Acta Biomaterialia 10 (2014) 2518–2528

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Study of bilineage differentiation of human-bone-marrow-derived

mesenchymal stem cells in oxidized sodium alginate/N-succinyl
chitosan hydrogels and synergistic effects of RGD modification
and low-intensity pulsed ultrasound
Yingying Wang a, Wenzhen Peng b, Xia Liu a, Minghua Zhu c, Tao Sun c, Qiang Peng c, Yi Zeng c, Bo Feng a,
Wei Zhi a, Jie Weng a, Jianxin Wang a,⇑
a
Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
b
Department of Biochemistry and Molecular Biology, College of Basic and Forensic Medicine, Sichuan University, Chengdu 610041, People’s Republic of China
c
Sichuan Centre for Disease Control and Prevention, Chengdu 610041, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is
Received 19 July 2013 considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxi-
Received in revised form 5 December 2013 dized sodium alginate/N-succinyl chitosan (RGD–OSA/NSC) hydrogel as a scaffold and low-intensity
Accepted 26 December 2013
pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation
Available online 3 January 2014
of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of
human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results
Keywords:
showed that RGD–OSA/NSC composite hydrogels presented good biological properties in attachment,
Bone tissue engineering
Hydrogel
proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of
LIPUS cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones;
Osteogenic differentiation similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule forma-
Endothelial differentiation tion assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive
synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was
observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one
approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering
scaffold implants.
Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Bone tissue engineering provides a potential approach for the
production of three-dimensional (3-D) implants [1–3]. However,
there are still some challenges associated with 3-D bone regenera-
tion, including creating regenerated tissue with similar structural
and mechanical properties to those of the natural bone and achiev-
ing successful integration of scaffolds with the host bone [4–6]. The
levels of formation of new bone and vascularization are considered
to be critical factors for the clinical application of bone tissue engi-
neering [7,8]. Both synthetic and natural materials have been ex-
plored as potential scaffolds for bone regeneration [9–12]. Of
these materials, the most commonly explored is hydrogels, which
are particularly attractive because they can be non-invasively
⇑ Corresponding author.
E-mail addresses: j.wang63@gmail.com, jwang@swjtu.edu.cn (J. Wang).
injected to fill defects of any size and can homogenously suspend
cells within a 3-D environment. Physically cross-linked hydrogels
do not readily meet the basic demands for tissue engineering due
to their rapid degradation property. Chemically cross-linked com-
posite hydrogels (prepared without using chemical cross-linking
agents like formaldehyde), which exhibit excellent properties
of biocompatibility, degradation and mechanical strength, are
regarded as optimal scaffold materials for tissue engineering
[13]. However, hydrophilic surfaces of hydrogels are not beneficial
for the adsorption of protein and are not conducive to the adhesion
and growth of cells [14] and, in turn, to the formation of new bone
and vascularization. One approach to overcome this is to use RGD
to modify hydrophilic surfaces. The Schiff base reaction between
amine groups and aldehyde groups offers the possibility of modifi-
cation of RGD on the hydrophilic surfaces of hydrogels, whereas
polysaccharides can provide aldehyde groups for the Schiff base
reaction by oxidization of linear polysaccharides, and their source

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.actbio.2013.12.052
 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528
is very rich [14]. It is for these reasons that polysaccharide hydro-
gels are receiving increasing attention. In this study, we proposed
to use an RGD-grafted oxidized sodium alginate/N-succinyl chito-
san (RGD–OSA/NSC) polysaccharide composite hydrogel as a scaf-
fold and to employ low-intensity pulsed ultrasound (LIPUS) as
mechanical stimulation so as to achieve good integration of scaf-
folds with the host bone by improving the formation of new bone
and vascularization in bone tissue engineering scaffolds [15–18].
Angiogenesis is a process of endothelial migration and prolifera-
tion to form a capillary network while endothelial differentiation
of marrow-derived mesenchymal stem cells (MSCs) is very impor-
tant for vascularization in scaffolds when implanted. Vasculariza-
tion in scaffolds will be conducive to the formation of new bone.
Additionally, the ability of scaffold materials to induce osteogenic
differentiation of MSCs determines the level of formation of new
bone in scaffold materials. In the present study, an in vitro study
of endothelial and osteogenic differentiation of MSCs in RGD–
OSA/NSC hydrogels is employed to evaluate the ability of scaffold
materials to induce the formation of new bone and vascularization.
The synergistic effect of LIPUS and RGD on the proliferation and
differentiation of human mesenchymal stem cells (hMSCs) is also
investigated. To the authors’ knowledge, there has been little pre-
vious research of this topic.
2. Materials and methods
A flow chart for the preparation of materials and cell culture in
this study is shown in Fig. 1.
Fig. 1. Flow chart for preparation of materials and cell culture.
2519
2.1. Materials
Sodium alginate (SA, Mw: 612 kDa) and chitosan (CS, Mw:
209 kDa) were purchased from Maichao Chemical Reagent (Shang-
hai, China) and Aokang Biological Technology Co. Ltd (Shandong,
China), respectively. All other chemicals and solvents were pur-
chased from Kelong Chemical Reagent Factory (Chengdu, China)
with reagent grade or better, without further purification.
2.2. Preparation of composite hydrogels
Oxidized sodium alginate (OSA) was prepared according to the
method previously reported [19,20]. Aqueous solutions of RGD
(4 ml, 10 mg ml–1) and OSA (2 ml, 10% w/v) were mixed and stirred
in the dark at room temperature for 36 h and then the obtained
RGD–OSA product was lyophilized at –80 °C for use. N-succinyl
chitosan (NSC) was prepared according to the previously reported
method [21,22]. The final formation of RGD-oxidized sodium algi-
nate (RGD–OSA)/N-succinyl chitosan (NSC) hydrogels was based
on the Schiff base reaction. 1 g of sterilized NSC was dissolved in
40 ml sterilized phosphate buffer solution (PBS, pH = 7.4). Simi-
larly, 2 g of sterilized RGD–OSA was dissolved in 20 ml PBS. Steril-
ized NSC and RGD–OSA solutions were mixed by stirring
vigorously. Subsequently, the mixed solution became highly vis-
cous and was then used to coat glass discs using a spinning coater.
The coating finally formed into an RGD–OSA/NSC gel on the glass
discs within a few minutes at 37 °C. All the hydrogel-coated glass
discs for cell culture were immersed in 75% ethanol for 2 h for fur-
ther sterilization and then rinsed three times with sterilized PBS
2520 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528
before cell culture. All the hydrogel-coated glass discs were placed
in 24-well plates for cell culture.
2.3. Cell culture
hMSCs were purchased from Sichuan University and cultured in
25 cm2 flasks in D-minimum essential medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS) and 100 U ml–1 penicil-
lin/streptomycin antibiotics. The cell culture medium was replaced
with fresh medium every 3 days. The hMSCs were purified by con-
trolling the passage time [23]. The average passage time was
2 days. The fifth passage cells, which grew stably and kept their
primary characteristics, were used for later induction experiments.
Passage cells were harvested with 0.25% trypsin-ethylenediamine-
tetraacetic acid solution and then they were resuspended in com-
plete medium for later seeding when reaching 80% confluence. The
viability of passage cells was measured by the Alamer Blue test.
When the activity of the cells reached 90%, the passage cells were
seeded on glass discs with OSA/NSC, RGD–OSA/NSC coatings and
blank glass discs in 24-well plates at a density of 0.5  105 cells
per well, and then they were cultured and differentiated. All the
culture experiments were divided into two groups with and with-
out LIPUS. The cells in the groups with LIPUS were exposed to the
pulsed ultrasound with an intensity of 200 mW cm–2, a duty cycle
of 20% and a repetition rate of 1 MHz for 10 min each day.
2.4. In vitro endothelial and osteogenic differentiation of hMSCs
2.4.1. Cell culture for endothelial and osteogenic differentiation of
hMSCs
Endothelial induction was performed using low glucose DMEM
(L-DMEM) supplemented with 10% FBS, 2 mM L-glutamine,
100 U ml–1 antibiotic, 50 ng ml–1 vascular endothelial growth fac-
tor (VEGF165), 100 U ml–1 antibiotics and 10 ng ml–1 bone morpho-
genetic protein-2 (BMP-2). While osteogenic induction was
conducted using high glucose DMEM (H-DMEM) supplemented
with 10% (v/v) FBS, 50 lM L-ascorbic acid, 10 mM b-glycerophos-
phate, 0.1 lM dexamethasone and 0.1 mM dexamethasone. All
the cells were cultured in a humidified atmosphere containing
5% CO2 and 95% air at 37 °C. The medium was renewed every
3 days [24,25].
2.4.2. Proliferation assay of cells during endothelial and osteogenic
differentiation of hMSCs
The proliferation of cells was determined using the MTT
(3-{4, 5-dimethylthiazol-2yl}-2, 5-diphenyl-2H-tetrazoliumbromide)
assay. The original medium was removed and 20 ll of 0.5% MTT
added to each well. Incubation of 4 h required a humidified
atmosphere containing 5% CO2 and 95% air at 37 °C until MTT
was taken up by active cells and reduced in the mitochondria to
insoluble purple formazan granules. Subsequently, the medium
was discarded and the precipitated formazan was dissolved in
dimethyl sulfoxide (50 ll per well). Optical density of the solution
was evaluated using an ELISA reader at a wavelength of 570 nm.
Three wells were examined for each type sample each time. The
investigated time points were days 1, 3, 7, 10, 14 and 21.
2.4.3. Morphological observation during endothelial and osteogenic
differentiation of hMSCs
Fluorescence staining using acridine orange (AO) was employed
for cell morphological analysis in our study. After 2, 3 and 4 weeks
of induction, cells were rinsed twice with PBS and fixed in 4% para-
formaldehyde solution for 2 h at room temperature. The fixed cells
were then incubated in 0.1 ml PBS containing 1% AO for 15 min at
37 °C and rinsed with PBS to remove excess dye. Microscopy obser-
vation was performed using an Olympus fluorescence microscope
equipped with a digital camera and Image Pro-plus (Radiance
2100; Biorad Laboratories, Hercules, CA, USA).
2.4.4. Endothelial differentiation assay
After endothelial induction of 28 days, endothelial differentia-
tion of hMSCs was examined by immunofluorescence staining, as
described by Wang et al. [26]. In brief, cells were rinsed twice with
PBS and fixed in 4% paraformaldehyde for 20 min at room temper-
ature and then incubated with 1% Triton X-100 in PBS to permea-
bilize cell membrane for 10 min at room temperature. After that,
these cells were incubated overnight at 4 °C with primary antibody
(Anti-CD31, 1:25, Beijing Biosynthesis Biotechnology Co. Ltd) and
then the non-specific bindings were blocked by washing three
times with the blocking buffer of PBS containing 4% bovine serum
albumin. After the removal of the blocking buffer, these cells were
incubated again with secondary antibodies (fluorescein isothiocy-
anate (FITC)-conjugated goat-anti-rabbit immunoglobulin G,
1:100, Beijing Zhongshan Glodenbridge Biotechnology Co. Ltd)
for 1 h at room temperature in the dark and then rinsed three
times with the blocking buffer. The nuclei were counterstained
with 40 -6-diamidino-2-phenylindole (DAPI) for visualization. For
CD34 staining, the process was the same as described above, ex-
cept that cells were incubated with the different primary antibody
(Anti-CD34, 1:50, Beijing Biosynthesis Biotechnology Co. Ltd).
Microscopy observation was performed using an Olympus fluores-
cence microscope equipped with a digital camera and Image Pro-
plus. Fluorescent intensity was also used to quantitatively measure
the expression of CD31 and CD34 by using Image Pro-plus.
2.4.5. Osteogenic differentiation assay
At days 3, 7, 10 and 14 of osteogenic induction, the qualitative
and quantitative examination of osteogenic differentiation of
hMSCs was conducted by monitoring the activity of alkaline phos-
phatase (ALP) using an alkaline phosphatase kit (Jiancheng Bioen-
gineering Institute, Nanjing, China) as well as staining with a BCIP/
NBT alkaline phosphatase kit (R&D Systems, Minneapolis, MN,
USA) according to the manufacturer’s instructions. Additionally,
at days 14, 21 and 28 of osteogenic induction, mineralization dur-
ing osteogenic differentiation was analyzed by von Kossa staining
according to the protocol described previously [27]. Before von
Kossa staining, cells were rinsed twice with PBS and then fixed in
4% paraformaldehyde for 2 h at room temperature. In the mean-
time, calcium content was measured quantitatively using a Cal-
cium C-Test kit (Wako Pure Chemical Industries, Ltd) as
described by Takayama et al. [28].
2.5. Statistical analysis
Each experiment was repeated three times. Statistical signifi-
cances of all data were determined using Student‘s t-test. The dif-
ference is considered significant if the p-value is less than 0.05.
Indicated error bars correspond to the standard deviation (SD).
3. Results
3.1. Observation of proliferative density and morphology of cells
Cell proliferation was determined by the optical density (OD)
values of the MTT assay in our study. The OD values of the MTT
assay for all the groups exhibited a similar increasing tendency
over time, as shown in Fig. 2. It can be seen that cell proliferation
was low during the first 3 days. The main reason for this might
be because that hydrophilic hydrogel surfaces were not beneficial
for the adsorption of proteins and this, in turn, was not conducive
to the adhesion and growth of cells. It can also be seen that LIPUS
 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528 2521

Fig. 2. Cell proliferation by MTT assays on days 1, 3, 7, 10, 14 and 21 during (A) endothelial induction and (B) osteogenic induction. Results were presented as the mean ± SD,
and experiments were performed in triplicate. ⁄Significantly different (p < 0.05, n = 3) to the control group at the same time point; ⁄⁄significantly different (p < 0.01, n = 3) to
the control group at the same time point. The OSA/NSC groups, RGD–OSA/NSC groups and blank control groups are denoted as ON, R-ON and Blank, respectively, whilst these
groups with LIPUS are named as LIPUS-ON, LIPUS-R-ON and LIPUS-Blank, respectively.

led to lower cell proliferation during the first 3 days compared
with the groups without LIPUS, demonstrating that LIPUS is not
beneficial for the early adsorption of adhesive proteins. However,
the groups with RGD modification showed higher cell proliferation
compared with that in the control groups and the groups without
RGD modification, indicating a positive effect of RGD on enhancing
the adhesion and growth of cells. A significant increase in cell pro-
liferation was observed in all the groups from day 7 to day 14 for
osteogenic induction and from day 7 to day 21 for endothelial
induction. The use of LIPUS or RGD promoted cell proliferation.
From Table 1, it can be seen that the enhancement of cell prolifer-
ation caused by the use of both LIPUS and RGD was much larger
than that caused by LIPUS only plus that caused by RGD only, dem-
onstrating the synergistic effect of LIPUS and RGD on the enhance-
ment of the adhesion and growth of cells. The strongest synergistic
effect of LIPUS and RGD on the enhancement of cell proliferation
occurred at day 14 for osteogenic induction and at day 21 for endo-
thelial induction.
It is generally accepted that MSCs are mainly fusiform and
fibroblast-like cells and have the ability to differentiate into a vari-
ety of tissue cells. The morphologies of cells have a close relation-
ship with stem cell differentiation and their mechanical properties.
Studies revealed that differentiation can result in the change of
morphologies of cells while they, in turn, affect differentiation
[29–33]. Thus, the change of morphologies of cells may be used
as evidence for differentiation of MSCs.
After cell culture of 5 days, the proliferation of the passage cells
reached up to 90% confluence. Their volume was larger than that of
the primary cells and they had an irregular polygon shape, like
fibroblastic cells; their nuclei were oval or round and consisted
of multiple nucleoli. After induction of 28 days, it was seen that
most of the hMSCs presented totally different morphologies from
those of mesenchymal stem cells, demonstrating that most of them
have differentiated. Most cells for endothelial induction presented
a typical flat polygonal appearance, like endothelial cells, as
shown in Fig. 3A, while those for osteogenic induction exhibited

Table 1
Cell proliferations determined by means of the MTT assay during osteogenic (endothelial) differentiation.

Time (D) ON R-ON LIPUS-ON LIPUS-R-ON LIPUS-Blank

1 D-value 0.0001 (–0.0001) 0.0004 (0.0003) –0.0004 (–0.0007) –0.0003 (–0.0003) –0.0006 (–0.0006)
D/B (%) 1.08 (–1.09) 4.35 (3.26) –4.35 (–7.61) 3.26 (3.26) –6.52 (–6.52)
p 0.4915 (0.4840) 0.0601 (0.4299) 0.3718 (0.0185) 0.1930 (0.0015) 0.3536 (0.1190)
3 D-value 0.0003 (–0.0005) 0.0025 (0.001) –0.0057 (–0.0013) 0.0029 (0.0029) –0.0109 (–0.0006)
D/B (%) 0.17 (–0.28) 1.42 (0.56) –3.23 (–0.73) 1.64 (0.85) –6.18 (–0.34)
p 0.2569 (0.4681) 0.4642 (0.4680) 0.4105 (0.3661) 0.3986 (0.2614) 0.0098 (0.4123)
7 D-value –0.0082 (–0.0003) 0.0154 (0.0131) 0.1208 (0.1089) 0.1618 (0.2011) 0.1234 (0.1285)
D/B (%) –1.72 (–0.06) 3.23 (2.74) 25.33 (22.73) 33.93 (42.05) 25.88 (26.87)
p 0.0646 (0.4065) 0.1507 (0.0783) 0.0062 (0.0153) 0.0107 (0.0122) 0.0192 (0.0120)
10 D-value –0.0105 (–0.0105) 0.0254 (0.0154) 0.1080 (0.0982) 0.1903 (0.1805) 0.1450 (0.1068)
D/B (%) –1.31 (–1.30) 3.18 (1.91) 13.52 (12.16) 23.81 (22.36) 18.15 (13.23)
p 0.0919 (0.1102) 0.0114 (0.0117) 0.0013 (0.0019) 0.0009 (0.0004) 0.0042 (0.0003)
14 D-value –0.011 (–0.0118) 0.0229 (0.0406) 0.2079 (0.1656) 0.3298 (0.2284) 0.2703 (0.1912)
D/B (%) –1.36 (–1.30) 2.83 (4.46) 25.69 (18.21) 40.75 (25.11) 33.40 (21.02)
p 0.0235 (0.0986) 0.0042 (0.0120) 0.0003 (0.0015) <0.0001 (0.0002) 0.0018 (0.0038)
21 D-value –0.0197 (–0.016) 0.0088 (0.0323) 0.2080 (0.165) 0.3267 (0.2287) 0.2182 (0.1982)
D/B (%) –2.49 (–1.70) 1.11 (3.41) 26.26 (17.40) 41.24 (24.12) 23.69 (20.90)
p 0.0478 (0.2220) 0.0579 (0.1006) 0.0006 (0.0017) 0.0002 (0.0026) 0.0053 (0.0044)

B: The blank control group; D-value: the difference in the OD between the samples and the blank control group; p: p-value determined using Student’s t-test (n = 3).
2522 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528
Fig. 3. Microscopic observation of the cell morphology that was stained with acridine orange (AO) after 28 days of (A) endothelial induction and (B) osteogenic induction.
Original magnification: 40; the insets represent a corresponding original magnification of 100.
spindle-shaped morphologies similar to osteoblasts, as shown in
Fig. 3B. Compared with the control groups, the cells in the groups
with LIPUS grew more intensively and showed stronger character-
istics of endothelial and osteoblasts cells, demonstrating that LIPUS
was conducive to proliferation and differentiation of hMSCs.
3.2. Endothelial induction
Angiogenesis is a process of endothelial migration and prolifer-
ation to form a capillary network. Endothelial differentiation of
MSCs is very important for vascularization in implant scaffolds
when implanted. In our study, endothelial differentiation of hMSCs
on OSA/NSC and RGD–OSA/NSC hydrogels were conducted to eval-
uate their ability to induce vascularization.
The immunofluorescence staining can provide a direct evidence
for endothelial differentiation of the cells. The differentiated cells
can be detected for the expression of endothelial cell markers such
as CD31 and CD34 can be visualized by FITC-conjugated secondary
antibody by immunofluorescence staining. We chose two endothe-
lial cell markers, CD31 and CD34, to assay the endothelial differen-
tiation of hMSCs and found that all the cells expressed both specific
endothelial markers of CD31 and CD34 at day 28 and could be visu-
alized by FITC-conjugated secondary antibody, demonstrating that
the hMSCs had been successfully induced to differentiate into
endothelial cells (Figs. 4 and 5). The cells in the groups with LIPUS
showed a much higher level of the positive expression of CD31 and
CD34 compared with those in the groups without LIPUS, revealing
that the use of LIPUS is very useful for promoting the endothelial
differentiation of hMSCs. Fluorescent intensity has been used to
quantitatively assay the expression of CD31 and CD34 (Fig. 6). Sim-
ilar results were observed. The expression of CD31 and CD34 was
remarkably enhanced when LIPUS was used. The highest expres-
sion of CD31 and CD34 was detected in the group with both LIPUS
and RGD and it was much higher than the totality caused by LIPUS
and RGD alone shown in Table 2, indicating the synergistic effect of
LIPUS and RGD on the enhancement of the endothelial differentia-
tion of hMSCs.
3.3. Osteogenic induction
ALP activity is the most widely recognized biochemical marker
for osteoblastic activity in the early stage. It is a typical protein for
osteoblast-phenotype and osteoblast differentiation [34].
Fig. 7A shows the results of ALP staining of the cultured cells at
day 3, 7, 10 and 14. The expression of alkaline phosphatase was
observed at day 7, which then increased with increasing the
cultivation time and reached a peak by day 10 and then started
to decrease [35]. The initial increase in ALP activity is a marker
of the commitment towards osteoblastic lineage while the
subsequent decrease should be attributed to the formation of the
advanced matrix mineralization and more mature phenotype
[36–40]. A repeatable positive expression of alkaline phosphatase
in the groups with RGD could be observed, although it was not
significant from a statistical point of view; however, it was
pronounced when LIPUS was used, indicating that there existed a
possible positive enhancement effect of RGD and LIPUS on the
osteogenic differentiation of hMSCs. Further work needs to be done
to prove it.
The similar results were also detected in the measurement of
ALP activity in Fig. 7B and Table 3. The use of LIPUS led to a much
higher level of the ALP activity compared with that in the controls,
revealing that LIPUS did enhance the differentiation of cultured
hMSCs towards osteoblasts, as indicated by the increase in the
ALP activity. Moreover, it can be seen from Table 3 that the syner-
gistic effect of LIPUS and RGD on the enhancement of the bone ma-
trix proteins ALP was relatively low compared with proliferation of
 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528 2523

Fig. 4. Images of immunofluorescence staining of cells after endothelial induction of 28 days. The differentiated cells were detected for the expression of (red) endothelial cell
markers CD31 and visualized by FITC-conjugated secondary antibody. DAPI counter stained nuclei in blue. Original magnification: 200.

hMSCs and a high level in the synergistic effect was observed at
day 14.
Alizarin red S (AR-S) as a dye can bind selectively to calcium
salts and hence is widely used for calcium mineral histochemistry
and the assay of formation of new bone. This kind of histological
staining is based on the capacity of alizarin red to specifically stain
matrix containing calcium and its positive appearance is consid-
ered an expression of bone matrix deposition [41]. The ability of
cells to form mineralized matrix is essential with regard to the
development of materials for the formation of new bone [41]. Nor-
mally, the measurement of ALP activity alone is not sufficient for
determining bone formation. Hence, calcium nodulation is consid-
ered as another specific marker of extracellular mineralized depos-
it in the late stage. We examined the formation of mineralized
nodules in the presence and absence of transient LIPUS stimulation
by staining with alizarin red, shown in Fig. 8A, and microscopy
observation of the living cells, shown in Fig. 8B. Alizarin red posi-
tive nodular aggregates have already been observed at day 14, indi-
cating the formation of bone mineralization matrix in all the
groups. At the same time point, the number and size of Alizarin
red positive nodular aggregates were larger in the groups with
RGD and with LIPUS compared with in the controls, demonstrating
the enhancement of both LIPUS and RGD on calcium deposition
and formation of bone matrix. The increased number and size of
Alizarin red positive nodules with increasing induction time indi-
cated that more and more calcium deposition had occurred.
The calcium content at different induction time points was
measured using a Calcium C-Test kit so as to quantitatively assay
the formation of bone matrix. It can be seen that the change of cal-
cium content presented the same tendency over time as Alizarin
red S staining shown in Fig. 8C and Table 4. It can be seen that
the use of LIPUS or RGD could induce a higher level of calcium
deposition compared with that in the controls, further revealing
that LIPUS and RGD can enhance the differentiation of cultured
hMSCs towards osteoblasts, as indicated by the increase in the cal-
cium content. In comparison, LIPUS led to a much higher level of
calcium deposition and hence had a much stronger enhancement
effect on osteogenic induction than RGD. The synergistic effect of
LIPUS and RGD on the enhancement of calcium nodules was also
observed but was not very singnificant compared with the prolifer-
ation of hMSCs, as shown in Tables 1 and 4.
4. Discussion
4.1. The effect of RGD modification on the proliferation and
differentiation of hMSCs
Due to good biocompatibility and the properties of degradation
and release, as well as appropriate mechanical properties, hydro-
gels have good prospects for use in biomedical applications. How-
ever, hydrophilic surfaces for hydrogels are not beneficial for the
2524 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528

Fig. 5. Images of immunofluorescence staining of cells after endothelial induction of 28 days. The differentiated cells were detected for the expression of (green) endothelial
cell markers CD34 and visualized by FITC-conjugated secondary antibody. DAPI counter stained nuclei in blue. Original magnification: 200.

Fig. 6. Quantitative analysis of fluorescence intensity of (A) CD31 and (B) CD34 weas performed and values are expressed as IOD. The results are presented as mean ± SD from
experiments performed in triplicate. ⁄Significantly different (p < 0.05, n = 3) to the control group at the same time point; ⁄⁄significantly different (p < 0.01, n = 3) to the control
group at the same time point. The OSA/NSC groups, RGD–OSA/NSC groups and blank control groups are denoted as ON, R-ON and Blank, respectively, whilst these groups with
LIPUS are named as LIPUS-ON, LIPUS-R-ON and LIPUS-Blank, respectively.
 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528 2525

Table 2
Integrated optical density (IOD) of images by CD31 (CD34) staining.

28D ON R-ON LIPUS-ON LIPUS-R-ON LIPUS-Blank

D-value D/B (%) p –37563 (–37518.6) 31481.7 (31509.5) 83754.4 (93800.9) 233853.5 (233936) 117030.8 (127092.4)
–14.98 (–14.90) 12.56 (12.52) 33.39 (37.26) 93.24 (92.93) 46.66 (50.49)
0.0058 (0.0003) 0.0002 (0.0025) <0.0001 (0.0014) 0.0001 (<0.0001) <0.0001 (0.0009)

B: The blank control group; D-value: the difference in the OD between the samples and the blank control group; p: p-value determined using Student’s t-test (n = 3).

Fig. 7. (A) ALP staining assay after osteogenic induction of 3, 7, 10 and 14 days. Original magnification: 100. (B) ALP activity assay after osteogenic induction of 3, 7, 10 and
14 days. Results are presented as mean ± SD from experiments were performed in triplicate. ⁄Significantly different (p < 0.05, n = 3) to the control group at the same time
point; ⁄⁄significantly different (p < 0.01, n = 3) to the control group at the same time point. The OSA/NSC groups, RGD–OSA/NSC groups and blank control groups are denoted
as ON, R-ON and Blank, respectively, whilst these groups with LIPUS are named as LIPUS-ON, LIPUS-R-ON and LIPUS-Blank, respectively.

Table 3
ALP activity assay of osteogenic induction.

Time (D) ON R-ON LIPUS-ON LIPUS-R-ON LIPUS-Blank

3 D-value – 0.0002 –0.0008 0.0026 0.0005
D/B (%) – 1.20 –1.30 4.21 0.81
p 0.4976 0.4914 0.4420 0.3338 0.4738
7 D-value –0.0038 0.0067 0.0316 0.0413 0.0338
D/B (%) –2.66 4.69 22.11 28.90 23.65
p 0.2732 0.1427 0.0132 0.0147 0.0081
10 D-value –0.0013 0.0066 0.0525 0.0618 0.0545
D/B (%) –0.46 2.31 18.41 21.68 19.11
p 0.4190 0.0877 <0.0001 0.0027 0.0026
14 D-value –0.0013 0.0040 0.0267 0.0364 0.0283
D/B (%) –0.71 2.18 14.56 19.85 15.43
p 0.1423 0.0558 0.0011 0.0059 0.0112

B: The blank control group; D-value: the difference in the OD between the samples and the blank control group; p: p-value determined using Student’s t-test (n = 3).

adsorption of protein and not conducive to the adhesion and
growth of cells; hence these will limit the biomedical applications
of hydrogels. The crucial factors for adherent cell survival are the
stability of the substrate’s adherent surface and its ability to sup-
port cell adhesion. Hence, the processes of material functionaliza-
tion and modification have become very important in tissue
engineering. In this study, the modification of hydrogels by graft-
ing adhesive molecules such as RGD onto them has been proved
to be a promising approach to overcome these limitations. The
results showed that the RGD modification of hydrogels could
improve the adhesion, growth and proliferation of hMSCs to a
certain extent, although not more significant than expected. RGD
was also found to promote the differentiation of hMSCs in this
study. Moreira et al.’s study showed that the seeded cell population
in the 3-D dense collagen scaffolds can affect the degree of osteo-
blastic cell proliferation, differentiation and some aspects of matrix
remodeling activity [42]. Wang et al.’s study found that a higher
density of human umbilical cord mesenchymal stromal cells can
result in better osteogenic differentiation and more matrix biosyn-
thesis than lower densities [43]. Hence, we speculate that the
promoted effect of RGD on the differentiation of hMSCs might be
attributed to a high cell density caused by RGD.
4.2. The effect of LIPUS with 1 MHz and 200 mW cm–2 on the
proliferation and differentiation of hMSCs
LIPUS led to lower cell proliferation during the first 3 days com-
pared with the groups without LIPUS, indicating that LIPUS is not
2526 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528

Fig. 8. (A) Images of mineralized nodule formation by staining with alizarin red S after osteogenic induction of 14, 21 and 28 days. Original magnification 100. (B) Images of
living cells after osteogenic induction of 28 days, showing mineralized nodule formation. Original magnification 400. (C) Quantitative analysis of calcium content after
osteogenic induction of 14, 21 and 28 days. Results are presented as the mean ± SD from experiments performed in triplicate. ⁄Significantly different (p < 0.05, n = 3) to the
control group at the same time point; ⁄⁄significantly different (p < 0.01, n = 3) to the control group at the same time point. The OSA/NSC groups, RGD–OSA/NSC groups and
blank control groups are denoted as ON, R-ON and Blank, respectively, whilst these groups with LIPUS are named as LIPUS-ON, LIPUS-R-ON and LIPUS-Blank, respectively.

Table 4
Mineralized nodule formation of osteogenic induction.

Time (D) ON R-ON LIPUS-ON LIPUS-R-ON LIPUS-Blank

14 D-value –0.1 0.4 1.1 2.0 1.2
D/B (%) –2.38 9.52 26.19 47.62 28.57
p 0.4075 0.0889 0.0030⁄⁄ 0.0162⁄ 0.0587
21 D-value –0.2 0.5 2.1 3.0 2.2
D/B (%) –2.06 5.15 21.65 30.93 22.68
p 0.0435 0.0189 0.0172 0.0044 0.0161
28 D-value –0.3 0.4 1.9 2.9 2.1
D/B (%) –2.04 2.72 12.93 19.73 14.29
p 0.3169 0.2346 0.0217 0.0101⁄ 0.0003

B: The blank control group; D-value: the difference in the OD between the samples and the blank control group; p: p-value determined using Student’s t-test (n = 3).

Significantly different (p < 0.05, n = 3) to the control group at the same time point.

Significantly different (p < 0.01, n = 3) to the control group at the same time point.

beneficial for the early adsorption of adhesive proteins. However,
the enhancement of cell proliferation caused by LIPUS was
detected after culture of 3 days and it was very strong.
Whether LIPUS can promote cell proliferation or not remains
debatable. Takayama et al. reported that transient LIPUS stimula-
tion (1.5 MHz, 30 mW cm–2, 20 min day–1) did not affect the rate
of cell proliferation [28]. Parvizi et al. reported that pulsed ultra-
sound stimulates (1 kHz) had no effect on cell proliferation [44].
However, Zhang et al. reported that LIPUS influenced chondrocyte
proliferation in an intensity-dependent manner [45]. We speculate
that bio-effects of cells caused by LIPUS depends on ultrasound
dosage and ultrasound stimulates have no effect on cell prolifera-
tion under too low an intensity and frequency and can inhibit pro-
liferation of cells under too high an intensity and frequency. Our
results showed that 1 MHz and 200 mW cm–2 might be a suitable
frequency and intensity beneficial for the proliferation of cells.
The present study shows that LIPUS can enhance osteogenic dif-
ferentiation of hMSCs by stimulating ALP activity and promoting
mineralized nodule formation. Similar results have been reported
elsewhere [16,46–50]. Warden et al. found that LIPUS could stim-
ulate the expression of the immediate early response genes c-fos
and COX-2, and elevate mRNA levels for the bone matrix proteins
ALP and OC, suggesting that LIPUS has a direct effect on bone for-
mation [16]. They also found that the cells could be induced by the
differentiation of hMSCs to osteoblasts in the absence of LIPUS.
Hence, these suggest that soluble factors secreted by hMSCs might
induce the differentiation of hMSCs in vitro while LIPUS only
enhanced this process rather than triggering it. Chen et al. found
 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528
Fig. 9. Schematic diagram (based on the model proposed by Chien [53]) showing the signaling pathways during the differentiation of hMSCs.
that bone marrow mesenchymal stem cells responded to LIPUS by
increasing extracellular signal-regulated kinase phosphorylation
and enhancing osteogenic differentiation [49]. Jiang et al.
investigated the osteogenic in vitro effect of LIPUS on SD rat adi-
pose-derived stem cells (ADSCs) and found that LIPUS-treated
ADSCs presented higher mRNA expression levels of runt-related
transcription factor 2 (Runx2), osteocalcin (OCN), ALP and bone
sialoprotein (BSP) genes and higher protein levels of Runx2 and
BSP than the controls, suggesting that LIPUS can also induce the
osteogenic differentiation of ADSCs in vitro [50]. These findings
are consistent with those observed by us.
In our study, the cells in the groups with LIPUS showed a much
higher level of the positive expression of CD31 and CD34 compared
with those in the groups without LIPUS, revealing that LIPUS can
also promote the endothelial differentiation of hMSCs. Reher
et al.’s study demonstrated that ultrasound stimulates the produc-
tion of angiogenic factors such as IL-8, bFGF and VEGF [47]. Hence,
we speculate that endothelial differentiation might be attributed
to the non-thermal effects of LIPUS via a similar approach.
Although in vitro investigations possess limitations, they do
provide important insight into the potential benefits of LIPUS and
the mechanisms by which these effects are produced for the
non-thermal effects of LIPUS [51,52]. We speculate that LIPUS
might stimulate the release of some growth factors and extracellu-
lar matrices (ECMs) from hMSCs during induction and as a result,
promoted the proliferation and differentiation of hMSCs. To further
investigate it, further in vitro study is required in the future.
4.3. Synergistic effect of LIPUS and RGD on the proliferation and
differentiation of hMSCs
In our study, LIPUS and RGD enhanced the proliferation and dif-
ferentiation of hMSCs. This is a little-studied phenomenon. We
found that the synergistic effect of LIPUS and RGD on the enhance-
ment of proliferation of hMSCs was different to the effect on hMSC
differentiation. We speculate that the synergistic effect on the
enhancement of proliferation is achieved by RGD promoting cell
proliferation to create a higher cell density and LIPUS stimulating
hMSCs to release ECMs, which further enhance the proliferation
of hMSCs. Similarly, for the synergistic effect on the enhancement
of differentiation of hMSCs, the use of RGD can promote cell prolif-
eration to achieve a high cell density for differentiation, thus en-
abling LIPUS to stimulate more hMSCs to release growth factors
to form a microenvironment with high concentrations of growth
factors where differentiation of hMSCs can be successfully induced.
Based on the model proposed by Chien [53], possible signaling
pathways of endothelial and osteogenic differentiation of hMSCs
were presented in Fig. 9. A series of signaling pathways plays a cru-
cial role in the differentiation of hMSCs, where in our experiments
there are three kinds of sources of extracellular signaling including
2527
RGD, cytokine and LIPUS, which may be regarded as different
‘‘chemical ligands’’ responsible for binding with different receptors.
Like chemical ligands, LIPUS can stimulate hMSCs by activating
some special mechanosensors while these sensors can trigger sig-
naling pathways to activate transcription factors to bind with the
appropriate cis elements so as to modulate the expression of the
gene and then the protein [53]. The changes in gene and then pro-
tein expressions, in turn, modulate the function of the hMSCs and
finally result in the differentiation of hMSCs [53]. As the receptors
of RGD, integrins are responsible for not only regulating the attach-
ment between cells and materials but also passing chemical and
mechanical signals of the ECM into the cells to activate the signal-
ing pathways [54,55]. During the differentiation of hMSCs, some of
the same receptors might simultaneously respond to a number of
different ‘‘chemical ligands’’; for example, integrins respond not
only to RGD but also to LIPUS. Moreover, LIPUS can increase the
permeability of the cell membrane and accelerate signal transmis-
sion and enhance the role of cytokines and RGD, promoting the dif-
ferentiation of hMSCs [56]. Hence, a positive synergistic effect of
LIPUS and RGD can be expected in this process, as we have seen
in our experiments.
5. Conclusion
An RGD–OSA/NSC hydrogel was fabricated. An in vitro study of
endothelial and osteogenic differentiation of marrow-derived
MSCs was conducted on hydrogels and the effect of RGD modifica-
tion and LIPUS on the proliferation and differentiation of hMSCs
was investigated. The results demonstrated that cell proliferation
was upregulated, and osteogenic and endothelial differentiation
of hMSCs was enhanced by the use of RGD modification and LIPUS,
which might be the result of LIPUS increasing the permeability of
the cell membrane and accelerating signal transmission and
enhancing the role of cytokines and RGD. The synergistic effect
of LIPUS and RGD on the enhancement of proliferation and
differentiation of hMSCs was observed. These findings suggest
the hybrid use of RGD modification and LIPUS might provide one
approach to achieve a high level of formation of new bone and
vascularization in bone tissue engineering scaffold implants.
Acknowledgements
This work was supported by the National Basic Research Pro-
gram of China (973 Program, 2012CB933600), the National Natural
Science Foundation of China (No. 51072167 and No. 31370966),
Funds from the Health Department of Sichuan Province
(No. 110281) and Fundamental Research Funds for the Central
Universities (SWJTU11ZT05).
2528 Y. Wang et al. / Acta Biomaterialia 10 (2014) 2518–2528

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