertrophic Cartilage Vascularization but not Osteoblast Differ-
entiation during Endochondral Bone Development. Bone Res. 2014,2,
No. 14004.
(13) Zhang, N.; Lock, J.; Sallee, A.; Liu, H. Magnetic Nanocomposite
Hydrogel for Potential Cartilage Tissue Engineering: Synthesis,
Characterization, and Cytocompatibility with Bone Marrow Derived
Mesenchymal Stem Cells. ACS Appl. Mater. Interfaces 2015,7, 20987−
20998.
(14) Thoniyot, P.; Tan, M. J.; Karim, A. A.; Young, D. J.; Loh, X. J.
Nanoparticle-Hydrogel Composites: Concept, Design, and Applica-
tions of These Promising, Multi-Functional Materials. Adv. Sci. 2015,
2, No. 1400010.
(15) Ren, K.; He, C.; Xiao, C.; Li, G.; Chen, X. Injectable
Glycopolypeptide Hydrogels as Biomimetic Scaffolds for Cartilage
Tissue Engineering. Biomaterials 2015,51, 238−249.
(16) Elisseeff, J. Injectable Cartilage Tissue Engineering. Expert Opin.
Biol. Ther. 2004,4, 1849−1859.
(17) Khetan, S.; Guvendiren, M.; Legant, W. R.; Cohen, D. M.;
Chen, C. S.; Burdick, J. A. Degradation-Mediated Cellular Traction
Directs Stem Cell Fate in Covalently Crosslinked Three-Dimensional
Hydrogels. Nat. Mater. 2013,12, 458−465.
(18) Söntjens, S. H.; Nettles, D. L.; Carnahan, M. A.; Setton, L. A.;
Grinstaff, M. W. Biodendrimer-based Hydrogel Scaffolds for Cartilage
Tissue Repair. Biomacromolecules 2006,7, 310−316.
(19) Kim, K. S.; Park, S. J.; Yang, J. A.; Jeon, J.-H.; Bhang, S. H.; Kim,
B. S.; Hahn, S. K. Injectable Hyaluronic Acid-tyramine Hydrogels for
the Treatment of Rheumatoid Arthritis. Acta Biomater. 2011,7, 666−
674.
(20) Chen, F.; Yu, S.; Liu, B.; Ni, Y.; Yu, C.; Su, Y.; Zhu, X.; Yu, X.;
Zhou, Y.; Yan, D. An Injectable Enzymatically Crosslinked
Carboxymethylated Pullulan/Condroitin Sulfate Hydrogel for Carti-
lage Tissue Engineering. Sci. Rep. 2016,6, No. 20014.
(21) Vickers, S. M.; Gotterbarm, T.; Spector, M. Cross-Linking
Affects Cellular Condensation and Chondrogenesis in Type II
Collagen-GAG Scaffolds Seeded with Bone Marrow-Derived Mesen-
chymal Stem Cells. J. Orthop. Res. 2010,28, 1184.
(22) Lee, C. R.; Grodzinsky, A. J.; Spector, M. The Effects of Cross-
Linking of Collagen-Glycosaminoglycan Scaffolds on Compressive
Stiffness, Chondrocyte-Mediated Contraction, Proliferation and
Biosynthesis. Biomaterials 2001,22, 3145−3154.
(23) Ko, C. S.; Huang, J. P.; Huang, C. W.; Chu, I. M. Type II
Collagen-Chondroitin Sulfate-Hyaluronan Scaffold Cross-Linked by
Genipin for Cartilage Tissue Engineering. J. Biosci. Bioeng. 2009,107,
177−182.
(24) Wieland, J. A.; Houchin-Ray, T. L.; Shea, L. D. Non-Viral Vector
Delivery from PEG-Hyaluronic Acid Hydrogels. J. Controlled Release
2007,120, 233−241.
(25) Ferretti, M.; Marra, K. G.; Kobayashi, K.; Defail, A. J.; Chu, C.
R. Controlled in vivo Degradation of Genipin Crosslinked Poly-
ethylene Glycol Hydrogels within Osteochondral Defects. Tissue Eng.
2006,12, 2657.
(26) Kehr, N. S.; Prasetyanto, E. A.; Benson, K.; Ergün, B.; Galstyan,
A.; Galla, H.-J. Periodic Mesoporous Organosilica-Based Nano-
composite Hydrogels as Three-Dimensional Scaffolds. Angew. Chem.,
Int. Ed. 2013,52, 1156−1160.
(27) Andrejecsk, J. W.; Cui, J.; Chang, W. G.; Devalliere, J.; Pober, J.
S.; Saltzman, W. M. Paracrine Exchanges of Molecular Signals Between
Alginate-Encapsulated Pericytes and Freely Suspended Endothelial
Cells within a 3D Protein Gel. Biomaterials 2013,34, 8899−8908.
(28) Markstedt, K.; Mantas, A.; Tournier, I.; Ávila, H. M.; Hägg, D.;
Gatenholm, P. 3D Bioprinting Human Chondrocytes with Nano-
cellulose-Alginate Bioink for Cartilage Tissure Engineering Applica-
tion. Biomacromolecules 2015,16, 1489−1496.
(29) Chung, H. J.; Kim, I. K.; Kim, T. G.; Gwan, T. Park Highly
Open Porous Biodegradable Microcarriers: in vitro Cultivation of
Chondrocytes for Injectable Delivery. Tissue Eng., Part A 2008,14,
607−615.
(30) Holland, T. A.; Tabata, Y.; Mikos, A. G. Dual Growth Factor
Delivery from Degradable Oligo(Poly(ethyleneglycol) Fumarate)
Hydrogel Scaffolds for Cartilage Tissue Engineering. J. Controlled
Release 2005,101, 111−125.
(31) Kim, H. K.; Chung, H. J.; Park, T. G. Biodegradable Polymeric
Microspheres with “Open/Closed”Pores for Sustained Release of
Human Growth Hormone. J. Controlled Release 2006,112, 167−174.
(32) Kim, I.; Byeon, H. J.; Kim, T. H.; Lee, E. S.; Oh, K. T.; Shin, B.
S.; Lee, K. C.; Youn, Y. S. Doxorubicin-Loaded Highly Porous Large
PLGA Microparticles as a Sustained-Release Inhalation System for the
Treatment of Metastatic Lung Cancer. Biomaterials 2012,33, 5574−
5583.
(33) Nukavarapu, S. P.; Kumbar, S. G.; Brown, J. L.; Krogman, N. R.;
Weikel, A. L.; Hindenlang, M. D.; Nair, L. S.; Allcock, H. R.;
Laurencin, C. T. Polyphosphazene/Nano-Hydroxyapatite Composite
Microsphere Scaffolds for Bone Tissue Engineering. Biomacromolecules
2008,9, 1818−1825.
(34) Zhang, Z.; Gupte, M. J.; Jin, X.; Ma, P. X. Injectable Peptide
Decorated Functional Nanofibrous Hollow Microspheres to Direct
Stem Cell Differentiation and Tissue Regeneration. Adv. Funct. Mater.
2015,25, 350−360.
(35) Kim, T. K.; Yoon, J. J.; Lee, D. S.; Park, T. G. Gas Foamed Open
Porous Biodegradable Polymeric Microspheres. Biomaterials 2006,27,
152−159.
(36) Lee, J.; Oh, Y. J.; Lee, S. K.; Lee, K. Y. Facile Control of Porous
Structures of Polymer Microspheres Using an Osmotic Agent for
Pulmonary Delivery. J. Controlled Release 2010,146,61−67.
(37) Fan, M.; Liao, J.; Guo, G.; Ding, Q.; Yang, Y.; Luo, F.; Qian, Z.
Dexamethasone-Loaded Poly(D, L-lactic acid) Microspheres/Poly-
(ethylene glycol)-Poly(ε-caprolactone)-Poly(ethylene glycol) Micelles
Composite for Skin Augmentation. J. Biomed. Nanotechnol. 2014,10,
592−602.
(38) Lee, C. H.; Rodeo, S. A.; Fortier, L. A.; Lu, C.; Erisken, C.; Mao,
J. J. Protein-Releasing Polymeric Scaffolds Induce Fibrochondrocytic
Differentiation of Endogenous Cells for Knee Meniscus Regeneration
in Sheep. Sci. Transl. Med. 2014,6, No. 266ra171.
(39) Liao, J.; Shi, K.; Ding, Q.; Qu, Y.; Luo, F.; Qian, Z. Recent
Developments in Scaffold-Guided Cartilage Tissue Regeneration. J.
Biomed. Nanotechnol. 2014,10, 3085−3104.
(40) Onoe, H.; Okitsu, T.; Itou, A.; Kato-Negishi, M.; Gojo, R.;
Kiriya, D.; Sato, K.; Miura, S.; Iwanaga, S.; Kuribayashi-Shigetomi, K.;
Matsunaga, Y. T.; Shimoyama, Y.; Takeuchi, S. Metre-Long Cell-
Laden Microfibres Exhibit Tissue Morphologies and Functions. Nat.
Mater. 2013,12, 584−590.
(41) Wang, P.; Zhao, L.; Liu, J.; Weir, M. D.; Zhou, X.; Xu, H. H. K.
Bone Tissue Engineering via Nanostructured Calcium Phosphate
Biomaterials and Stem Cells. Bone Res. 2014,2, No. 14017.
(42) Cogan, F.; Booth, A.; Gough, J. E.; Webb, S. J. Conversion of
Magnetic Impulses into Cellular Responses by Self-Assembled
Nanoparticle-Vesicle Hydrogels. Angew. Chem., Int. Ed. 2011,50,
12290−12293.
(43) Morhenn, V. B.; Lemperle, G.; Gallo, R. L. Phagocytosis of
Different Particulate Dermal Filler Substances by Human Macro-
phages and Skin Cells. Dermatol. Surg. 2002,28, 484−490.
(44) Altman, R. D.; Kates, J.; Chun, L. E.; Dean, D. D.; Eyre, D.
Preliminary Observations of Chondral Abrasion in a Canine Model.
Ann. Rheum. Dis. 1992,51, 1056−1062.
(45) Dai, M.; Xu, X.; Song, J.; Fu, S. Z.; Gou, M. L.; Luo, F.; Qian, Z.
Y. Preparation of Camptothecin-Loaded PCEC Microspheres for the
Treatment of Colorectal Peritoneal Carcinomatosis and Tumor
Growth in Mice. Cancer Lett. 2011,312, 189−196.
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DOI: 10.1021/acsomega.6b00495
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