Received: 21 February 2019 Revised: 10 May 2019 Accepted: 13 May 2019

DOI: 10.1002/jbm.a.36723

ORIGINAL ARTICLE

An injectable scaffold based on temperature-responsive

hydrogel and factor-loaded nanoparticles for application in
vascularization in tissue engineering

Dan He1 | An-Sha Zhao1 | Hong Su1 | Yan Zhang3 | Ya-Nan Wang1 | Dan Luo1 |
Yuan Gao1 | Jing-An Li1,2 | Ping Yang1

1
Key Laboratory for Advanced Technologies of
Materials, School of Material Science and Abstract
Engineering, Southwest Jiaotong University, Controlled release of functional factors contributes to target migration of therapeutic
Ministry of Education, Chengdu, People’s
Republic of China cells and plays a crucial role in the in situ vascularization of tissue repair and regener-
2
School of Material Science and Engineering, ation. A biomedical application requires the selective release of multiple factors
Zhengzhou University, Zhengzhou, People’s
which will guide the synergy of the cells. Here, we developed an injectable system
Republic of China
3
Department of Cardiology, Chengdu Military based on a temperature-responsive hydrogel and stromal cell-derived factor-1 (SDF-
General Hospital, Chengdu, People’s Republic 1)/vascular endothelial growth factor (VEGF) loaded into two types of nanoparticles
of China
to induce migration and rapid proliferation of mesenchymal stem cells (MSCs) and
Correspondence endothelial cells (ECs) via selective SDF-1/VEGF release. Series of in vitro and in vivo
An-Sha Zhao and Jing-An Li, Key Laboratory
for Advanced Technologies of Materials, experiments demonstrate that our composited system can accurately guide MSCs
School of Material Science and Engineering, and ECs for vascularization. In addition, the properties of the nanoparticles and
Southwest Jiaotong University, Ministry of
Education, Chengdu 610031, People’s hydrogel, including micro/nanoscales, characteristic of charge, and biocompatibility,
Republic of China. played crucial roles for the selective release and cells behavior (target migration and
Emails: anshazhao@home.swjtu.edu.cn,
4828713@qq.com rapid proliferation).

Funding information KEYWORDS

Key Project and Special Foundation of
hydrogels, nanoparticles, selective release, tissue engineering scaffolds, vascularization
Research, Development and Promotion in
Henan province, Grant/Award Number:
182102310076; National Natural Science
Foundation of China, Grant/Award Number:
NSFC 81771988; The Joint Fund for Fostering
Talents of NCIR-MMT & HNKL-MMT, Grant/
Award Number: MMT2017-01; Top Doctor
Program of Zhengzhou University, Grant/
Award Number: 32210475

1 | I N T RO D UC T I O N However, only a few engineered tissues or organs have clinical appli-

Nowadays, tissue engineering is widely recognized as a hot topic in
the field of biomedical science, and it is playing more and more crucial
roles for organ/tissue repair and replacement (Caccavo, Cascone,
Lamberti, & Barba, 2018). Current technologies have achieved the
aims of implanting the biomaterials loaded with the massive in vitro
cultured cells into the human body in order to restore, replace, main-
tain, or improve the function of tissues or organs (Zhang et al., 2018).
cation. The successful examples, such as skin, cartilage, and trachea,
are thin-walled tissues that require less vascularization; the thicker
essential organs, such as the liver and kidney, require a functional vas-
cular network to achieve graft survival in vivo (Griffith & Naughton,
2002). Thus, insufficient vascularization has become a bottleneck hin-
dering the clinical application of the tissue engineering.
Stem cells homing and vascular endothelial cells (VECs) target
migration, as well as their rapid proliferation in situ play important

J Biomed Mater Res. 2019;107A:2123–2134. wileyonlinelibrary.com/journal/jbma © 2019 Wiley Periodicals, Inc. 2123
2124
parts in the vascularization process: wherein stem cells homing, rapid
proliferation, and oriented differentiation mainly contribute to the
regeneration of vascular tissue structure. VECs migration and prolifer-
ation are inclined to vascular functions like relaxation, patency, and
signal molecule release (e.g., nitric oxide and prostatic cyclin), and so
forth (Laiva, Raftery, Keogh, & O'Brien, 2018; Li et al., 2014). Stromal
cell-derived factor-1 (SDF-1) is a cytokine with small molecule weight,
which affects homing of mesenchymal stem cells (MSCs) to the lesion
location via SDF-1/CXCR4 axis (Park, Lee, Kim, Park, & Song, 2018).
Vascular endothelial growth factor (VEGF) is a specific protein which
promotes the VECs migration and proliferation, and it is also the pref-
erable factor on inducing angiogenesis in vivo (Hu et al., 2018). Cer-
tain studies also suggest a positive contribution of VEGF to the
oriented differentiation of MSCs in the specific microenvironment,
such as bone, vascular, and nervous (Lü, Deegan, Musa, Xu, & Yang,
2018; Luo, Zhang, Zhang, & Jin, 2012; Wang et al., 2018).
Hence, loading and sustained release of SDF-1 and VEGF at the
focus of the lesion are effective methods for in situ vascularization in
tissue engineering scaffolds. Yet, the traditional method of direct load-
ing of the factors into the scaffolds has a disadvantage of uncontrolled
release, which may result in disturbed biological response or loss of
function of the loaded factors (Davoodi et al., 2018).
Nanoparticles have been successfully used as carriers for the
delivery of bioactive substances to cells and in tissue engineering (Liu,
Jiang, & Hunziker, 2016). Co-loading of nanoparticles with molecules
of different charge, VEGF (negative charge), and SDF-1 (positive
charge) can improve the controllable and selective release of the
whole delivery system. The development of environmental-responsive
hydrogels with response to temperature, pH, enzymes, and so forth
allows for better targeting of tissue engineering scaffolds (Bao et al.,
2017; Tang et al., 2017; Zhong et al., 2018). In particular, injectable
hydrogel scaffolds may avoid surgical trauma and infection as much as
possible, to further reduce the risk of surgery, and simultaneously
improve in situ the precision of the gel at the focus of the lesion
(Rose & Laura, 2018).
In this contribution, we developed an injectable hydrogel with the
natural cationic polysaccharide chitosan, sodium β-glycerophosphate,
and gelatin as a matrix, to co-load chitosan/fucoidan/VEGF nano-
particles and chitosan/fucoidan/SDF-1 nanoparticles for in situ vascu-
larization. This composite system was temperature responsive and
improved MSCs homing/VEC migration as well as their proliferation.
The chick chorioallantoic membrane (CAM) model was applied to
observe the vascular regeneration via injecting the composite system.
We hope this study may provide a novel research direction for regener-
ative medical biomaterials that require vascularization in situ during
implantation.
2 | MATERIALS AND METHODS
2.1 | Nanoparticles preparation and characterization
Chitosan (degree of deacetylation 9 ≥95%; Aladdin) was added to
0.2% (v/v) aqueous acetic acid with stirring to get a 0.05% (w/v)
HE ET AL.
chitosan solution. The fucoidan solution 0.05% (w/v) was prepared in
distilled water (dH2O) in the same way. The nanoparticles were
obtained by mixing chitosan and fucoidan solutions and then collected
at 14,000 rpm for 4 min. Nanoparticles prepared by chitosan and
fucoidan with volume ratios of 5:5 were labeled as C5F5 and selected
for loading SDF-1 (the SDF-1-loaded C5F5 was labeled as C5F5@SDF-
1), while nanoparticles prepared by chitosan and fucoidan with vol-
ume ratios of 2:5 were labeled as C2F5 and selected for loading VEGF
(the VEGF-loaded C2F5 was labeled as C2F5@VEGF).
The chemical compositions of the C5F5 and C2F5 were examined
by transmission mode of Fourier-transform infrared (FTIR) spectrome-
ter (ST-IR20SX, Nicolet, Madison, Wisconsin, US) after freeze-drying
for 12 hr. The particle size, zeta potential, and polymer dispersity
index (PDI) of C5F5, C2F5, C5F5@SDF-1, and C2F5@VEGF were mea-
sured by a nanoscale laser particle size analyzer (Zetasizer Nano-
ZS90, Malvern Ltd, United Kingdom). The microstructures of C5F5,
C2F5, C5F5@SDF-1, and C2F5@VEGF nanoparticles were observed by
transmission electron microscopy (TEM, JEM-2100F, Hitachi, Japan).
2.2 | Preparation and characterization of the
temperature-responsive hydrogel
Chitosan was dissolved in 0.1 mol/L aqueous acetic acid at room temper-
ature (RT) using a magnetic stirrer (85-1, Shanghai Sile Instrument Co.,
Ltd, China) for 5 h to obtain a 2.2% (w/v) solution. Gelatin (Sigma-Aldrich,
Germany) was dissolved in the dH2O at 60
C to give a 1% solution (w/v).
β-Glycerophosphate (Sigma) was also dissolved in the dH2O at RT to give
a 56% (w/v) solution. Gelatin solution was introduced to the chitosan
solution with a volume ratio of 1:10, then the β-glycerophosphate solu-
tion was added drop by drop with stirring on ice. The volume ratio of the
chitosan solution and the β-glycerophosphate solution was 5:1. After this
process, the hydrogel was obtained.
The entire hydrogel at 20
C and 37
C was directly inspected and
photographed with a Huawei Mate10 camera equipment. A stress-
controlled rheometer (DRH-1, TA Instrument) was applied to detect
the storage modulus (G0 ), loss modulus (G00 ), phase angle, and complex
viscosity of the hydrogel during the gelatination process. A temperature
scan with a heating rate of 2/min from 17
C to 90
C was performed,
the analysis frequency was 1 rad/s, and the change curves of the G0 ,
G00 , phase angle, and complex viscosity were recorded; the inter-
section of G0 and G00 is defined as gelation temperatures (Tgel, Chenite,
2000). The morphology of the gelling hydrogel was observed by scan-
ning electron microscopy (QUANTA200, Holland FEI, The Netherlands)
after freeze-drying and gold spraying (Zhang, Shi, et al., 2018).
2.3 | SDF-1 and VEGF release from the
nanocomposite system
C5F5@SDF-1 and C2F5@VEGF nanoparticles were distributed into the
hydrogel to prepare the nanocomposite system (the concentration of
both SDF-1 and VEGF solution was 50 ng/mL, the samples were
labeled as hydrogel@NPVEGF&SDF-1), and then placed onto a desktop
concentrator at 37
C for gelling. A control was prepared with SDF-1
HE ET AL.
and VEGF directly introduced into the hydrogel, labeled as hydro-
gel@VEGF&SDF-1 (the concentration of both SDF-1 and VEGF solu-
tion was also 50 ng/mL). Hydrogels loaded with single C5F5@SDF-1
and C2F5@VEGF were also fabricated as control samples, and they
were labeled as hydrogel@NPSDF-1 and hydrogel@NPVEGF, respectively.
All the gelling hydrogels were immersed in phosphate buffer solu-
tion (PBS, pH 7.4) at 37
C and the concentration of the released SDF-1
and VEGF from each hydrogel was detected via a typical enzyme-linked
immuno sorbent assay at the first, third, fifth, and seventh days, respec-
tively (Li et al., 2015).
2.4 | MSCs homing and proliferation
The MSCs were isolated from bone marrow of stromal cell-derived
rats as described in our previous work and its third passage was
applied for investigating the ability of each sample on improving hom-
ing and proliferation (Li et al., 2016). The protocol of MSCs homing
was carried out using a Millipore transwell (pore size: 8.0 μm). In this
experiment, 200 μL of each hydrogel was filled in a 12-well plate and
incubated at 37
C for gelling. For the test, fresh culture medium was
initially added to the hydrogels in the 12-well plate (700 μL/well). The
transwell was subsequently inserted into the plate, and 500 μL of
MSCs suspension (5 × 104 cells/mL) was added. After culturing at
37
C and 5% CO2 for 12 hr, the MSCs adhering to the upper surface
of the insert were removed using a swab, and the MSCs across the
bottom and those that had migrated to the undersurface were stained
using 0.1% crystal violet (Liu et al., 2017). The homing MSCs were
observed using optical microscopy (Carl Zeiss A2). The numbers of the
homing MSCs of each group were counted from at least 15 visual
fields (Li et al., 2015).
For the MSCs proliferation assay, the third passage of MSCs was
suspended in the hydrogel@NPVEGF&SDF-1, hydrogel@VEGF&SDF-1,
hydrogel@NPSDF-1, hydrogel@NPVEGF, and the single hydrogel con-
trol, respectively, with a concentration of 5 × 104 cells/mL at 20
C,
and cultured using fresh Dulbecco's Modified Eagle Media/Nutrient
Mixture F-12 (DMEM/F12) complete medium (with 10% fetal bovine
serum) at 37
C in a humidified atmosphere containing 95% air and 5%
CO2. Cells were fed with freshly prepared growth medium every
24 hr. After culturing for 1 and 3 days, the MSCs-loaded hydrogels
were stained with acridine orange (AO) solution (DMEM/F12 = 1:50)
at 37
C for 10 min and observed under a confocal laser scanning
microscope (CLSM, Nikon C2 Plus, Japan) in Z-axis scanning mode to
investigate the MSCs proliferation, and a Cell Counting Kit-8 (CCK-8)
assay was also applied for the quantitative characterization (Zhang,
Shi, et al., 2018).
2.5 | Endothelial cells migration, proliferation, and
apoptosis
In this experiment, the 12-well plate was also modified with each
hydrogel as descripbed in Section 2.4. Human umbilical vein endothe-
lial cells (HUVECs) obtained from newborn umbilical cord (Huaxi Hos-
pital, Chengdu, China) were applied in this study: for the proliferation
2125
investigation, the fifth passage of the HUVEC was seeded onto the
samples with a density of 5 × 104 cells/mL and then incubated at
37
C for 1 and 3 days; after fixation with 4% paraformaldehyde
(Sigma) for 2 hr at RT, the HUVECs of each group were stained by
phalloidin (Sigma) and for 15 min and 40 ,6-diamino-2-phenylindole
(DAPI, Sigma) for 5 min, and finally observed by a fluorescence micro-
scope (DMRX, Leica, Germany). A typical CCK-8 assay was performed
to investigate the HUVEC proliferation on each sample (Chen et al.,
2018). The HUVEC migration experiment was performed according to
the protocol of MSCs homing which was described in Section 2.4.
To investigate the apoptosis of HUVEC of each group, cell-
permeable AO) dye in combination with propidium iodide (PI), a
plasma membrane-impermeable Deoxyribonucleic acid (DNA)-binding
dye, was applied. AO and PI excite green and orange-red fluores-
cence, respectively, after intercalation into DNA. The HUVEC apopto-
sis assay was carried out as follows: after cultured for 1 and 3 days,
respectively, the cells were stained with a 1:1 mixture of AO
(100 mg/mL) and PI (100 mg/mL) at 37
C for 3 min, and then immedi-
ately inspected with a fluorescence microscope. The vital ratios of
HUVEC on each sample were calculated from at least 15 fields
(Li et al., 2017).
2.6 | Establishment of Chick embryo CAM model
Chick embryo CAM is a thin and transparent membrane attached to
the eggshell, and it will be in large quantities during the development of
chick embryos. The CAM model was used in the study of angiogenesis
for the first time by Auerbach, Kubai, Knighton, & Folkman, (1974) for
CAM takes on abundant blood vessels, enabling observation of micro-
vascular structure changes without special equipment (Auerbach et al.,
1974). In this study, we applied the CAM model to investigate the con-
tribution of each hydrogel group to the angiogenesis. In brief, fertilized
chicken eggs (ChengDu Muxing Poultry Industry Limited Liability Com-
pany, China) were incubated at the standard conditions of 37
C and
55% humidity and turned 3–5 times daily. After 7 days of incubation,
the eggs were gently cleaned with a 2% benzalkonium bromide solu-
tion, and a 1.5 cm × 1.5 cm hole was opened at the end of the chick
embryo chamber by tweezers to remove eggshell and inner eggshell
membrane. Then, the gas chamber membrane was picked up by twee-
zers, the upper chamber membrane was removed, and the CAM film on
the lower layer was finally exposed. An amount of 100 μL of hydroge-
l@NPVEGF&SDF-1, hydrogel@VEGF&SDF-1, hydrogel@NPSDF-1, hydro-
gel@NPVEG, and the single hydrogel control were placed on the large
vascular branches of the CAM (each group has five parallel CAM, n = 5)
at 20
C; the hole was sealed with medical wound dressing and the eggs
were returned to the incubator for continuous culture. After 3 days cul-
ture, 4% paraformaldehyde was added from the hole for a 15-min fixed
step, and then the CAM covered by the hydrogels were cut off and
rinsed with PBS. Paraffin-embedded sections were stained with
hematoxylin-eosin (HE) to observe each hydrogel of blood vessel
ingrowth (Zou et al., 2018). The operation process of the CAM model is
displayed in Figure 1.
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2.7 | Statistical analysis
The data were statistically evaluated using ANOVA by homogeneity
test of variances firstly, and the post hoc test was subsequently per-
formed using the least significant difference method for comparison.
The data are expressed as mean ± SD. The probability value p < 0.05
was considered as a significant difference. The data analysis was car-
ried out using the software SPSS 11.5 (Chicago, IL).
3 | RESULTS
3.1 | Characterization of the factor-loaded
chitosan/fucoidan nanoparticles
To confirm the successful preparation of the chitosan/fucoidan
nanoparticles, the chemical structures of chitosan/fucoidan nanoparticles,
as well as the chitosan and fucoidan controls, were analyzed by FTIR
spectrophotometry. As shown in Figure 2a, the chitosan/fucoidan
nanoparticles, chitosan, and fucoidan controls had a peak around
1,560/cm ascribed to the bending vibration of N─H, while the
chitosan/fucoidan nanoparticles and the chitosan control presented a
characteristic peak at 1150/cm ascribed to the stretching vibration of
C─O─C. Only the chitosan/fucoidan nanoparticles and the fucoidan
control displayed the peaks at 1160–1260/cm (stretching vibration of
S─O) and 845/cm (stretching vibration of C─O─S), indicating that the
chitosan/fucoidan nanoparticles were successfully prepared.
The formation of chitosan/fucoidan nanoparticles was mainly
driven by the electrostatic interaction between negatively charged
fucoidan and positively charged chitosan. The charge properties of
the chitosan/fucoidan nanoparticles could be controlled by changing
the ratios of these two molecules, wherein the C5F5 nanoparticles
were positively charged and selected for loading SDF-1 (negative
charge), while the C2F5 nanoparticles possessed negative charge and
were selected for loading VEGF (positive charge). Figure 2b–d
HE ET AL.
F I G U R E 1 The operation process of
the chorioallantoic membrane (CAM)
model aiming at studying angiogenesis
indicates that both the factors-loaded nanoparticles (C5F5@SDF-1
and C2F5@VEGF) and the single nanoparticles (C5F5 and C2F5) had
moderately stable structure and distribution because the absolute
values of their zeta potentials were more than 30 mV, and this param-
eter point is considered as a minimum value for the stability of
nanoparticles. As Liu et al., (2017) reported, PDI is an important indi-
cator to evaluate the size distribution of the particles, and PDI values
smaller than 0.2 indicate good uniformity. The C5F5@SDF-1 and
C2F5@VEGF not only showed PDI values below 0.2 but also
maintained their original charge property (C5F5@SDF-1: positive
charge; C2F5@VEGF: negative charge), which may contribute to their
better uniformity and orderly delivery.
3.2 | Characterization of the temperature-responsive
hydrogels
Figure 3a displays the entire chitosan/gelatin/β-glycerophosphate
hydrogel at different temperatures: the hydrogel presented as a limpid
solution at 20
C, and when the temperature went up to 37
C, it
converted into a milky white colloid. Figure 3b shows the dynamic vis-
coelasticity of thermosensitive hydrogel: G0 represents elastic proper-
ties, G00 represents viscous properties, and the tangent of the phase
angle equals to G00 /G0 and represents the degree of viscoelasticity of
the system. At 37
C, the hydrogel became solid, its G00 was less than G0
and it, therefore, had a lower phase angle that indicated elastic property
in the range of frequency measurement. Figure 3c presented the G0
and G00 changes of the thermosensitive hydrogel during the tempera-
ture rise: G0 reflects the solid-state properties of the hydrogel's rheolog-
ical properties, while G00 reflects the fluid properties of the hydrogel's
rheological properties. So, at the low temperature, the G0 maintained a
relatively low value, while it rose rapidly when the temperature went
up to a certain point, and G0 even exceeded G00 as the temperature con-
tinued to rise. The intersection point of G00 and G00 is defined as gelation
temperatures (Tgel), and the hydrogel's Tgel was 35
C from
HE ET AL.
F I G U R E 2 (a) Fourier-transform infrared
spectra of chitosan/fucoidan nanoparticles,
chitosan, and fucoidan controls;
(b) transmission electron microscopy images
and (c) size measurements of C5F5, C2F5,
C5F5@SDF-1, and C2F5@VEGF
nanoparticles; (d) sizes, polymer dispersity
index and zeta potential values of different
nanoparticles
Figure 3c. The composite viscosity reflects the injectable properties of
the thermosensitive hydrogel (Figure 3d): smaller composite viscosity
indicated better flow property of the hydrogel which meant more con-
ductivity for the in situ injection, while larger composite viscosity
expressed gelling. Figure 3d shows that the composite viscosity
maintained a lower and stable value when the temperature was lower
than the Tgel, suggesting the hydrogel's injectable property; however,
when the temperature exceeded the Tgel, the composite viscosity rap-
idly increased, and this meant the gelation of the hydrogel.
3.3 | SDF-1 and VEGF release
Figure 4a depicts the morphology of the gelling single hydrogel and
the hydrogel@NPVEGF&SDF-1 samples. Both the hydrogel control and
the hydrogel@NPVEGF&SDF-1 samples had a porous network structure
2127
with uniform pore diameters and the pore size in the range from 30 to
50 μm; this structure is conducive to the transportation of water and
nutrients; besides, introducing nanoparticles did not change the
microstructure of the single hydrogel. Figure 4b shows the VEGF
release of the hydrogel@NPVEGF&SDF-1, hydrogel@NPVEGF, and hydro-
gel@VEGF&SDF-1 samples: obviously, the hydrogel@NPVEGF&SDF-1
samples released less VEGF compared with the hydrogel@NPVEGF and
hydrogel@VEGF&SDF-1 samples, suggesting better stability of the
VEGF loaded in the hydrogel@NPVEGF&SDF-1 samples. VEGF plays a
crucial role in EC proliferation and angiogenesis. Thus, its better stabil-
ity in the hydrogel scaffold will help for rapid vascularization in the
tissue/organ repair and regeneration. Figure 4c presents the SDF-1
release curves of the hydrogel@NPVEGF&SDF-1, hydrogel@NPSDF-1, and
hydrogel@VEGF&SDF-1 samples within 7 days, and displays an oppo-
site trend compared to the VEGF release: the hydrogel@NPVEGF&SDF-1
2128 HE ET AL.

F I G U R E 3 (a) The entire

object of the hydrogel at 20
C
and 37
C; (b) G0 , G00 and phase
angle of the hydrogel at 37
C;
(c) G0 and G00 change of the
hydrogel with the temperature
changing; (d) complex viscosity
change of the hydrogel with the
temperature changing

F I G U R E 4 (a) Scanning
electron microscopy images of
the gelling single hydrogel and
the hydrogel@NPVEGF&SDF-1
samples; (b) VEGF release of
hydrogel@NPVEGF&SDF-1,
hydrogel@NPVEGF and
hydrogel@VEGF&SDF-1
samples (mean ± SD, n = 5);
(c) SDF-1 release of
hydrogel@NPVEGF&SDF-1,
hydrogel@NPSDF-1 and
hydrogel@VEGF&SDF1 samples
(mean ± SD, n = 5). NP,
nanoparticle; SDF-1, stromal
cell-derived factor-1; VEGF,
vascular endothelial growth
factor

samples released more SDF-1 compared with the hydrogel@NPSDF-1 3.4 | MSCs homing and proliferation
and hydrogel@VEGF&SDF-1 samples, indicating that more MSCs
The chemotactic effect of each hydrogel on MSCs is shown in
might be induced by the nanocomposite system because SDF-1 is a
Figure 5. The crystal violet stains showed that all the hydrogels
chemokine for the MSCs homing.
HE ET AL. 2129

F I G U R E 5 (a) Crystal violet staining of MSCs migrated to the underside of bottom membrane; (b) amounts of migrated MSCs (mean ± SD,
n = 3, *p < .05 compared with the other samples, **p < .05 compared with hydrogel@NPSDF-1, hydrogel@NPVEGF, and the single hydrogel control,
***p < .05 compared with hydrogel@NPVEGF and the single hydrogel control); (c) CLSM images of MSCs distributed in different hydrogels;
(d) proliferation of MSCs loaded in different hydrogels detected by CCK-8 assay (mean ± SD, n = 3, *p < .05 compared with the other samples,
**p < .05 compared with hydrogel@NPSDF-1, hydrogel@NPVEGF and the single hydrogel control, ***p < .05 compared with hydrogel@NPSDF-1 and
the single hydrogel control, ****p < .05 compared with the single hydrogel control). CCK-8, Cell Counting Kit-8; CLSM, confocal laser scanning
microscope; MSCs, mesenchymal stem cell; NP, nanoparticle; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor

induce MSCs migration through the semipermeable membrane due
to their good biocompatibility (Figure 5a). Wherein the hydrogel@
NPVEGF&SDF-1 presented the largest MSCs number, suggesting better
ability on inducing MSCs homing compared with other samples, and
the hydrogel@VEGF&SDF-1 and hydrogel@NPSDF-1 exhibited slightly
inferior capability successively, but they were still superior to the
hydrogel@NPVEGF and single hydrogel control (Figure 5b). The MSCs
homing displayed a consistent result with the SDF-1 release,
indicating that the amounts of the SDF-1 released from the samples
determined their functions on inducing MSCs homing.
Figure 5c,d presents the MSCs distribution and proliferation in the
hydrogel@NPVEGF&SDF-1, hydrogel@VEGF&SDF-1, hydrogel@NPSDF-1,
hydrogel@NPVEGF, and single hydrogel control. The CLSM images
in a Z-axis mode showed that the hydrogel@NPVEGF&SDF-1 and
hydrogel@VEGF&SDF-1 samples possessed more homogeneous
MSCs distribution and higher MSCs density compared with other
2130 HE ET AL.

samples (Figure 5c). The CCK-8 results showed that all the hydrogels 3.5 | HUVEC migration, proliferation, and apoptosis
promoted MSCs proliferation, wherein the designed hydroge-
The effect of different hydrogels on HUVEC migration behavior was
l@NPVEGF&SDF-1 system exhibited better functions (Figure 5d). Among
investigated via corresponding to the MSCs homing, and the results
the single-nanoparticle hydrogels, the hydrogel@NPVEGF contributed
are shown in Figure 6a,b. Consistent with the MSCs homing, the
significantly more to MSCs proliferation then the hydrogel@NPSDF-1,
which may be due to the direct promotion of the loaded VEGF factor hydrogel@NPVEGF&SDF-1 possessed the largest migrated HUVEC num-

and the preferable scale of the VEGF nanoparticles (154 nm). Our pre- ber; the second was the hydrogel@VEGF&SDF-1, followed by the

vious work proved that the nanoscale around 100 nm contributed to hydrogel@NPSDF-1, hydrogel@NPVEGF, and single hydrogel control.

better proliferation and oriented proliferation of the MSCs According to reports elsewhere, VEGF promotes angiogenesis and EC
(Li et al., 2016). growth (Ferrara, Gerber, & LeCouter, 2003; Leung, Cachianes, Kuang,

F I G U R E 6 (a) Crystal violet staining of HUVEC migrated to the underside of bottom membrane; (b) amounts of migrated HUVEC (mean ± SD,
n = 3, *p < .05 compared with the other samples, **p < .05 compared with hydrogel@NPSDF-1, hydrogel@NPVEGF, and the single hydrogel control,
***p < .05 compared with hydrogel@NPVEGF and the single hydrogel control, ****p < .05 compared with the single hydrogel control);
(c) phalloidin and DAPI staining of HUVEC on different samples surfaces; (d) HUVEC number detection via a typical CCK-8 assay (mean ± SD,
n = 3, *p < .05 compared with the other samples, **p < .05 compared with hydrogel@NPSDF-1, hydrogel@NPVEGF and the single hydrogel control,
***p < .05 compared with hydrogel@NPSDF-1 and the single hydrogel control, ****p < .05 compared with the single hydrogel control). CCK-8, Cell
Counting Kit-8; DAPI, 40 ,6-diamino-2-phenylindole; HUVEC, human umbilical vein endothelial cell, NP, nanoparticle
HE ET AL.
Goeddel, & Ferrara, 1989; Shweiki, Itin, Soffer, & Keshet, 1992), how-
ever, according to the results in Figure 6a,b, the migrated HUVEC
number on hydrogel@NPVEGF samples was less than the HUVEC num-
ber on the single hydrogel control. The hydrogel@NPSDF-1 samples
presented larger HUVEC numbers than the hydrogel@NPVEGF sam-
ples. In this experiment, two major factors may influence the HUVEC
migration: SDF-1 also promoted the HUVEC migration, and simulta-
neously it induced the migrated HUVEC-secreted VEGF (Liu et al.,
2017); SDF-1 is a chemotactic factor, but VEGF is a growth factor.
Thus, the former plays an important role in directed migration,
homing, and mobilization of cells (Orimo et al., 2005), while the
latter mainly contributes to cell proliferation and activity (Ferrara &
DavisSmyth, 1997).
To investigate the effect of different hydrogels on HUVEC prolifer-
ation, fluorescence staining and CCK-8 characterization were per-
formed for HUVEC after 1 and 3 days culture on each hydrogel
(Figure 6c,d). Figure 6c shows that all the hydrogels supported the
HUVEC growth, wherein the hydrogel@NPVEGF&SDF-1 obviously pres-
ented larger HUVEC coverage compared with the other controls,
suggesting better endothelial compatibility. In addition, more blue dots
stained by DAPI appeared on the hydrogel@NPVEGF&SDF-1 group, which
meant more DNA production in this group, and this phenomenon indi-
cated that the hydrogel@NPVEGF&SDF-1 sample had the better ability on
improving HUVEC proliferation. The CCK-8 result (Figure 6d) showed
a consistent trend with the fluorescence images: the hydrogel@
NPVEGF&SDF-1 group exhibited larger HUVEC number compared with
the other groups, suggesting better functions of improving HUVEC pro-
liferation. Although the HUVECs on all hydrogels presented increased
number after 3 days culture, which indicated improved proliferation,
the HUVEC number showed a significant order on each sample: hydro-
gel@NPVEGF&SDF-1 > hydrogel@VEGF&SDF-1 > hydrogel@
NPVEGF > hydrogel@NPSDF-1 > single hydrogel control. Compared with
the VEGF release curve in Figure 4b, this result showed a negative cor-
relation, that is, more HUVEC grew on the less VEGF release
(or more VEGF remaining in the hydrogel) samples. As mentioned previ-
ously, VEGF is the specific growth factor for endothelial cells (ECs), thus
more VEGF loading contributed to quicker HUVEC growth. In addition,
the SDF-1-loaded hydrogels (hydrogel@NPVEGF&SDF-1, hydrogel@
VEGF&SDF-1, and hydrogel@NPSDF-1) also possessed the better func-
tion of improving HUVEC proliferation compared with single hydrogel
control which probably did from the VEGF released by the HUVEC via
an SDF-1/CXCR4 pathway.
The HUVEC apoptosis rate on each sample was investigated by a
live/dead staining method and further calculated. In the fluorescence
images, the green dots indicate the live cells and the red dots indicate
the dead cells. Figure 7a shows that all the hydrogels had lots of green
cells and very few red cells, suggesting good ability to inhibit HUVEC
apoptosis. Quantification of vital cells from at least 15 images showed
that the rate of vital HUVEC on each sample was beyond 90%, the
VEGF and SDF-1-loaded hydrogel had a higher rate, and the hydroge-
l@NPVEGF&SDF-1 further enhanced the values, which indicated a better
inhibition of HUVEC apoptosis in the composite system (Figure 7b).
2131
3.6 | Blood vessel ingrowth
To further investigate the stimulating effect of the composite system
on in situ vascularization, we injected the hydrogel@NPVEGF&SDF-1,
hydrogel@VEGF&SDF-1, hydrogel@NPSDF-1, hydrogel@NPVEGF, and
the single hydrogel control in the CAM model to observe the induc-
tion of blood vessel ingrowth. Figure 8 shows the HE staining of each
group: the purple area represents vascular tissue, and the red area
indicates the hydrogels. There was a clear boundary between the pur-
ple area and red area in the single hydrogel control, suggesting little
blood vessel ingrowth. The hydrogel@NPSDF-1 group showed more
blood vessel ingrowth compared with the single hydrogel control, but
still less than the hydrogel@NPVEGF group. Obviously, VEGF plays a
critical role in in situ vascularization. The hydrogel@NPVEGF&SDF-1
group showed most blood vessel of ingrowth; the blood vessels
filled almost the whole hydrogels. On the contrary, the hydrogel@
VEGF&SDF-1 group induced very little blood vessel ingrowth, merely
as much as the hydrogel@NPSDF-1 group. The blood vessel ingrowth
in the CAM model demonstrated that the hydrogel@NPVEGF&SDF-1
system induced better in situ vascularization depending on its selec-
tive release of VEGF/SDF-1, which was controlled by the co-loaded
amphoteric nanoparticle carriers.
4 | DISCUSSION
Tissue repair and regeneration has always been the key technology of
clinical treatment and the focus of basic medical research (Wei, Zhan,
Yu, & Chen, 2017). During this process, tissue vascularization plays a
crucial role for both the structure and function of vascular networks,
as well as nutrition and signal transmission through the blood flow
(Zhang et al., 2018). The amount and behavior of stem cells and VECs
decide the vascularization speed: numerous and active cells will
undoubtedly accelerate vascularization in tissue engineering, thereby
contributing to tissue repair and regeneration (Rafii & Lyden, 2003).
Thus, the rapid recruitment and proliferation of stem cells and VECs
to the lesion site induced by the scaffold are the key to the vasculari-
zation of early tissue repair and regeneration.
Temperature-responsive injectable hydrogels recently attracted
high attention for biomedical application, because their handling is
more straightforward, minimally invasive, they can adapt to various
kinds of damaged tissues and organs, and they allow better contact
with the host tissue (Ekenseair et al., 2012; Park et al., 2009). Our
study also indicated that the pore sizes of the injectable hydrogels
was controlled by the materials parameters (such as feed ratios or
molecular weight) and allowed tuning of the cell behaviors. Figure 4a
shows that the hydrogels had a porous network structure with uni-
form pore size between 30 and 50 μm. The previous studies found
that pore sizes between 20 and 30 μm regulate single VECs to main-
tain their physiological functions (Li et al., 2013; Wu et al., 2015), but
multicellular regulation may need a little bigger sizes.
Selective release of multiple biological factors from scaffolds may
guide multicellular synergy and precise therapy in clinical application.
2132 HE ET AL.

F I G U R E 7 (a) HUVEC viability

on the hydrogel@NPVEGF&SDF-1,
hydrogel@NPSDF-1,
hydrogel@NPVEGF,
hydrogel@VEGF&SDF-1, and
single hydrogel samples. (b) Ratio
of vital HUVEC grown on the
hydrogel@NPVEGF&SDF-1,
hydrogel@NPSDF-1,
hydrogel@NPVEGF,
hydrogel@VEGF&SDF-1, and
single hydrogel samples, which
were calculated from at least
15 images (*p < 0.05 compared
with all the other samples,
**p < 0.05 compared with the
single hydrogel control, mean
± SD, n = 15); green color indicates
vital cells, red color indicate dead
cells. HUVEC, human umbilical
vein endothelial cell; SDF-1,
stromal cell-derived factor-1;
VEGF, vascular endothelial growth
factor

Clinical scientists always hold in esteem on stem cells which are usually
chemotactic for SDF-1 to the scaffold to support more rapid recruit-
ment and proliferation in vascularization (Tachibana et al., 1998; Tang
et al., 2015), while VECs tend to migrate slowly to the target location.
The VEGF release may accelerate cell migration (Mohsen, Hatef,
Hamid, Ali, & Ghasem, 2018; Torio-Padron et al., 2007). However, it
seems that keeping VEGF in the scaffold supports vascularization
because it not only enhances the proliferation of cells (Carpenter et al.,
2005; Zhou et al., 2018), but also regulates the interaction between
The MSCs migration experiment (Figure 5a,b) proved that the scaf-
fold with selective SDF-1/VEGF release (hydrogel@NPVEGF&SDF-1)
induced better MSCs homing compared with the nonselective one
(hydrogel@VEGF&SDF-1). The scaffold with selective SDF-1/VEGF
release also enhanced cell proliferation (Figures 5c,d and 6c,d) and cau-
sed less cell apoptosis (Figure 7) compared to the nonselective scaffold.
In the typical CAM model, the SDF-1/VEGF selective-released scaffold
obviously possessed more areas of blood vessel ingrowth compared
with the nonselective scaffold, which confirmed better vascularization.

stem cells and ECs (such as directional differentiation of stem cells

induced by ECs and pore size in the scaffold) and promotes angiogene-
sis (Adibfar et al., 2018; Cho, Moon, Park, Lee, & Seo, 2018; Zhou et al.,
2018). Thus, in the present study, we designed two kinds of
nanoparticles to load SDF-1 and VEGF, respectively. These molecules
had distinct charge properties (C5F5@SDF-1 nanoparticles were posi-
tively charged, and C2F5@VEGF nanoparticles were negatively charged,
Figure 2d) and regulated the scaffold to release more SDF-1 (Figure 4c)
and less VEGF (Figure 4b) via electrostatic interaction with the hydrogel
(the chitosan-based hydrogel was positively charged).
5 | C O N CL U S I O N S
In this study, we developed an injectable system composed of a
temperature-responsive hydrogel and factors-loaded nanoparticles for
potential application of vascularization in tissue engineering. This
composite system can control the selective release of MSCs
chemokines (SDF-1) and cell growth factor (VEGF; more SDF-1
release and less VEGF release) via co-loading with two different
nanoparticles. The SDF-1 nanoparticles possessed positive charge and
HE ET AL.
F I G U R E 8 Histological analysis results: typical optical photographs of the HE-stained cross-sectional slices of the chick embryo blood vessel
with hydrogel@NPVEGF&SDF-1, hydrogel@NPSDF-1, hydrogel@NPVEGF, hydrogel@VEGF&SDF-1, and single hydrogel samples (the area between
two black dotted lines is the blood vessels in growth; the purple area indicates chick embryo blood vessel, and the red area indicates each
hydrogel). HE, hematoxylin-eosin; HUVEC, human umbilical vein endothelial cell; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial
growth factor
bigger scales (304 nm), while the VEGF nanoparticles possessed nega-
tive charge and smaller scales (154 nm); wherein the smaller dimen-
sion and the loaded VEGF contributed to in situ cell growth, while the
sustained SDF-1 release supported the MSCs homing. The CAM
model further demonstrated that the composite system significantly
improved angiogenesis of the hydrogel scaffold. We expect that our
research facilitates the in situ vascularization during application in tis-
sue or organ repair and regeneration. Future studies will focus on fur-
ther fine-tuning the parameters of the functional nanoparticles, such
as dimensions, degradability, and more factors, aiming at more rapid
vascularization.
ACKNOWLEDGMENTS
We sincerely thank Professor Manfred F. Maitz for his help in this
work. This work was supported by the National Natural Science Foun-
dation of China (NSFC 81771988), Key Project and Special Founda-
tion of Research, Development and Promotion in Henan province
(No. 182102310076), the Joint Fund for Fostering Talents of NCIR-
MMT and HNKL-MMT (No. MMT2017-01), and Top Doctor Program
of Zhengzhou University (No. 32210475).
CONF LICT OF IN TE RE ST
The authors declare no potential conflict of interest.
ORCID
Jing-An Li https://orcid.org/0000-0002-0305-6929
2133
RE FE RE NCE S
Adibfar, A., Amoabediny, G., Eslaminejad, M. B., Mohamadi, J.,
Bagheri, F., & Doulabi, B. Z. (2018). VEGF delivery by smart polymeric
PNIPAM nanoparticles affects both osteogenic and angiogenic capaci-
ties of human bone marrow stem cells. Materials Science and Engineer-
ing: C, 93, 790–799.
Auerbach, R., Kubai, L., Knighton, D., & Folkman, J. (1974). A simple proce-
dure for the long-term cultivation of chicken embryos. Developmental
Biology, 41(2), 391–394.
Bao, B., Hao, J., Bian, X., Zhu, X., Xiao, K., Liao, J., … Jiang, L. (2017). 3D
porous hydrogel/conducting polymer heterogeneous membranes with
electro-/pH-modulated ionic rectification. Advanced Materials, 29(44),
1702926.
Caccavo, D., Cascone, S., Lamberti, G., & Barba, A. A. (2018). Hydrogels:
Experimental characterization and mathematical modelling of their
mechanical and diffusive behaviour. Chemical Society Reviews, 47(7),
2357–2373.
Carpenter, B., Lin, Y. K., Stoll, S., Raffai, R. L., McCuskey, R., & Wang, R.
(2005). VEGF is crucial for the hepatic vascular development required
for lipoprotein uptake. Development, 132(14), 3293–3303.
Chen, L., Li, J. A., Chang, J. W., Jin, S. B., Wu, D., Yan, H. H., … Guan, S. K.
(2018). Mg-Zn-Y-Nd coated with citric acid and dopamine by layer-by-
layer self-assembly to improve surface biocompatibility. Science China
Technological Sciences, 61(8), 1228–1237.
Cho, H. D., Moon, K. D., Park, K. H., Lee, Y. S., & Seo, K. I. (2018). Effects
of auriculasin on vascular endothelial growth factor (VEGF)-induced
angiogenesis via regulation of VEGF receptor 2 signaling pathways
in vitro and in vivo. Food and Chemical Toxicology, 121, 612–621.
Davoodi, P., Lee, L. Y., Xu, Q., Sunil, V., Sun, Y., Soh, S., & Wang, C. H.
(2018). Drug delivery systems for programmed and on-demand
release. Advanced Drug Delivery Reviews, 132, 104–138.
Ekenseair, A. K., Boere, K. W. M., Tzouanas, S. N., Vo, T. N.,
Kasper, F. K., & Mikos, A. G. (2012). Synthesis and characterization of
thermally and chemically gelling injectable hydrogels for tissue engi-
neering. Biomacromolecules, 13(6), 1908–1915.
Ferrara, N., & DavisSmyth, T. (1997). The biology of vascular endothelial
growth factor. Endocrine Reviews, 18(1), 4–25.
2134
Ferrara, N., Gerber, H. P., & LeCouter, J. (2003). The biology of VEGF and
its receptors. Nature Medicine, 9(6), 669–676.
Griffith, L. G., & Naughton, G. (2002). Tissue engineering-current chal-
lenges and expanding opportunities. Science, 295(5557), 1009–1014.
Hu, H. T., Hao, L. X., Yan, B., Li, X. M., Zhu, Y. X., Yao, J., … Jiang, Q.
(2018). Gefitinib inhibits retina angiogenesis by affecting VEGF signal-
ing pathway. Biomedicine & Pharmacotherapy, 102, 115–119.
Laiva, A. L., Raftery, R. M., Keogh, M. B., & O'Brien, F. J. (2018). Pro-
angiogenic impact of SDF-1α gene-activated collagen-based scaffolds
in stem cell driven angiogenesis. International Journal of Pharmaceutics,
544(2), 372–379.
Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., & Ferrara, N.
(1989). Vascular endothelial growth factor is a secreted angiogenic
mitogen. Science, 246(4935), 1306–1309.
Li, J. A., Qin, W., Zhang, K., Wu, F., Yang, P., He, Z. K., … Huang, N. (2016).
Controlling mesenchymal stem cells differentiate into contractile
smooth muscle cells on a TiO2 micro/nano interface: Towards benign
pericytes environment for endothelialization. Colloids and Surfaces B:
Biointerfaces, 145, 410–419.
Li, J. A., Zhang, K., Wu, F., He, Z. K., Yang, P., & Huang, N. (2015). Con-
structing bio-functional layers of hyaluronan and type IV collagen on
titanium surface for improving endothelialization. Journal of Materials
Science, 50, 3226–3236.
Li, J. A., Zhang, K., Wu, J. J., Zhang, L. J., Yang, P., Tu, Q. F., & Huang, N.
(2015). Tailoring of the titanium surface by preparing cardiovascular
endothelial extracellular matrix layer on the hyaluronic acid micro-
pattern for improving biocompatibility. Colloids and Surfaces B: Bio-
interfaces, 128, 201–210.
Li, J. A., Zhang, K., Xu, Y., Chen, J., Yang, P., Zhao, Y. C., … Huang, N. (2014).
A novel co-culture models of human vascular endothelial cells and
smooth muscle cells by hyaluronic acid micro-pattern on titanium sur-
face. Journal of Biomedical Materials Research: Part A, 102A, 1950–1960.
Li, J. A., Zhang, K., Yang, P., Qin, W., Li, G. C., Zhao, A. S., & Huang, N.
(2013). Human vascular endothelial cell morphology and functional
cytokine secretion influenced by different size of HA micro-pattern on
titanium substrate. Colloids and Surfaces B: Biointerfaces, 110, 199–207.
Li, J. A., Zou, D., Zhang, K., Luo, X., Yang, P., Jing, Y. Y., … Huang, N.
(2017). Strong multi-functions based on conjugating chondroitin sul-
fate on amine-rich surface direct vascular cells fate for cardiovascular
implanted devices. Journal of Materials Chemistry B, 5, 8299–8313.
Liu, K., Jiang, X., & Hunziker, P. (2016). Carbohydrate-based amphiphilic
nano delivery systems for cancer therapy. Nanoscale, 8, 16091–16156.
Liu, T., Wang, X., Tang, X., Gong, T., Ye, W., Pan, C., … Wang, Q. M. (2017).
Surface modification with ECM-inspired SDF-1α/laminin-loaded
nanocoating for vascular wound healing. ACS Applied Materials & Inter-
faces, 9, 30373–30386.
Lü, L., Deegan, A., Musa, F., Xu, T., & Yang, Y. (2018). The effects of bio-
mimetically conjugated VEGF on Osteogenesis and angiogenesis of
MSCs (human and rat) and HUVECs co-culture models. Colloids and
Surfaces B: Biointerfaces, 167, 550–559.
Luo, H., Zhang, Y., Zhang, Z., & Jin, Y. (2012). The protection of MSCs from
apoptosis in nerve regeneration by TGFβ1 through reducing inflamma-
tion and promoting VEGF-dependent angiogenesis. Biomaterials, 33
(17), 4277–4287.
Mohsen, K., Hatef, S. A., Hamid, A., Ali, G., & Ghasem, M. (2018). Mesen-
chymal stem cells differentiate to endothelial cells using recombinant
vascular endothelial growth factor-A. Reports of Biochemistry & Molec-
ular Biology, 6(2), 144.
Orimo, A., Gupta, P. B., Sgroi, D. C., Arenzana-Seisdedos, F., Delaunay, T.,
Naeem, R., … Weinberg, R. A. (2005). Stromal fibroblasts present in inva-
sive human breast carcinomas promote tumor growth and angiogenesis
through elevated SDF-1/CXCL12 secretion. Cell, 121(3), 335–348.
Park, H. J., Lee, W. Y., Kim, J. H., Park, C., & Song, H. (2018). Expression
patterns and role of SDF-1/CXCR4 axis in boar spermatogonial stem
cells. Theriogenology, 113, 221–228.
HE ET AL.
Park, K. M., Lee, S. Y., Joung, Y. K., Na, J. S., Lee, M. C., & Park, K. D. (2009).
Thermosensitive chitosan-pluronic hydrogel as an injectable cell delivery
carrier for cartilage regeneration. Acta Biomaterialia, 5(6), 1956–1965.
Rafii, S., & Lyden, D. (2003). Therapeutic stem and progenitor cell transplantation
for organ vascularization and regeneration. Nature Medicine, 9(6), 702–712.
Rose, J. C., & Laura, L. D. (2018). Hierarchical design of tissue regenerative
constructs. Advanced Health Care Materials, 7(6), 1701067.
Shweiki, D., Itin, A., Soffer, D., & Keshet, E. (1992). Vascular endothelial
growth factor induced by hypoxia may mediate hypoxia-initiated
angiogenesis. Nature, 359(6398), 843–845.
Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y., …
Nagasawa, T. (1998). The chemokine receptor CXCR4 is essential for vas-
cularization of the gastrointestinal tract. Nature, 393(6685), 591–594.
Tang, J., Cui, X., Caranasos, T. G., Hensley, M. T., Vandergriff, A. C.,
Hartanto, Y., … Cheng, K. (2017). Heart repair using nanogel-
encapsulated human cardiac stem cells in mice and pigs with myocar-
dial infarction. ACS Nano, 11(10), 9738–9749.
Tang, T., Jiang, H., Yu, Y., He, F., Ji, S. Z., Liu, Y. Y., … Xia, Z. F. (2015). A
new method of wound treatment: Targeted therapy of skin wounds
with reactive oxygen species-responsive nanoparticles containing
SDF-1 alpha. International Journal of Nanomedicine, 10, 6571–6585.
Torio-Padron, N., Borges, J., Momeni, A., Mueller, M. C.,
Tegtmeier, F. T., & Stark, G. B. (2007). Implantation of VEGF trans-
fected preadipocytes improves vascularization of fibrin implants on
the cylinder chorioallantoic membrane (CAM) model. Minimally Inva-
sive Therapy & Allied Technologies, 16(3), 155–162.
Wang, C., Li, Y., Yang, M., Zou, Y., Liu, H., Liang, Z., … Zhang, B. (2018).
Efficient differentiation of bone marrow mesenchymal stem cells into
endothelial cells in vitro. European Journal of Vascular and Endovascular
Surgery, 55(2), 257–265.
Wei, T., Zhan, W., Yu, Q., & Chen, H. (2017). Smart biointerface with photo-
switched functions between bactericidal activity and bacteria-releasing
ability. ACS Applied Materials & Interfaces, 9(31), 25767–25774.
Wu, J. J., Li, J. A., Wu, F., He, Z. K., Yang, P., & Huang, N. (2015). Effect of
micropatterned TiO2 nanotubes thin film on the deposition of endo-
thelial extracellular matrix: For the purpose of enhancing surface bio-
compatibility. Biointerphases, 10, 04A302.
Zhang, H., Zhou, Y., Zhang, W., Wang, K., Xu, L., Ma, H., & Deng, Y. (2018).
Construction of vascularized tissue-engineered bone with a double-
cell sheet complex. Acta Biomaterialia, 77, 212–227.
Zhang, K., Shi, Z. Q., Zhou, J. K., Xing, Q., Ma, S. S., Li, Q. H., … Guan, F. X.
(2018). Potential application of an injectable hydrogel scaffold loaded
with mesenchymal stem cells for treating traumatic brain injury. Jour-
nal of Materials Chemistry B, 6, 2982–2992.
Zhong, R., Tang, Q., Wang, S., Zhang, H., Zhang, F., Xiao, M., … Pei, H. (2018).
Self-assembly of enzyme-like nanofibrous G-molecular hydrogel for printed
flexible electrochemical sensors. Advanced Materials, 30(12), 1706887.
Zhou, L., Niu, X., Liang, J., Li, J., Li, J., Cheng, Y., … Zhang, K. (2018). Effi-
cient differentiation of vascular endothelial cells from dermal-derived
mesenchymal stem cells induced by endothelial cell lines conditioned
medium. Acta Histochemica, 120(8), 734–740.
Zou, D., Luo, X., Li, J. A., Wang, S., Zhang, K., Sun, J. Y., … Zhang, C. J.
(2018). Investigating blood compatibility and tissue compatibility of a
biomimetic ECM layer on cardiovascular biomaterials. Journal of Bio-
materials and Tissue Engineering, 8(5), 640–646.
How to cite this article: He D, Zhao A-S, Su H, et al. An
injectable scaffold based on temperature-responsive hydrogel
and factor-loaded nanoparticles for application in vascularization
in tissue engineering. J Biomed Mater Res. 2019;107A:
2123–2134. https://doi.org/10.1002/jbm.a.36723