Biomaterials 44 (2015) 173e185

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Electrospun scaffolds for multiple tissues regeneration in vivo through

topography dependent induction of lineage specic differentiation
Zi Yin a, b, 1, Xiao Chen a, b, 1, Hai-xin Song e, Jia-jie Hu a, b, Qiao-mei Tang a, b, Ting Zhu a, b,
Wei-liang Shen d, Jia-lin Chen a, b, Huanhuan Liu a, b, Boon Chin Heng f,
Hong-Wei Ouyang a, b, c, *
a

Department of Sports Medicine, School of Medicine, Zhejiang University, Hangzhou, China

Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, Hangzhou, China
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious
Diseases, The First Afliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
d
Department of Orthopedic Surgery, 2nd Afliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
e
Department of Rehabilitation, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
f
Department of Biosystems Science & Engineering (D-BSSE), ETH-Zurich, Basel, Switzerland
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 10 September 2014
Accepted 20 December 2014
Available online

Physical topographic cues from various substrata have been shown to exert profound effects on the
growth and differentiation of stem cells due to their niche-mimicking features. However, the biological
function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously
characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells
(MSCs) and neo-tissue formation trigged by aligned and randomly-oriented brous scaffolds, both
in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the
Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical
staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent
bone tissue formation through ossication. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced
tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of
osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on
randomly-oriented ber scaffolds displayed enhanced osteogenic differentiation compared with cells
cultured on aligned ber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these
ndings illustrate the relationship between topographic cues of the scaffold and their inductive role in
tissue regeneration; thus providing an insight into future development of smart functionalized bioscaffold design and its application in tissue engineering.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Bio-scaffold topography
Tissue engineering
Tendon regeneration
Bone formation
Mesenchymal stem cells

1. Introduction

by

Stem cell survival, self-renewal and differentiation are governed

local biochemical and mechanical factors within their

* Corresponding author. Center for Stem Cell and Tissue Engineering, School of
Medicine, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China.
Tel./fax: 86 571 88208262.
E-mail address: hwoy@zju.edu.cn (H.-W. Ouyang).
1
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.biomaterials.2014.12.027
0142-9612/ 2014 Elsevier Ltd. All rights reserved.

microenvironmental niche [1]. The key niche components include

soluble factors, other cells, and extracellular matrix molecules. While
the role of biochemical signals is well-documented, the importance
of biophysical cues has received more recognition and attention only
in the last decade. Current advances in microfabrication technologies
have enabled the generation of substrates with nano/micro-scale
topographies to study the effects of biophysical signals on cellular
function. A number of studies have demonstrated that the physical
properties of substrata have profound effects on the cellular functions of pluripotent and multipotent stem cells, including cell

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Z. Yin et al. / Biomaterials 44 (2015) 173e185

adhesion, morphology, proliferation, migration and differentiation

[2e6]. Based on the concept of contact guidance, we designed a
biomimetic aligned nanober scaffold modelled on the parallel
collagenous bers of tendon extracelluar matrix; and subsequently
demonstrated that alignment within the scaffold regulate tendon
stem cell orientation and induce specic teno-lineage differentiation
[7]. Meanwhile, both random orientation and nanoscale disorder
have been demonstrated to induce ossication of human multipotent stem cells in vitro, even in the absence of osteogenic media
[6]. Although there are promising results in various studies that have
attempted to control stem cell fate in vitro through modication of
substrate physical properties, there is a dire need to move from
culture substrate to implantable scaffolds with direct applications in
tissue engineering [8].
Conventional scaffolds are designed and fabricated according to
the basic requirements of biocompatibility, structural support as
well as cell delivery, and have already been widely utilized in
various tissue engineering applications [9]. Modern bio-scaffolds
not only just serve as a carrier for seed cells, but also provide an
appropriate microenvironment for stem cells and mediates biological functions. Further microstructural renement of current
scaffold biotechnology will enhance the progress of tissue engineering in the future [10]. However, the three-dimensional
microenvironment in vivo represents a much more complicated
milieu that encompasses a much more diverse multitude of
signaling cues compared to an in vitro culture system. Under
physiological conditions, stem cells naturally encounter a variety of
different signaling cues that can potentially inuence cell fate. It is
essential and necessary to use the results of in vitro studies to aid
the rigorous characterization of the functionality of tissue engineered scaffolds in vivo. This prompted our investigation on the
inductive effects of scaffold topographic cues on stem cell differentiation pathways and lineage fate.
This study aims to characterize the biophysical effects of scaffold
topography on tissue regeneration in vivo within a 3D microenvironment, utilizing aligned and randomly-oriented brous scaffolds.
We tested the hypothesis that topographic cues from the aligned
brous scaffold can enhance tendon-like tissue formation, and that
there would be a higher degree of osteogenesis and tissue ossication with the randomly-oriented ber scaffold. Additionally, the
role of cytoskeletal organization in topography driven differentiation of mesenchymal stem cells was also investigated in vitro. We
believe that the data presented here would be benecial to the
design and application of future biomaterials.
2. Materials and methods
2.1. Fabrication of PLLA scaffolds
Both aligned (1068 190 nm) and randomly-oriented PLLA scaffolds
(739 129 nm) were fabricated using the electrospinning technique as previous
reported. The polymer solution was prepared by dissolving PLLA (Ji'nan Daigang
Biomaterial Co., Ltd) in a mixture of chloroform/ethanol (3:1) at a concentration of
4% (aligned) or 3% (random). The solution was then fed into a 12-ml plastic syringe,
which was controlled by a syringe pump at a rate of 2 ml/h. A high voltage (12 kV)
was applied to the needle tip, which was placed 10 cm above the collector. A at
aluminum plate was used to collect the random bers. The collector for aligned bers was a disk rotating at 4000 rpm. The resulting scaffolds were then transferred
to cover slips and sterilized with ethanol and UV overnight before they were utilized
for cell culture. Nanobers were collected for 2e3 h, resulting in a ber mat ranging
in thickness from 0.14 to 0.17 mm. The aligned and randomly-oriented scaffolds
utilized in this study were of similar thickness and distribution.

2.3. SEM imaging

C3H10T1/2 cells (mouse multipotent mesenchymal stem cell line) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
Cells were seeded onto PLLA scaffolds at 2  104 cells/cm2 and cultured in Dulbecco's
modied Eagle's medium (DMEM, low glucose; Gibco, Grand Island, NY, http://
www.invitrogen.com) with 10% (v/v) fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, http://www.invitrogen.com-Gibco) and 1% (v/v) penicillin-streptomycin
(Gibco). The medium was changed once every 3 days. Three days after seeding,
the cell morphology and distribution were visualized using SEM. Specimens were
xed in 0.25% glutaraldehyde solution, and then rinsed 3 times in PBS, 30 min each
time. The specimens were immersed in OsO4 for 40 min and then rinsed 3 times in
PBS, 30 min each time, followed by dehydration in increasing concentrations of
acetone (30e100% v/v). After drying, the specimens were mounted on aluminum
stubs and coated with gold, then viewed under a Hitachi S-3000N SEM at an
accelerating voltage of 15 kV. For quantication of cell morphology on different
scaffolds, a minimum of fty cells for each SEM image were selected randomly as ROI
(Regions of Interest). The area and radius ratio were then quantied using the
Image-Pro Plus software.
2.4. Alkaline phosphatase (ALP) staining
C3H10T1/2 cells (104/cm2) were seeded onto scaffolds and cultured in osteogenic induction medium, in the presence of 10 mM b-glycerol phosphate (Sigma),
0.1 mM dexamethasone (Sigma), and 50 mg/ml ascorbic acid (Sigma) supplemented
in DMEM-high glucose medium containing 10% (v/v) FBS and 1% (v/v) penicillinestreptomycin. After 7 days, ALP activity was assayed using a BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology). DAPI
(Beyotime Institute of Biotechnology) was used to stain nuclei and observed under a
light microscope (Olympus IX71).
2.5. Quantitative PCR
Total cellular RNA was isolated by lysis in TRIzol (Invitrogen). The expression
levels of tendon-specic genes and osteogenic markers in cells cultured on aligned
and randomly-aligned brous scaffolds were assessed by quantitative PCR. PCR was
performed using a Brilliant SYBR Green qPCR Master Mix (TakaRa) on a Light Cycler
apparatus (ABI 7900HT). The PCR cycling consisted of 40 cycles of amplication of
the template DNA with primer annealing at 60  C. The relative expression levels of
each target gene was then calculated using the 2-DDCt method. The amplication
efciencies of primer pairs were validated to enable quantitative comparison of gene
expression. All primers (Generay) were designed using primer 5.0 software and are
summarized in the Supplementary Table 1.
2.6. Animal model
The Zhejiang University Institutional Animal Care and Use Committee approved
the study protocol. In situ rat Achilles tendon repair model: Twenty hind limbs of
skeletally mature female rats weighing 200e220 g were utilized for this experiment.
Under general anesthesia, a gap wound was created and the Achilles tendon was
removed to create a defect of 6 mm in length. Aligned and random brous scaffolds
(8 mm  8 mm, thickness 100 um) were folded about 2 mm from the bottom of
the membrane upwards. This was followed with fan-folding the next 2 mm to the
back. Fan-folding of the scaffold was continued until it was completely folded. This
was followed by binding the center of the strip using a suture and subsequent
placement into the gap wound. Suturing to the remaining Achilles tendon was then
carried out using a non-resorbable suture material (Nylon6). The wound was then
irrigated and the skin was closed. The animals were allowed free cage activity after
surgery. At 2, 4, and 8 weeks post-implantation, samples from each group were
harvested for the evaluation of histology, transmission electron microscopy imaging,
mechanical testing, as well as collagen content determination (Supplementary
Table 2).
2.7. Immunouorescence
Briey, cells were xed in 4% (w/v) paraformaldehyde for 10 min at room
temperature, permeabilized, and blocked for 30 min with 1% (w/v) bovine serum
albumin, and then permeabilized with 0.1% (w/v) Triton X-100. Fixed cells were
washed and incubated with a primary antibody against SCX (Abcam Inc.), Vinculin
(Millipore), or control IgG (BD) at 4  C overnight. Cells were then incubated with
Alexa uor 488-conjugated secondary antibody (Invitrogen) for 2 h and the nuclei
were stained with DAPI. TRITC-phalloidin (Millipore) staining was used to visualize
the cytoskeleton. The imaging was then performed with confocal microscopy (Zeiss
LSM-510).

2.2. Morphology of PLLA scaffolds

2.8. Histological evaluation and staining

The scaffold samples were sputter-coated with gold, and then their structure
was observed under scanning electron microscopy (SEM) (Hitachi S3000N) at an
accelerating voltage of 15 kV. After the micrographs were obtained, image analysis
software (Image-Pro Plus) was used to measure the average diameter of the nanobers (n 3). For each sample, an average of 50 bers were counted.

Harvested specimens were immediately xed in 10% (v/v) neutral buffered

formalin, dehydrated through an alcohol gradient, cleaned, and then embedded
within parafn blocks. Histological sections (7 um) were prepared using a microtome and subsequently stained with hematoxylin and eosin. In addition, Masson
trichrome staining was performed according to standard procedures to examine the

Z. Yin et al. / Biomaterials 44 (2015) 173e185

general appearance of the collagen bers. To detect proteoglycan synthesis as an
indicator of cartilage formation, sections were stained with 0.1% Safranin O (Sangon,
Shanghai, China) for 8 min, followed by counter staining with 0.02% Fast Green for
4 min. Polarizing microscopy was employed to detect mature collagen brils. General histological scoring was performed using hematoxylin and eosin staining. Six
parameters (ber structure, ber arrangement, rounding of nuclei, inammation,
vascularity, cell population) were semi-quantitatively assessed. These six parameters were semi-quantitatively graded on a four-point scale (0eIII), with 0 being
normal and 3 being maximally abnormal. Therefore, a normal tendon would score 0,
while a maximally abnormal tendon would score 18. The detailed scoring system
was summarized in Supplementary Table 3.
2.9. Immunohistochemistry
Parafn sections (7 mm) were incubated in antigen retrieval buffer (100 mM Tris,
5% (w/v) urea, pH 9.5) at 95  C for 10 min for antigen retrieval. Endogenous
peroxidase was blocked by incubation with 3% (v/v) hydrogen peroxide in methanol
for 10 min. Non-specic protein binding was blocked by incubation with 10% (v/v)
goat serum. After overnight incubation at 4  C with primary antibodies against
Collagen (Abcam Inc.) or osteocalcin (Millipore), sections were washed and then
incubated with goat anti-mouse (Beyotime Institute of Biotechnology Inc., Jiangsu,
China) or goat anti-rabbit (Beyotime Institute of Biotechnology Inc., Jiangsu, China)
secondary antibodies for 2 h at room temperature. The DAB substrate system (Zsbio,
Beijing, China) was used for color development. Hematoxylin staining was used to
reveal the nuclei.
2.10. Mechanical testing
Mechanical testing was performed using an Instron tension/compression system with Fast-Track software (Model 5543, Instron, Canton, MA). Measurements of
the tendon cross-sectional area were performed using two Vernier calipers at 5 mm
proximal to the conjunction of bone and tendon. The bone end of the tendon was
secured by a specially designed restraining jig and the tendon end was pinched with
a clamp [11]. The AT-calcaneus complex (ACC) was then rigidly xed to custommade clamps. After applying a preload of 0.1 N, each ACC underwent preconditioning by cyclic elongation of between 0 and 0.5 mm for 20 cycles at 5 mm/
min. This was followed by a load to failure test at an elongation rate of 5 mm/min.
The loadeelongation behavior of the ACCs and failure modes were recorded. The
structural properties of the ACC were represented by stiffness (N/mm), ultimate load
(N), energy absorbed at failure (mJ) and stress at failure. For each ACC, the greatest
slope in the linear region of the loadeelongation curve over a 0.5 mm elongation
interval was used to calculate the stiffness.
2.11. Determination of collagen content
The amount of deposited collagen in the scaffold was quantied by using a
collagen assay kit according to the manufacturer's protocol (Jiancheng Ltd., Nanjing,
China). This kit is a hydroxyproline assay, which is widely used to determine total
collagen content, and a conversion factor of 1:7.46 was used to convert hydroxyproline to collagen [12], lyophilized tendons were digested with a hydrolysis regent
at 95  C for 20 min. Serial dilutions of acid-soluble collagen type I provided by the kit
were utilized as standards. Following the assay, collagen concentration was determined through absorbance measurements at 550 nm using a microplate reader
(Molecular Devices).
2.12. Transmission electron microscopy
Tissue specimens were xed by standard procedures for TEM to assess collagen
bril diameter and alignment. Briey, samples were pre-xed in 2% (w/v) glutaraldehyde for 2 h at 4  C and then washed twice in PBS at 4  C followed by postxation with 1% (v/v) osmic acid for 2 h at 4  C. After two washes in PBS, the
samples were dehydrated with an ethanol gradient and dried to a critical point. The
samples were then mounted and sputter-coated with gold for viewing under TEM
(Quanta 10 FEI). Approximately 500 collagen brils were measured for each sample
to obtain an accurate representation of the bril diameter distribution.
2.13. Radiographic evaluation
The X-ray photographs of whole animals were captured with a non-invasive
Kodak-FX in vivo imaging system (Kodak, Inc.) to evaluate ectopic bone formation.
At 8 weeks post-implantation, captured images were analyzed with the Image-pro
plus software to quantify the area, mean density, max density, and sum density of
ectopic bone formation.
2.14. Statistical analysis
All quantitative data sets are expressed as mean SD. The Student's t-test was
performed to assess statistically signicant differences in the results of different
experimental groups. Values of p < 0.05 were considered to be signicantly
different.

175

3. Results
3.1. Fabrication and morphological characterization of scaffolds
Electrospinning is used to fabricate aligned brous scaffolds, as
well as randomly-oriented brous membranes with similar ber
diameters, which served as controls. The surface topography of
both aligned and randomly-oriented brous scaffolds was examined using SEM (Fig. 1A and B). Majority of the bers in the aligned
scaffolds were parallel to each other and formed angles from 0 to
10 with respect to the horizontal axis, while the randomlyoriented nanober scaffold exhibited nearly equal distributions at
all angles.
3.2. The effects of aligned and randomly-oriented scaffolds on neotissue formation
3.2.1. Histology of repaired tendons
After 2 weeks post-surgery, histological analysis showed that
there was a greater number of cells exhibiting spindle-shaped
morphology on the aligned scaffolds (Fig. 1C). While, the cell
morphology in the randomly-oriented groups displayed relatively
round shapes (Fig. 1D). Masson trichrome staining showed that the
tissue matrix was denser in the aligned versus randomly-oriented
scaffold group, which means that more collagen bers have been
deposited compared to the control group (Fig. 1 E and F). Collagen I
immunohistochemical staining showed organized collagen deposition on the aligned bers (Fig. 1 G and H), while the randomlyoriented scaffolds were lled with loose, disarranged matrix.
At 4 weeks post-surgery, the histological results showed that the
aligned scaffold-induced spindle-shaped cells and tendon-like tissue formation in vivo, as evidenced by the Masson trichrome
staining. In addition, the aligned scaffold implantation showed
improved tendon repair quality was compared to the histology of
blank group without scaffold implantation, which displayed limited
tissue regeneration (Supplementary Fig.S1). The collagen I immunohistochemical staining reveals enhanced collagen I matrix production and arrangement in the aligned scaffold group (Fig. 2A).
Histology scoring also conrmed the histological results that the
quality of repaired tendons in the aligned scaffold group were
signicantly better than the control group (p < 0.05, Fig. 2E),
particularly in ber structure, ber arrangement and nuclei shape
aspects. The ultrastructural morphology based on TEM imaging
from transection revealed larger bril formation within the aligned
scaffold, as compared to the thin brils observed within the
randomly-oriented scaffold (Fig. 2B). The average diameter of
collagen brils in the aligned group was 52.88 nm (373 bers),
which was 121.4% of the random group (43.57 nm, 266 bers) at 4
weeks post-implantation (Fig. 2D). Analysis of the distribution of
bril diameters within the two groups (Fig. 2C) revealed that the
ATs in the aligned scaffold treatment group formed signicantly
larger brils compared to ATs in the random group at 4 weeks postimplantation. The collagen content assay demonstrated that the
aligned group had signicantly more collagen deposition,
compared to the random group (0.281 0.039 mg/mg vs.
0.198 0.072 mg/mg, p < 0.05, Supplementary Fig.S2).
We also utilized polarized light microscopy to compare the
collagen ber maturation levels within the two groups. The aligned
scaffold group exhibited more continuous collagen bers at the
repair site (Fig. 3B). Furthermore, the expression of tendon-specic
marker scleraxis (Scx) was signicantly higher in the aligned versus
randomly-oriented group at 2 weeks and 8 weeks post-surgery
(Fig. 3A). The other teno-lineage marker gene tenomodulin
(Tnmd) also displayed signicantly higher levels in the aligned
scaffold group, thus indicating that aligned topographic cues have

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Fig. 1. (A) and (B) SEM micrographs (1000  ) of electrospun PLLA with aligned (A) and randomly-oriented (B) brous scaffold surface topography. Scale bars, 50 mm. Histological
results of repaired rat Achilles tendon in section of aligned group (upper panel) and randomly-oriented group (lower panel) at 2 weeks post-surgery, (C) and (D) are typical
hematoxylin and eosin staining, (E) and (F) are Masson trichrome staining, (G) and (H) are immunohistochemical staining of collagen type I, respectively. Arrows indicated the
remaining brous scaffolds. Scale bars, 50 mm, 100 mm (inset).

Fig. 2. Repaired rat Achilles tendon at 4 weeks post-surgery. (A) Typical hematoxylin and eosin staining, Masson trichrome staining, immunohistochemical staining of collagen type
I in repaired zones within sections of aligned group (upper panel) and randomly-oriented group (lower panel). (B) Transmission Electron Microscopy images show ultrastructure of
repaired tendons after 4 weeks post-surgery. (C) Histogram and distribution of collagen bril diameters of aligned group (upper panel) and randomly-oriented group (lower panel).
(D) Collagen bril diameters. Data are mean SD, n 3. (E) The overall histology score is the sum of six parameters (ber structure, ber arrangement, rounding of nuclei,
inammation, vascularity, cell population). Statistically signicant at *p < 0.05. Scale bars, 100 mm (HE and Masson stained images), 200 mm (immunohistochemical staining of
collagen type I, left images), 50 mm (immunohistochemical staining of collagen type I, right images).

Z. Yin et al. / Biomaterials 44 (2015) 173e185

177

Fig. 3. (A) Gene expression levels of tendon-related markers assessed by quantitative PCR at 2, 4 and 8 months post-surgery. (B) Polarized microscopy images of aligned group
(upper panel) and randomly-oriented group (lower panel) at 2, 4 and 8 months post-surgery. Statistically signicant at *p < 0.05, **p < 0.01. Scale bars, 100 mm.

more potential in tendon tissue repair and regeneration. Msx-2 [13],

which has been reported to play a central role in preventing tendons from mineralizing, exhibited elevated expression in the
aligned versus randomly-oriented groups at all time-points
(Fig. 3A).
3.2.2. Histology of bone formation
On the other hand, chondrocyte-like cells appeared within the
injury site implanted with the randomly-oriented scaffold at 4
weeks post-surgery (Fig. 2A). Furthermore, Safranin O staining
showed signicantly more chondrocyte-like cells in the randomlyoriented versus aligned scaffold group (Fig. 4A). It is obvious with
the randomly-oriented group that the ossied deposits surrounded
by chondrocyte-like cells were localized at the tendon mid-section
inside the wound, which caused disruption to the organization of
collagen bers (Fig. 4A). Additionally, bone marrow was also
formed in the randomly-oriented scaffold group at 8 weeks
(Fig. 4A) Nevertheless, no ossication was detected in all samples
with X-ray scanning at 4 weeks post-surgery (data no shown),
whereas all samples of the randomly-oriented group exhibited
spontaneous ectopic bone formation at 8 weeks post-surgery
(Fig. 5A). Upon quantication of the area and density of ectopic
bone formed, it was found that the randomly-oriented scaffold
group displayed signicantly larger area (Fig. 5B, p < 0.05), higher
mean density (Fig. 5B), max density (Fig. 5B, p < 0.05) and integrated optical density (IOD) (Fig. 5B, p < 0.05) of ectopic bone, as
compared to the aligned scaffold group. The results thus indicated

that randomly-oriented topographic scaffolds had signicantly

higher potential to induce ectopic bone formation compared to
aligned scaffolds.
Expression of osteochondral-lineage marker genes were
analyzed to further evaluate tissue formation. It was observed that
the expression of growth factors BMP4, chondrogenic transcription
factor Sox9 and chondrocyte specic matrix Col II were signicantly
higher in the randomly-oriented versus aligned groups at 2 weeks
post-surgery (Fig. 4B). The matrix osteocalcin expression was
signicantly much higher in the randomly-oriented versus aligned
groups, while expression of the osteogenic transcription factor
Runx2 exhibited the largest difference at 8 weeks post-surgery
(Fig. 4B). This suggests that randomly-oriented topographic cues
play a critical role in initiating cartilage and bone formation at the
early repair stage (2 weeks) with neo-bone being formed as a result
of cartilage ossication at the later repair stage (8 weeks). The
expression of collagen type X, a representative marker of chondrocyte hypertrophy was detected by immunohistochemical
staining. Comparison of the two groups showed signicantly
denser and larger positively-stained areas within the randomlyoriented versus aligned group (Fig. 6), which is consistent with
increased Safranin-O staining (Fig. 4A). This indicated that the
ossied deposits were formed by endochondral ossication.
Expression of the bone formation marker osteocalcin was examined by immunohistochemical staining as well, and the expression
levels were obviously much higher in the randomly-oriented
versus aligned group at 8 weeks post-surgery (Fig. 6). These data

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Z. Yin et al. / Biomaterials 44 (2015) 173e185

Fig. 4. (A) Safranin O staining images of aligned group (upper panel) and randomly-oriented group (lower panel) at 4 and 8 months post-surgery. (B) Gene expression levels of
chondrogenic (aggrecan, Sox9, collagen type II) and osteogenic markers (Bmp4, Ocn, Runx2) assessed by quantitative PCR at 2, 4 and 8 months post-surgery. Statistically signicant at
*p < 0.05, **p < 0.01. Scale bars, 200 mm (100X), 100 mm (200X).

collectively suggested that randomly-oriented scaffolds can induce

more endochondral bone tissue formation by 8 weeks postimplantation.

3.2.3. Mechanical properties of repaired tendons

To further correlate tissue structural features with their mechanical properties, harvested tendons (n 5 for each group)
were subjected to mechanical testing at 8 weeks post-surgery. The
aligned group had better mechanical properties than the
randomly-oriented controls (Fig. 7 and Supplementary Fig.S3). The
modulus (24.42 2.20 MPa vs. 20.86 3.56 MPa) of the aligned
group were better than the control group (Fig. 7). The energy in
the aligned group was 20% higher than that of the control group
(179.65 9.29 mJ vs. 149.23 38.49 mJ, Fig. 7). The maximum
force in the aligned group was higher than that of the control
group (87.12 5.61 N vs. 77.09 13.27 N, p > 0.05). The stress at
failure and stiffness were consistently higher in the aligned group
than that of the control group, but these difference was not statistically signicant (Fig. 7), probably due to the small sampling
size.

3.3. The effects of topographical cues on MSCs

The SEM micrographs indicated that MSCs were well attached to
both scaffolds, but displayed distinct morphology. On the aligned
nanober scaffold, the cells exhibited an elongated spindle-shaped
morphology and were oriented parallel to the substrate alignment,
whereas cells cultured on the randomly-oriented scaffolds were
spread out and exhibited a polygonal phenotype (Fig. 8A and B).
The bar graphs illustrate the size and aspect ratios of MSCs cultured
on different topographic substrates. MSCs grown on the aligned
scaffold displayed relatively smaller size, but with a signicantly
much higher aspect ratio than their counterparts on the randomlyoriented scaffold (Fig. 8C and D). Under confocal uorescence microscopy, alignment of cell orientation was also apparent under
TRITC-phalloidin staining, with the cytoskeleton being more uniformly oriented towards the alignment of nanobers within the
aligned scaffold (Fig. 9A). However, the MSCs cultured on the
randomly-oriented scaffolds exhibited different arrangement of the
F-actin network (Fig. 9A). The immunouorescence staining of
vinculin, which is a component of the focal adhesion complex,
showed the presence of focal adhesions and their distribution

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179

Fig. 5. (A) X-ray images of repaired tendons of mice at 8 weeks post-transplantation and evaluation of the extent of ossication (n 5). (B) The distribution and medians of area,
max density, sum density and integrated optical density of ectopic bone in aligned group (dot) and randomly-oriented group (square). Arrows depict exactly the locations of the
ectopic bone formation. Statistically signicant at *p < 0.05.

within cells cultured on scaffolds. Vinculin was localized mainly at

the peripheral region of MSCs cultured on the randomly-oriented
scaffold, at the end of F-actin ber bundles in the lopodia or
lamellipodia. A higher density of vinculin was observed at the poles
of elongated spindle-shaped MSCs cultured on the aligned scaffold.
Consistent with the great differences in cell morphology and
orientation observed on the two scaffolds, the focal adhesion and
cytoskeleton distribution also displayed distinctively different
patterns between the two groups (Fig. 9A). The expression of the
tenogenic transcription factor gene Scx was signicantly more
highly expressed by MSCs on aligned versus randomly-oriented
nanobers (Fig. 9C). The expression of the osteogenic transcription factor Runx2 was signicantly lower in MSCs cultured on
aligned versus randomly-oriented scaffold (Fig. 9D).
3.4. Topography-induced lineage commitment of MSCs is
dependent on cytomyosin cytoskeleton
To further understand the phenomena of topographic induced
differentiation of MSCs, we examined the effects of two small
molecules on cytoskeletal reorganization and mechanotransduction
- cytochalasin D (cyto D) and the Rho kinase (ROCK) inhibitor Y27632. The addition of cyto D to the medium attenuated the contact
guidance response by suppressing cell elongation in the aligned
scaffold group, while reducing projected surface area of cells in the
randomly-oriented group (Fig. 9B). We observed that cytochalasin D

treatment caused cells to become rounded without any notable

differences in cell morphology between the two groups (Fig. 9B).
Meanwhile, we also found that random orientation-induced
osteogenesis and alignment-induced tenogenesis were attenuated
as well by cyto D, as evidenced by downregulation of osteogenic
marker Runx2 and tenogenic mark Scx expression levels (Fig. 9C and
D).These results indicated that the actin cytoskeleton might be
important to the MSC lineage commitment process. Furthermore,
we treated the cell with Rho kinase (ROCK) inhibitor Y-27632 to
inhibit myosin-generated cytoskeletal tension. In the presence of Y27632, cells remained well-spread and morphologically similar on
the two different topographical substrates. Additionally, vinculin
expression was diminished signicantly in both groups (Fig. 9A).
Moreover, the quantitative PCR results revealed that there were no
signicant differences in either tenogenesis or osteogenesis of MSCs
cultured on different topographic substrates (Fig. 9C and D). The SCX
immunouorescence images showed that before addition of Y27632, SCX expression was higher and more concentrated in the
nuclei of MSCs cultured on aligned bers. Subsequent exposure to Y27632 not only blocked cell alignment but also eliminated differences in SCX expression between the two groups (Fig. 10). Similarly,
the ALP staining results also demonstrated that osteogenesis
induced by randomly-oriented nanober topography was abrogated
upon treatment with Y-27632 (Fig. 11). These ndings collectively
suggest that topography-induced MSC differentiation and
morphological change are dependent on cytoskeletal tension.

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Fig. 6. Immunohistochemical staining of collagen type X and osteocalcin in repaired zones within sections of aligned group and randomly-oriented group after 4 weeks and 8
weeks post-surgery. Scale bars, 100 mm (200X), 50 mm (400X).

4. Discussion
This study is based on our previous report on alignmentinduced tenogenesis of tendon stem cells, as well as osteogenesis being enhanced by randomly-aligned ber scaffolds [7].

This inspired us to evaluate the long term efcacy of aligned and

randomly-oriented brous scaffolds for tissue engineering in vivo
and to investigate the effects of topographic cues on mesenchymal
stem cells, which are more likely to be utilized clinically. In this
study, both the aligned and randomly-oriented brous scaffolds

Fig. 7. Mechanical properties of repaired tendons at 8 weeks post-surgery. n 5.

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181

Fig. 8. (A) and (B) show MSCs cultured on the aligned and randomly-oriented nanobrous scaffolds respectively. Scale bars, 100 mm. Quantitative data of cell size (C) and radius ratio
(D) on different substrates. Statistically signicant at *p < 0.05, **p < 0.01.

were utilized as tissue engineering platforms in vivo. The aligned

brous scaffolds promoted tendon-like tissue formation and yielded much more mature tendon tissue at 4 weeks postimplantation. In contrast, we observed substantial chondrogenesis and subsequent tissue ossication at the implantation
sites of the randomly-oriented brous scaffold group. The
observed signicant up-regulation of tendon-specic markers in
the aligned versus randomly-oriented brous scaffold group,
provided further evidence that aligned topography initiated tenolineage differentiation. Consistent with the topographic effect
in vivo, the random orientation of bers within the scaffold promoted osteogenesis of MSCs in vitro, as evidenced by elevated
osteogenic marker expression and positive ALP staining. Finally,
the results of further experiments showed distinct differences in
the distribution of focal adhesion complexes and cytoskeletal organization of MSCs on the aligned and randomly-oriented brous
scaffolds. Treatment with cyto D and Y-27632 led to the loss of
spindle-shaped morphology of MSCs cultured on the aligned
brous scaffold and abrogated the effects of topography-induced
differentiation. These results thus highlight the important role of
the actomyosin cytoskeleton in lineage commitment of MSCs.
Collectively, these ndings illustrate the relationship between
topographic cues of the scaffold and tissue formation, as well as
suggesting the possible risks of scaffold-induced ectopic ossication in tendon tissue repair.
Although there are numerous studies demonstrating that
various physical characteristics of scaffolds can profoundly inuence stem cell biological functions, particularly cell fate decision
[14e17], it is still unknown whether any topography-induced effects observed in vitro can be faithfully recapitulated in vivo. This
study compared the function of two microstructurally distinct
brous scaffolds in an Achilles tendon injury model to evaluate
neo-tissue regeneration potential. Our in vivo study showed that

tendon-like tissue formation, assessed by histological examination

and immunohistochemical analysis, were signicantly increased in
the aligned versus randomly-oriented brous scaffold group. In
contrast, histological analysis clearly revealed much tissue ossication and bone formation in the randomly-oriented brous scaffold group. At two weeks post-implantation, there were signicant
increases in the expression levels of chondro-lineage specic genes
such as collagen type II, Sox9 and aggrecan in the randomlyoriented versus aligned brous scaffold group, thus suggesting
that chondrogenesis was initiated during the early repair stage. The
biological microenvironment of injured tendons is very different to
that of normal healthy tendons. Transforming growth factor-b2
(TGF-b2), TGF-b3, bone morphogenetic protein-2 (BMP-2), BMP-4
BMP-7 and vascular endothelial growth factor (VEGF) were significantly up-regulated [18]. These growth factors have been implicated in the regulation of cartilage and bone development and have
been widely utilized in various chondrogenic and osteogenic differentiation protocols [19,20]. We reported that alignment-induced
lineage commitment could even override the effects of osteogenic
induction medium, in contrast to the synergetic effect of randomlyaligned topographic cues on osteogenesis [7]. Consistent with the
in vitro data, the randomly-oriented brous scaffold group displayed more mature bone tissue formation during healing.
Regarding the possible source of endogenous progenitor cells for
tendon repair, it is likely to be either MSCs that have migrated into
the wound or tendon-derived stem cells, which was identied
recently [21]. These cells possess multipotent differentiation capacity and would likely undergo aberrant differentiation within the
tendon injury niche. A comparative study of tenocytes and
mesenchymal stem cells seeded on polyglycolic acid (PGA) and
collagen type I scaffolds in a full-size tendon defect model showed
that the transplantation of tenocytes results in a lower degree of
tissue ossication and better extracellular matrix organization, in

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Z. Yin et al. / Biomaterials 44 (2015) 173e185

Fig. 9. (A) Immunouorescence staining of vinculin and F-actin in MSCs cultured on the aligned and randomly-oriented nanobrous scaffolds with or without Y-27632 for 3 days,
with the right column being the merged images with DAPI staining. Scale bars, 20 mm. (B) F-actin staining in MSCs cultured on the aligned and randomly-oriented nanobrous
scaffolds, with or without exposure to cyto D for 48 h, with the right column being the merged images with DAPI staining. Scale bars, 20 mm. Quantitative PCR analysis of tenogenic
(C) and osteogenic-specic gene (D) expression of MSCs seeded on different scaffolds with or without Y-27632 and cyto D on day 3. Gene expression levels are normalized to the
housekeeping gene, Gapdh. n 5. Statistically signicant at *p < 0.05.

comparison to the use of MSCs alone or just scaffold materials [22].

Our ndings indicated that ectopic tendon ossication, which may
develop following surgical trauma deserves more attention, as this
could shed light on the development of novel bio-scaffolds with
appropriate micro/nanoscaled structures.
In recent years, the physical properties of scaffolds have been
given more attention and were explored by a variety of different
techniques, such as lithography, nano/micro-pattern, electrospinning, as well as decellularization. Electrochemically aligned
collagen threads have also been used to keep the cells oriented
parallel to aligned collagen bers and it was also found that
anisotropic orientation promotes tenogenic differentiation of human MSCs in the absence of bio-inductive cues in vitro [14]. Zhu
et al. reported that simply maintaining the cultured tenocytes in an

elongated form by culturing them on microgrooved silicone

membrane could maintain their phenotype [23]. Besides physical
cues from surface topography, Sharma et al. also investigated
hydrogels with varying gradients of mechanical compliances and
found the appropriate stiffness range that was conducive for
tenogensis [24]. Furthermore, the comparative study of collagenous
decellurized matrices of different origins showed that tendonderived matrix possessed native mechanical properties and
topography, which is conducive for tenogenesis of tendon stem
cells [25]. Although the decellularized matrix is more complicated
than articial scaffolds, the role of physical architecture was successfully investigated by comparing the inductive effects of crosscut and longitudinally-cut tendon sections on MSCs, so as to
discern the biochemical cues within bio-matrix [26]. The difference

Z. Yin et al. / Biomaterials 44 (2015) 173e185

183

Fig. 10. Immunouorescence staining of SCX and F-actin in MSCs cultured on the aligned and randomly-oriented nanobrous scaffolds with or without exposure to Y-27632 for 3
days, with the right column being the merged images with DAPI staining. Scale bars, 20 mm.

between nanometer scale and micrometer scale architecture inuence cell behavior signicantly in terms of proliferation and
differentiation [27e29]. The diameters of around 700 nm and
1000 nm bers used in this study are belongs to similar micro-scale
and would not cause signicant effect on cell activities. Further
comparison studies of topography need to be carried out on
nanometer scale bers if they can be manufactured with sophisticate control of diameters and alignment. Based on the aforementioned studies, elongated morphology and alignment orientation is
always associated with tenogenic differentiation, whereas spread
cell shape and random orientation is associated with osteogenesis
[6]. Since the cytoskeleton is known to play important roles in
maintaining cell morphology, this study demonstrated that cyto D
and Y-27632 treatment caused signicant cell morphological
changes followed by loss of lineage commitment. Divergent patterns of MSC adhesion on different substrate topography reected
varying cytoskeletal tension, as focal adhesions form the anchor
points of the cytoskeleton [30]. The ber topography altered the
pattern of cellesubstrate interaction and such changes in cytoskeletal rearrangements will in turn lead to changes in intracellular
mechanotransductive pathways, such as demonstrated by changes
in the Rho A-ROCK pathway of stem cells in response to material

stiffness and alignment [23,31]. Just like cells in tendon tissue, we

believe that spindle-shape morphology is essential for full tenogenesis but is not sufcient by itself. Tong et al. used nanoimprinting to replicate the physical topography and elasticity of
tendon matrix so that the resulting shape and alignment of
cultured MSCs were similar to that seeded on longitudinally-cut
section of tendons [26]. Nevertheless, this was not accompanied by
signicantly increased Tnmd expression [26]. However, collagen I
coating of the bioimprint could effectively induce Tnmd expression.
It indicated that alignment directly inuenced early teno-lineage
commitment but an additional second signal is required to complete the full differentiation process [32]. In our study, the in vivo
microenvironment provides signals for further differentiation and
regeneration. The synergistic benecial effect of growth factors,
such as broblast growth factor-2 (FGF2), bone morphogenetic
protein-12 (BMP12), platelet-derived growth factors (PDGFs),
together with mechanical stimulation can potentially be put to
good use in tendon differentiation [14,32e35]. Collectively, these
data strongly suggests that the integration of topographical cues
with chemical stimuli can facilitate novel scaffold fabrication and
their application in tissue engineering to achieve functional
healing.

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Z. Yin et al. / Biomaterials 44 (2015) 173e185

Fig. 11. ALP staining of MSCs cultured on the aligned and randomly-oriented nanobrous scaffolds with or without exposure to Y-27632 for 7 days, respectively. The middle column
shows MSC nuclei being stained with DAPI to quantify the cell number. The right columns are merged images of the left and middle images. Scale bars, 100 mm.

5. Conclusion
This study contributes to understanding of the biological effects
of physical cues from aligned and randomly-oriented nanobrous
scaffolds, not only on the aspect of cellular behavior, but also on
tissue formation in vivo. The aligned brous scaffold displays
promising results in tendon-like tissue regeneration at early repair
stage, while in the randomly-oriented brous scaffold group, we
observed the development of bone formation at the injury site. The
two topographically-different scaffolds not only support MSC
adhesion and spreading, but also induced tenogenesis and osteogenesis respectively, both in vitro and in vivo. Moreover, we found
that this topography-induced lineage commitment is dependent on
cytoskeleton-mediated mechanotransduction. These ndings thus
provide vital information for the development of the nextgeneration of stem cell and bio-scaffold interfaces in future tissue
engineering applications.

(LR14H060001).
The
National
Key
Scientic
Program
(2012CB966604), the National High Technology Research and
Development
Program
of
China
(863
Program)
(No.2012AA020503). Sponsored by Regenerative Medicine in
Innovative Medical Subjects of Zhejiang Province. Medical and
health science and technology plan of Department of Health of
Zhejiang Province (2013RCA010). The Postdoctoral Foundation of
China (2014M561775, 2014M551759). The Technology Development project (CXZZ20130320172336579) from the Science Technology and Innovation Committee of Shenzhen Municipality. We
are grateful to The Core Facilities of Zhejiang University School of
Medicine for technical assistance.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2014.12.027.

Acknowledgment
This work was supported by NSFC grants (81330041, 81125014,
31271041, 81401781, 81201396, J1103603). The Project Supported
by Zhejiang Provincial Natural Science Foundation of China

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