-
Electrospun scaffolds for multiple tissues regeneration in vivo
through
Zi Yin , Xiao Chen , Hai-xWei-liang Shen d, Jia-lin Chen a,
b
Hong-Wei Ouyang a, b, c, *
a Department of Sports Medicine, School of Medicine, Zhb
Zhejiang Provincial Key Laboratory of Tissue Engineerc State Key
Laboratory for Diagnosis and Treatment of IDiseases, The First
Afliated Hospital, College of Medicind Department of Orthopedic
Surgery, 2nd Afliated Hose Department of Rehabilitation, Sir Run
Run Shaw Hospf Department of Biosystems Science & Engineering
(D-B
ology and enhancedqRT-PCR analysis ofat MSCs cultured onompared
with cellsension release abro-. Collectively, theseeir inductive
role infunctionalized bio-
. All rights reserved.
Stem cell survival, self-renewal and differentiation are
governedby local biochemical and mechanical factors within
their
mponents includesoluble factors, other cells, and extracellular
matrix molecules.Whilethe role of biochemical signals is
well-documented, the importanceof biophysical cues has received
more recognition and attention onlyin the last decade. Current
advances inmicrofabrication technologieshave enabled the generation
of substrates with nano/micro-scaletopographies to study the
effects of biophysical signals on cellularfunction. A number of
studies have demonstrated that the physicalproperties of substrata
have profound effects on the cellular func-tions of pluripotent and
multipotent stem cells, including cell
* Corresponding author. Center for Stem Cell and Tissue
Engineering, School ofMedicine, Zhejiang University, 866 Yu Hang
Tang Road, Hangzhou 310058, China.Tel./fax: 86 571 88208262.
E-mail address: [email protected] (H.-W. Ouyang).1
Contents lists availab
Biomat
journal homepage: www.elsev
Biomaterials 44 (2015) 173e185These authors contribute equally
to this work.raphy. Furthermore, MSCs on the aligned substrate
exhibited tenocyte-like morphtenogenic differentiation compared to
cells grown on randomly-oriented scaffold.osteogenic marker genes
and alkaline phosphatase (ALP) staining demonstrated
thrandomly-oriented ber scaffolds displayed enhanced osteogenic
differentiation ccultured on aligned ber scaffolds. Finally, it was
demonstrated that cytoskeletal tgated the divergent differentiation
pathways on different substrate topographyndings illustrate the
relationship between topographic cues of the scaffold and thtissue
regeneration; thus providing an insight into future development of
smartscaffold design and its application in tissue engineering.
2014 Elsevier Ltd
1. Introduction microenvironmental niche [1]. The key niche
coBone formationMesenchymal stem cells
bone tissue formation through ossication. Additionally, X-ray
imaging and osteocalcin immunohisto-chemical staining also
demonstrated that osteogenesis in vivo is driven by randomly
oriented topog-a r t i c l e i n f o
Article history:Received 10 September 2014Accepted 20 December
2014Available online
Keywords:Bio-scaffold topographyTissue engineeringTendon
regenerationhttp://dx.doi.org/10.1016/j.biomaterials.2014.12.0270142-9612/
2014 Elsevier Ltd. All rights reserved.in Song , Jia-jie Hu ,
Qiao-mei Tang , Ting Zhu ,, Huanhuan Liu a, b, Boon Chin Heng
f,
ejiang University, Hangzhou, Chinaing and Regenerative Medicine,
Hangzhou, Chinanfectious Diseases, Collaborative Innovation Center
for Diagnosis and Treatment of Infectiouse, Zhejiang University,
Hangzhou, China
pital, School of Medicine, Zhejiang University, Hangzhou,
Chinaital, School of Medicine, Zhejiang University, Hangzhou,
ChinaSSE), ETH-Zurich, Basel, Switzerland
a b s t r a c t
Physical topographic cues from various substrata have been shown
to exert profound effects on thegrowth and differentiation of stem
cells due to their niche-mimicking features. However, the
biologicalfunction of different topographic materials utilized as
bio-scaffolds in vivo have not been rigorouslycharacterized. This
study investigated the divergent differentiation pathways of
mesenchymal stem cells(MSCs) and neo-tissue formation trigged by
aligned and randomly-oriented brous scaffolds, bothin vitro and in
vivo. The aligned group was observed to form more mature
tendon-like tissue in theAchilles tendon injury model, as evidenced
by histological scoring and collagen I immunohistochemicalstaining
data. In contrast, the randomly-oriented group exhibited much
chondrogenesis and subsequenta, b, 1 a, b, 1 e a, b a, b a,
btopography dependent induction of lineage specic differentiationle
at ScienceDirect
erials
ier .com/locate/biomater ia ls
-
rialsadhesion, morphology, proliferation, migration and
differentiation[2e6]. Based on the concept of contact guidance, we
designed abiomimetic aligned nanober scaffold modelled on the
parallelcollagenous bers of tendon extracelluar matrix; and
subsequentlydemonstrated that alignment within the scaffold
regulate tendonstem cell orientation and induce specic teno-lineage
differentiation[7]. Meanwhile, both random orientation and
nanoscale disorderhave been demonstrated to induce ossication of
human multi-potent stem cells in vitro, even in the absence of
osteogenic media[6]. Although there are promising results in
various studies that haveattempted to control stem cell fate in
vitro through modication ofsubstrate physical properties, there is
a dire need to move fromculture substrate to implantable scaffolds
with direct applications intissue engineering [8].
Conventional scaffolds are designed and fabricated according
tothe basic requirements of biocompatibility, structural support
aswell as cell delivery, and have already been widely utilized
invarious tissue engineering applications [9]. Modern
bio-scaffoldsnot only just serve as a carrier for seed cells, but
also provide anappropriate microenvironment for stem cells and
mediates bio-logical functions. Further microstructural renement of
currentscaffold biotechnology will enhance the progress of tissue
engi-neering in the future [10]. However, the
three-dimensionalmicroenvironment in vivo represents a much more
complicatedmilieu that encompasses a much more diverse multitude
ofsignaling cues compared to an in vitro culture system.
Underphysiological conditions, stem cells naturally encounter a
variety ofdifferent signaling cues that can potentially inuence
cell fate. It isessential and necessary to use the results of in
vitro studies to aidthe rigorous characterization of the
functionality of tissue engi-neered scaffolds in vivo. This
prompted our investigation on theinductive effects of scaffold
topographic cues on stem cell differ-entiation pathways and lineage
fate.
This study aims to characterize the biophysical effects of
scaffoldtopography on tissue regeneration in vivo within a 3D
microenvi-ronment, utilizing aligned and randomly-oriented brous
scaffolds.We tested the hypothesis that topographic cues from the
alignedbrous scaffold can enhance tendon-like tissue formation, and
thatthere would be a higher degree of osteogenesis and tissue
ossi-cation with the randomly-oriented ber scaffold. Additionally,
therole of cytoskeletal organization in topography driven
differentia-tion of mesenchymal stem cells was also investigated in
vitro. Webelieve that the data presented here would be benecial to
thedesign 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 previousreported. The polymer solution was prepared by
dissolving PLLA (Ji'nan DaigangBiomaterial Co., Ltd) in a mixture
of chloroform/ethanol (3:1) at a concentration of4% (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 ataluminum 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
transferredto cover slips and sterilized with ethanol and UV
overnight before theywere utilizedfor cell culture. Nanobers were
collected for 2e3 h, resulting in a ber mat rangingin thickness
from 0.14 to 0.17 mm. The aligned and randomly-oriented
scaffoldsutilized in this study were of similar thickness and
distribution.
2.2. Morphology of PLLA scaffolds
The scaffold samples were sputter-coated with gold, and then
their structurewas observed under scanning electron microscopy
(SEM) (Hitachi S3000N) at anaccelerating voltage of 15 kV. After
the micrographs were obtained, image analysis
Z. Yin et al. / Biomate174software (Image-Pro Plus) was used to
measure the average diameter of the nano-bers (n 3). For each
sample, an average of 50 bers were counted.2.3. SEM imaging
C3H10T1/2 cells (mouse multipotent mesenchymal stem cell line)
were ob-tained from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China).Cells were seeded onto PLLA scaffolds at
2104 cells/cm2 and cultured in Dulbecco'smodied Eagle's medium
(DMEM, low glucose; Gibco, Grand Island, NY,
http://www.invitrogen.com) with 10% (v/v) fetal bovine serum (FBS;
Invitrogen, Carls-bad, 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 werexed in 0.25%
glutaraldehyde solution, and then rinsed 3 times in PBS, 30 min
eachtime. The specimens were immersed in OsO4 for 40 min and then
rinsed 3 times inPBS, 30 min each time, followed by dehydration in
increasing concentrations ofacetone (30e100% v/v). After drying,
the specimens were mounted on aluminumstubs and coated with gold,
then viewed under a Hitachi S-3000N SEM at anaccelerating voltage
of 15 kV. For quantication of cell morphology on
differentscaffolds, aminimum of fty cells for each SEM imagewere
selected randomly as ROI(Regions of Interest). The area and radius
ratio were then quantied using theImage-Pro Plus software.
2.4. Alkaline phosphatase (ALP) staining
C3H10T1/2 cells (104/cm2) were seeded onto scaffolds and
cultured in osteo-genic induction medium, in the presence of 10 mM
b-glycerol phosphate (Sigma),0.1 mM dexamethasone (Sigma), and 50
mg/ml ascorbic acid (Sigma) supplementedin DMEM-high glucose medium
containing 10% (v/v) FBS and 1% (v/v) pen-icillinestreptomycin.
After 7 days, ALP activity was assayed using a BCIP/NBT alka-line
phosphatase color development kit (Beyotime Institute of
Biotechnology). DAPI(Beyotime Institute of Biotechnology) was used
to stain nuclei and observed under alight microscope (Olympus
IX71).
2.5. Quantitative PCR
Total cellular RNA was isolated by lysis in TRIzol (Invitrogen).
The expressionlevels of tendon-specic genes and osteogenic markers
in cells cultured on alignedand randomly-aligned brous scaffolds
were assessed by quantitative PCR. PCR wasperformed using a
Brilliant SYBR Green qPCR Master Mix (TakaRa) on a Light
Cyclerapparatus (ABI 7900HT). The PCR cycling consisted of 40
cycles of amplication ofthe template DNA with primer annealing at
60 C. The relative expression levels ofeach target gene was then
calculated using the 2-DDCt method. The amplicationefciencies of
primer pairs were validated to enable quantitative comparison of
geneexpression. All primers (Generay) were designed using primer
5.0 software and aresummarized in the Supplementary Table 1.
2.6. Animal model
The Zhejiang University Institutional Animal Care and Use
Committee approvedthe study protocol. In situ rat Achilles tendon
repair model: Twenty hind limbs ofskeletally mature female rats
weighing 200e220 gwere utilized for this experiment.Under general
anesthesia, a gap wound was created and the Achilles tendon
wasremoved 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 ofthe membrane upwards. This was followed with
fan-folding the next 2 mm to theback. Fan-folding of the scaffold
was continued until it was completely folded. Thiswas followed by
binding the center of the strip using a suture and
subsequentplacement into the gap wound. Suturing to the remaining
Achilles tendon was thencarried out using a non-resorbable suture
material (Nylon6). The wound was thenirrigated and the skin was
closed. The animals were allowed free cage activity aftersurgery.
At 2, 4, and 8 weeks post-implantation, samples from each group
wereharvested for the evaluation of histology, transmission
electronmicroscopy imaging,mechanical testing, as well as collagen
content determination (SupplementaryTable 2).
2.7. Immunouorescence
Briey, cells were xed in 4% (w/v) paraformaldehyde for 10 min at
roomtemperature, permeabilized, and blocked for 30 min with 1%
(w/v) bovine serumalbumin, and then permeabilized with 0.1% (w/v)
Triton X-100. Fixed cells werewashed and incubated with a primary
antibody against SCX (Abcam Inc.), Vinculin(Millipore), or control
IgG (BD) at 4 C overnight. Cells were then incubated withAlexa uor
488-conjugated secondary antibody (Invitrogen) for 2 h and the
nucleiwere stained with DAPI. TRITC-phalloidin (Millipore) staining
was used to visualizethe cytoskeleton. The imaging was then
performed with confocal microscopy (ZeissLSM-510).
2.8. Histological evaluation and staining
Harvested specimens were immediately xed in 10% (v/v) neutral
bufferedformalin, dehydrated through an alcohol gradient, cleaned,
and then embeddedwithin parafn blocks. Histological sections (7 um)
were prepared using a micro-
44 (2015) 173e185tome and subsequently stained with hematoxylin
and eosin. In addition, Massontrichrome staining was performed
according to standard procedures to examine the
-
rialsgeneral appearance of the collagen bers. To detect
proteoglycan synthesis as anindicator 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 for4
min. Polarizing microscopy was employed to detect mature collagen
brils. Gen-eral histological scoring was performed using
hematoxylin and eosin staining. Sixparameters (ber structure, ber
arrangement, rounding of nuclei, inammation,vascularity, cell
population) were semi-quantitatively assessed. These six
parame-ters were semi-quantitatively graded on a four-point scale
(0eIII), with 0 beingnormal and 3 being maximally abnormal.
Therefore, a normal tendonwould score 0,while a maximally abnormal
tendon would score 18. The detailed scoring systemwas 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. Endogenousperoxidase was blocked by incubation
with 3% (v/v) hydrogen peroxide in methanolfor 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
againstCollagen (Abcam Inc.) or osteocalcin (Millipore), sections
were washed and thenincubated 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 toreveal the nuclei.
2.10. Mechanical testing
Mechanical testing was performed using an Instron
tension/compression sys-tem with Fast-Track software (Model 5543,
Instron, Canton, MA). Measurements ofthe tendon cross-sectional
area were performed using two Vernier calipers at 5 mmproximal to
the conjunction of bone and tendon. The bone end of the tendon
wassecured by a specially designed restraining jig and the tendon
end was pinched witha clamp [11]. The AT-calcaneus complex (ACC)
was then rigidly xed to custom-made clamps. After applying a
preload of 0.1 N, each ACC underwent pre-conditioning 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. Thestructural properties of the ACCwere represented
by stiffness (N/mm), ultimate load(N), energy absorbed at failure
(mJ) and stress at failure. For each ACC, the greatestslope in the
linear region of the loadeelongation curve over a 0.5 mm
elongationinterval was used to calculate the stiffness.
2.11. Determination of collagen content
The amount of deposited collagen in the scaffold was quantied by
using acollagen assay kit according to the manufacturer's protocol
(Jiancheng Ltd., Nanjing,China). This kit is a hydroxyproline
assay, which is widely used to determine totalcollagen content, and
a conversion factor of 1:7.46 was used to convert hydroxy-proline
to collagen [12], lyophilized tendons were digested with a
hydrolysis regentat 95 C for 20min. Serial dilutions of
acid-soluble collagen type I provided by the kitwere utilized as
standards. Following the assay, collagen concentration was
deter-mined 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 collagenbril diameter and alignment. Briey, samples were
pre-xed in 2% (w/v) glutar-aldehyde for 2 h at 4 C and then washed
twice in PBS at 4 C followed by post-xation with 1% (v/v) osmic
acid for 2 h at 4 C. After two washes in PBS, thesamples were
dehydrated with an ethanol gradient and dried to a critical point.
Thesamples were then mounted and sputter-coated with gold for
viewing under TEM(Quanta 10 FEI). Approximately 500 collagen brils
were measured for each sampleto 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-invasiveKodak-FX in vivo imaging system (Kodak, Inc.) to
evaluate ectopic bone formation.At 8 weeks post-implantation,
captured images were analyzed with the Image-proplus software to
quantify the area, mean density, max density, and sum density
ofectopic bone formation.
2.14. Statistical analysis
All quantitative data sets are expressed as mean SD. The
Student's t-test wasperformed to assess statistically signicant
differences in the results of different
Z. Yin et al. / Biomateexperimental groups. Values of p <
0.05 were considered to be signicantlydifferent.3. Results
3.1. Fabrication and morphological characterization of
scaffolds
Electrospinning is used to fabricate aligned brous scaffolds,
aswell as randomly-oriented brous membranes with similar
berdiameters, which served as controls. The surface topography
ofboth aligned and randomly-oriented brous scaffolds was exam-ined
using SEM (Fig. 1A and B). Majority of the bers in the
alignedscaffolds were parallel to each other and formed angles from
0 to10 with respect to the horizontal axis, while the
randomly-oriented nanober scaffold exhibited nearly equal
distributions atall angles.
3.2. The effects of aligned and randomly-oriented scaffolds on
neo-tissue formation
3.2.1. Histology of repaired tendonsAfter 2 weeks post-surgery,
histological analysis showed that
there was a greater number of cells exhibiting
spindle-shapedmorphology on the aligned scaffolds (Fig. 1C). While,
the cellmorphology in the randomly-oriented groups displayed
relativelyround shapes (Fig. 1D). Masson trichrome staining showed
that thetissue matrix was denser in the aligned versus
randomly-orientedscaffold group, which means that more collagen
bers have beendeposited compared to the control group (Fig. 1 E and
F). Collagen Iimmunohistochemical staining showed organized
collagen depo-sition on the aligned bers (Fig. 1 G and H), while
the randomly-oriented scaffolds were lled with loose, disarranged
matrix.
At 4weeks post-surgery, the histological results showed that
thealigned scaffold-induced spindle-shaped cells and tendon-like
tis-sue formation in vivo, as evidenced by the Masson
trichromestaining. In addition, the aligned scaffold implantation
showedimproved tendon repair quality was compared to the histology
ofblank groupwithout scaffold implantation, which displayed
limitedtissue regeneration (Supplementary Fig.S1). The collagen I
immu-nohistochemical staining reveals enhanced collagen I matrix
pro-duction and arrangement in the aligned scaffold group (Fig.
2A).Histology scoring also conrmed the histological results that
thequality of repaired tendons in the aligned scaffold group
weresignicantly better than the control group (p < 0.05, Fig.
2E),particularly in ber structure, ber arrangement and nuclei
shapeaspects. The ultrastructural morphology based on TEM
imagingfrom transection revealed larger bril formation within the
alignedscaffold, as compared to the thin brils observed within
therandomly-oriented scaffold (Fig. 2B). The average diameter
ofcollagen 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 4weeks
post-implantation (Fig. 2D). Analysis of the distribution ofbril
diameters within the two groups (Fig. 2C) revealed that theATs in
the aligned scaffold treatment group formed signicantlylarger brils
compared to ATs in the random group at 4 weeks post-implantation.
The collagen content assay demonstrated that thealigned 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
thecollagen ber maturation levels within the two groups. The
alignedscaffold group exhibited more continuous collagen bers at
therepair site (Fig. 3B). Furthermore, the expression of
tendon-specicmarker scleraxis (Scx)was signicantly higher in the
aligned versusrandomly-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
44 (2015) 173e185 175scaffold group, thus indicating that
aligned topographic cues have
-
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. Histologicalresults 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 typicalhematoxylin and eosin staining, (E) and (F) are
Masson trichrome staining, (G) and (H) are immunohistochemical
staining of collagen type I, respectively. Arrows indicated
theremaining 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 typeI in
repaired zones within sections of aligned group (upper panel) and
randomly-oriented group (lower panel). (B) Transmission Electron
Microscopy images show ultrastructure ofrepaired 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 ofcollagen type I, left images), 50
mm (immunohistochemical staining of collagen type I, right
images).
Z. Yin et al. / Biomaterials 44 (2015) 173e185176
-
rialsZ. Yin et al. / Biomatemore potential in tendon tissue
repair and regeneration.Msx-2 [13],which has been reported to play
a central role in preventing ten-dons from mineralizing, exhibited
elevated expression in thealigned versus randomly-oriented groups
at all time-points(Fig. 3A).
3.2.2. Histology of bone formationOn the other hand,
chondrocyte-like cells appeared within the
injury site implanted with the randomly-oriented scaffold at
4weeks post-surgery (Fig. 2A). Furthermore, Safranin O
stainingshowed signicantly more chondrocyte-like cells in the
randomly-oriented versus aligned scaffold group (Fig. 4A). It is
obvious withthe randomly-oriented group that the ossied deposits
surroundedby chondrocyte-like cells were localized at the tendon
mid-sectioninside the wound, which caused disruption to the
organization ofcollagen bers (Fig. 4A). Additionally, bone marrow
was alsoformed in the randomly-oriented scaffold group at 8
weeks(Fig. 4A) Nevertheless, no ossication was detected in all
sampleswith X-ray scanning at 4 weeks post-surgery (data no
shown),whereas all samples of the randomly-oriented group
exhibitedspontaneous ectopic bone formation at 8 weeks
post-surgery(Fig. 5A). Upon quantication of the area and density of
ectopicbone formed, it was found that the randomly-oriented
scaffoldgroup displayed signicantly larger area (Fig. 5B, p <
0.05), highermean density (Fig. 5B), max density (Fig. 5B, p <
0.05) and inte-grated optical density (IOD) (Fig. 5B, p < 0.05)
of ectopic bone, ascompared to the aligned scaffold group. The
results thus indicated
Fig. 3. (A) Gene expression levels of tendon-related markers
assessed by quantitative PCR(upper panel) and randomly-oriented
group (lower panel) at 2, 4 and 8 months post-surge44 (2015)
173e185 177that randomly-oriented topographic scaffolds had
signicantlyhigher potential to induce ectopic bone formation
compared toaligned scaffolds.
Expression of osteochondral-lineage marker genes wereanalyzed to
further evaluate tissue formation. It was observed thatthe
expression of growth factors BMP4, chondrogenic transcriptionfactor
Sox9 and chondrocyte specic matrix Col IIwere signicantlyhigher in
the randomly-oriented versus aligned groups at 2 weekspost-surgery
(Fig. 4B). The matrix osteocalcin expression wassignicantly much
higher in the randomly-oriented versus alignedgroups, while
expression of the osteogenic transcription factorRunx2 exhibited
the largest difference at 8 weeks post-surgery(Fig. 4B). This
suggests that randomly-oriented topographic cuesplay a critical
role in initiating cartilage and bone formation at theearly repair
stage (2 weeks) with neo-bone being formed as a resultof cartilage
ossication at the later repair stage (8 weeks). Theexpression of
collagen type X, a representative marker of chon-drocyte
hypertrophy was detected by immunohistochemicalstaining. Comparison
of the two groups showed signicantlydenser and larger
positively-stained areas within the randomly-oriented versus
aligned group (Fig. 6), which is consistent withincreased
Safranin-O staining (Fig. 4A). This indicated that theossied
deposits were formed by endochondral ossication.Expression of the
bone formation marker osteocalcin was exam-ined by
immunohistochemical staining as well, and the expressionlevels were
obviously much higher in the randomly-orientedversus aligned group
at 8 weeks post-surgery (Fig. 6). These data
at 2, 4 and 8 months post-surgery. (B) Polarized microscopy
images of aligned groupry. Statistically signicant at *p < 0.05,
**p < 0.01. Scale bars, 100 mm.
-
rialsZ. Yin et al. / Biomate178collectively suggested that
randomly-oriented scaffolds can inducemore endochondral bone tissue
formation by 8 weeks post-implantation.
3.2.3. Mechanical properties of repaired tendonsTo further
correlate tissue structural features with their me-
chanical properties, harvested tendons (n 5 for each group)were
subjected to mechanical testing at 8 weeks post-surgery. Thealigned
group had better mechanical properties than therandomly-oriented
controls (Fig. 7 and Supplementary Fig.S3). Themodulus (24.42 2.20
MPa vs. 20.86 3.56 MPa) of the alignedgroup were better than the
control group (Fig. 7). The energy inthe 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 maximumforce in the aligned group was higher
than that of the controlgroup (87.12 5.61 N vs. 77.09 13.27 N, p
> 0.05). The stress atfailure and stiffness were consistently
higher in the aligned groupthan that of the control group, but
these difference was not sta-tistically signicant (Fig. 7),
probably due to the small samplingsize.
Fig. 4. (A) Safranin O staining images of aligned group (upper
panel) and randomly-orientchondrogenic (aggrecan, Sox9, collagen
type II) and osteogenic markers (Bmp4, Ocn, Runx2) a*p < 0.05,
**p < 0.01. Scale bars, 200 mm (100X), 100 mm (200X).44 (2015)
173e1853.3. The effects of topographical cues on MSCs
The SEMmicrographs indicated thatMSCswerewell attached toboth
scaffolds, but displayed distinct morphology. On the alignednanober
scaffold, the cells exhibited an elongated spindle-shapedmorphology
and were oriented parallel to the substrate alignment,whereas cells
cultured on the randomly-oriented scaffolds werespread out and
exhibited a polygonal phenotype (Fig. 8A and B).The bar graphs
illustrate the size and aspect ratios of MSCs culturedon different
topographic substrates. MSCs grown on the alignedscaffold displayed
relatively smaller size, but with a signicantlymuch higher aspect
ratio than their counterparts on the randomly-oriented scaffold
(Fig. 8C and D). Under confocal uorescence mi-croscopy, alignment
of cell orientation was also apparent underTRITC-phalloidin
staining, with the cytoskeleton being more uni-formly oriented
towards the alignment of nanobers within thealigned scaffold (Fig.
9A). However, the MSCs cultured on therandomly-oriented scaffolds
exhibited different arrangement of theF-actin network (Fig. 9A).
The immunouorescence staining ofvinculin, which is a component of
the focal adhesion complex,showed the presence of focal adhesions
and their distribution
ed group (lower panel) at 4 and 8 months post-surgery. (B) Gene
expression levels ofssessed by quantitative PCR at 2, 4 and 8
months post-surgery. Statistically signicant at
-
rialsZ. Yin et al. / Biomatewithin cells cultured on scaffolds.
Vinculin was localized mainly atthe peripheral region of MSCs
cultured on the randomly-orientedscaffold, at the end of F-actin
ber bundles in the lopodia orlamellipodia. A higher density of
vinculinwas observed at the polesof elongated spindle-shaped MSCs
cultured on the aligned scaffold.Consistent with the great
differences in cell morphology andorientation observed on the two
scaffolds, the focal adhesion andcytoskeleton distribution also
displayed distinctively differentpatterns between the two groups
(Fig. 9A). The expression of thetenogenic transcription factor gene
Scx was signicantly morehighly expressed by MSCs on aligned versus
randomly-orientednanobers (Fig. 9C). The expression of the
osteogenic transcrip-tion factor Runx2 was signicantly lower in
MSCs cultured onaligned versus randomly-oriented scaffold (Fig.
9D).
3.4. Topography-induced lineage commitment of MSCs isdependent
on cytomyosin cytoskeleton
To further understand the phenomena of topographic
induceddifferentiation of MSCs, we examined the effects of two
smallmolecules on cytoskeletal reorganization
andmechanotransduction- cytochalasin D (cyto D) and the Rho kinase
(ROCK) inhibitor Y-27632. The addition of cyto D to the medium
attenuated the contactguidance response by suppressing cell
elongation in the alignedscaffold group, while reducing projected
surface area of cells in therandomly-oriented group (Fig. 9B). We
observed that cytochalasin D
Fig. 5. (A) X-ray images of repaired tendons of mice at 8 weeks
post-transplantation and evmax density, sum density and integrated
optical density of ectopic bone in aligned group (ectopic bone
formation. Statistically signicant at *p < 0.05.44 (2015)
173e185 179treatment caused cells to become rounded without any
notabledifferences in cell morphology between the two groups (Fig.
9B).Meanwhile, we also found that random
orientation-inducedosteogenesis and alignment-induced tenogenesis
were attenuatedas well by cyto D, as evidenced by downregulation of
osteogenicmarker Runx2 and tenogenic mark Scx expression levels
(Fig. 9C andD).These results indicated that the actin cytoskeleton
might beimportant to the MSC lineage commitment process.
Furthermore,we treated the cell with Rho kinase (ROCK) inhibitor
Y-27632 toinhibit myosin-generated cytoskeletal tension. In the
presence of Y-27632, cells remained well-spread and morphologically
similar onthe two different topographical substrates. Additionally,
vinculinexpression was diminished signicantly in both groups (Fig.
9A).Moreover, the quantitative PCR results revealed that there were
nosignicant differences in either tenogenesis or osteogenesis of
MSCscultured on different topographic substrates (Fig. 9C andD).
The SCXimmunouorescence images showed that before addition of
Y-27632, SCX expression was higher and more concentrated in
thenuclei ofMSCs cultured on aligned bers. Subsequent exposure to
Y-27632 not only blocked cell alignment but also eliminated
differ-ences in SCX expression between the two groups (Fig. 10).
Similarly,the ALP staining results also demonstrated that
osteogenesisinduced by randomly-oriented nanober topographywas
abrogatedupon treatment with Y-27632 (Fig. 11). These ndings
collectivelysuggest that topography-induced MSC differentiation
andmorphological change are dependent on cytoskeletal tension.
aluation of the extent of ossication (n 5). (B) The distribution
and medians of area,dot) and randomly-oriented group (square).
Arrows depict exactly the locations of the
-
Z. Yin et al. / Biomaterials 44 (2015) 173e1851804.
Discussion
This study is based on our previous report on alignment-induced
tenogenesis of tendon stem cells, as well as osteo-genesis being
enhanced by randomly-aligned ber scaffolds [7].
Fig. 6. Immunohistochemical staining of collagen type X and
osteocalcin in repaired zoneweeks post-surgery. Scale bars, 100 mm
(200X), 50 mm (400X).
Fig. 7. Mechanical properties of repaired teThis inspired us to
evaluate the long term efcacy of aligned andrandomly-oriented brous
scaffolds for tissue engineering in vivoand to investigate the
effects of topographic cues on mesenchymalstem cells, which are
more likely to be utilized clinically. In thisstudy, both the
aligned and randomly-oriented brous scaffolds
s within sections of aligned group and randomly-oriented group
after 4 weeks and 8
ndons at 8 weeks post-surgery. n 5.
-
us sc
rialsFig. 8. (A) and (B) showMSCs cultured on the aligned and
randomly-oriented nanobro(D) on different substrates. Statistically
signicant at *p < 0.05, **p < 0.01.
Z. Yin et al. / Biomatewere utilized as tissue engineering
platforms in vivo. The alignedbrous scaffolds promoted tendon-like
tissue formation and yiel-ded much more mature tendon tissue at 4
weeks post-implantation. In contrast, we observed substantial
chondro-genesis and subsequent tissue ossication at the
implantationsites of the randomly-oriented brous scaffold group.
Theobserved signicant up-regulation of tendon-specic markers inthe
aligned versus randomly-oriented brous scaffold group,provided
further evidence that aligned topography initiated teno-lineage
differentiation. Consistent with the topographic effectin vivo, the
random orientation of bers within the scaffold pro-moted
osteogenesis of MSCs in vitro, as evidenced by elevatedosteogenic
marker expression and positive ALP staining. Finally,the results of
further experiments showed distinct differences inthe distribution
of focal adhesion complexes and cytoskeletal or-ganization of MSCs
on the aligned and randomly-oriented brousscaffolds. Treatment with
cyto D and Y-27632 led to the loss ofspindle-shaped morphology of
MSCs cultured on the alignedbrous scaffold and abrogated the
effects of topography-induceddifferentiation. These results thus
highlight the important role ofthe actomyosin cytoskeleton in
lineage commitment of MSCs.Collectively, these ndings illustrate
the relationship betweentopographic cues of the scaffold and tissue
formation, as well assuggesting the possible risks of
scaffold-induced ectopic ossica-tion in tendon tissue repair.
Although there are numerous studies demonstrating thatvarious
physical characteristics of scaffolds can profoundly inu-ence stem
cell biological functions, particularly cell fate decision[14e17],
it is still unknown whether any topography-induced ef-fects
observed in vitro can be faithfully recapitulated in vivo.
Thisstudy compared the function of two microstructurally
distinctbrous scaffolds in an Achilles tendon injury model to
evaluateneo-tissue regeneration potential. Our in vivo study showed
thataffolds respectively. Scale bars, 100 mm. Quantitative data of
cell size (C) and radius ratio
44 (2015) 173e185 181tendon-like tissue formation, assessed by
histological examinationand immunohistochemical analysis, were
signicantly increased inthe aligned versus randomly-oriented brous
scaffold group. Incontrast, histological analysis clearly revealed
much tissue ossi-cation and bone formation in the randomly-oriented
brous scaf-fold group. At two weeks post-implantation, there were
signicantincreases in the expression levels of chondro-lineage
specic genessuch as collagen type II, Sox9 and aggrecan in the
randomly-oriented versus aligned brous scaffold group, thus
suggestingthat chondrogenesis was initiated during the early repair
stage. Thebiological microenvironment of injured tendons is very
different tothat of normal healthy tendons. Transforming growth
factor-b2(TGF-b2), TGF-b3, bone morphogenetic protein-2 (BMP-2),
BMP-4BMP-7 and vascular endothelial growth factor (VEGF) were
signif-icantly up-regulated [18]. These growth factors have been
impli-cated in the regulation of cartilage and bone development and
havebeen widely utilized in various chondrogenic and osteogenic
dif-ferentiation protocols [19,20]. We reported that
alignment-inducedlineage commitment could even override the effects
of osteogenicinduction medium, in contrast to the synergetic effect
of randomly-aligned topographic cues on osteogenesis [7].
Consistent with thein vitro data, the randomly-oriented brous
scaffold group dis-played more mature bone tissue formation during
healing.Regarding the possible source of endogenous progenitor
cells fortendon repair, it is likely to be either MSCs that have
migrated intothe wound or tendon-derived stem cells, which was
identiedrecently [21]. These cells possess multipotent
differentiation ca-pacity and would likely undergo aberrant
differentiationwithin thetendon injury niche. A comparative study
of tenocytes andmesenchymal stem cells seeded on polyglycolic acid
(PGA) andcollagen type I scaffolds in a full-size tendon defect
model showedthat the transplantation of tenocytes results in a
lower degree oftissue ossication and better extracellular matrix
organization, in
-
rialsZ. Yin et al. / Biomate182comparison to the use of MSCs
alone or just scaffold materials [22].Our ndings indicated that
ectopic tendon ossication, which maydevelop following surgical
trauma deserves more attention, as thiscould shed light on the
development of novel bio-scaffolds withappropriate micro/nanoscaled
structures.
In recent years, the physical properties of scaffolds have
beengiven more attention and were explored by a variety of
differenttechniques, such as lithography, nano/micro-pattern,
electro-spinning, as well as decellularization. Electrochemically
alignedcollagen threads have also been used to keep the cells
orientedparallel to aligned collagen bers and it was also found
thatanisotropic orientation promotes tenogenic differentiation of
hu-man MSCs in the absence of bio-inductive cues in vitro [14].
Zhuet al. reported that simply maintaining the cultured tenocytes
in an
Fig. 9. (A) Immunouorescence staining of vinculin and F-actin in
MSCs cultured on the aligwith the right column being the merged
images with DAPI staining. Scale bars, 20 mm. (B)scaffolds, with or
without exposure to cyto D for 48 h, with the right column being
the merg(C) and osteogenic-specic gene (D) expression of MSCs
seeded on different scaffolds withhousekeeping gene, Gapdh. n 5.
Statistically signicant at *p < 0.05.44 (2015) 173e185elongated
form by culturing them on microgrooved siliconemembrane could
maintain their phenotype [23]. Besides physicalcues from surface
topography, Sharma et al. also investigatedhydrogels with varying
gradients of mechanical compliances andfound the appropriate
stiffness range that was conducive fortenogensis [24]. Furthermore,
the comparative study of collagenousdecellurized matrices of
different origins showed that tendon-derived matrix possessed
native mechanical properties andtopography, which is conducive for
tenogenesis of tendon stemcells [25]. Although the decellularized
matrix is more complicatedthan articial scaffolds, the role of
physical architecture was suc-cessfully investigated by comparing
the inductive effects of cross-cut and longitudinally-cut tendon
sections on MSCs, so as todiscern the biochemical cues within
bio-matrix [26]. The difference
ned and randomly-oriented nanobrous scaffolds with or without
Y-27632 for 3 days,F-actin staining in MSCs cultured on the aligned
and randomly-oriented nanobroused images with DAPI staining. Scale
bars, 20 mm. Quantitative PCR analysis of tenogenicor without
Y-27632 and cyto D on day 3. Gene expression levels are normalized
to the
-
rialsZ. Yin et al. / Biomatebetween nanometer scale and
micrometer scale architecture in-uence cell behavior signicantly in
terms of proliferation anddifferentiation [27e29]. The diameters of
around 700 nm and1000 nm bers used in this study are belongs to
similar micro-scaleand would not cause signicant effect on cell
activities. Furthercomparison studies of topography need to be
carried out onnanometer scale bers if they can be manufactured with
sophisti-cate control of diameters and alignment. Based on the
aforemen-tioned studies, elongated morphology and alignment
orientation isalways associated with tenogenic differentiation,
whereas spreadcell shape and random orientation is associated with
osteogenesis[6]. Since the cytoskeleton is known to play important
roles inmaintaining cell morphology, this study demonstrated that
cyto Dand Y-27632 treatment caused signicant cell
morphologicalchanges followed by loss of lineage commitment.
Divergent pat-terns of MSC adhesion on different substrate
topography reectedvarying cytoskeletal tension, as focal adhesions
form the anchorpoints of the cytoskeleton [30]. The ber topography
altered thepattern of cellesubstrate interaction and such changes
in cyto-skeletal rearrangements will in turn lead to changes in
intracellularmechanotransductive pathways, such as demonstrated by
changesin the Rho A-ROCK pathway of stem cells in response to
material
Fig. 10. Immunouorescence staining of SCX and F-actin in MSCs
cultured on the aligned andays, with the right column being the
merged images with DAPI staining. Scale bars, 20 mm44 (2015)
173e185 183stiffness and alignment [23,31]. Just like cells in
tendon tissue, webelieve that spindle-shape morphology is essential
for full teno-genesis but is not sufcient by itself. Tong et al.
used nano-imprinting to replicate the physical topography and
elasticity oftendon matrix so that the resulting shape and
alignment ofcultured MSCs were similar to that seeded on
longitudinally-cutsection of tendons [26]. Nevertheless, this was
not accompanied bysignicantly increased Tnmd expression [26].
However, collagen Icoating of the bioimprint could effectively
induce Tnmd expression.It indicated that alignment directly
inuenced early teno-lineagecommitment but an additional second
signal is required to com-plete the full differentiation process
[32]. In our study, the in vivomicroenvironment provides signals
for further differentiation andregeneration. The synergistic
benecial effect of growth factors,such as broblast growth factor-2
(FGF2), bone morphogeneticprotein-12 (BMP12), platelet-derived
growth factors (PDGFs),together with mechanical stimulation can
potentially be put togood use in tendon differentiation [14,32e35].
Collectively, thesedata strongly suggests that the integration of
topographical cueswith chemical stimuli can facilitate novel
scaffold fabrication andtheir application in tissue engineering to
achieve functionalhealing.
d randomly-oriented nanobrous scaffolds with or without exposure
to Y-27632 for 3.
-
rialsZ. Yin et al. / Biomate1845. Conclusion
This study contributes to understanding of the biological
effectsof physical cues from aligned and randomly-oriented
nanobrousscaffolds, not only on the aspect of cellular behavior,
but also ontissue formation in vivo. The aligned brous scaffold
displayspromising results in tendon-like tissue regeneration at
early repairstage, while in the randomly-oriented brous scaffold
group, weobserved the development of bone formation at the injury
site. Thetwo topographically-different scaffolds not only support
MSCadhesion and spreading, but also induced tenogenesis and
osteo-genesis respectively, both in vitro and in vivo. Moreover, we
foundthat this topography-induced lineage commitment is dependent
oncytoskeleton-mediated mechanotransduction. These ndings
thusprovide vital information for the development of the
next-generation of stem cell and bio-scaffold interfaces in future
tissueengineering applications.
Acknowledgment
This work was supported by NSFC grants (81330041,
81125014,31271041, 81401781, 81201396, J1103603). The Project
Supportedby Zhejiang Provincial Natural Science Foundation of
China
Fig. 11. ALP staining of MSCs cultured on the aligned and
randomly-oriented nanobrous scashows MSC nuclei being stained with
DAPI to quantify the cell number. The right columns44 (2015)
173e185(LR14H060001). The National Key Scientic
Program(2012CB966604), the National High Technology Research
andDevelopment Program of China (863 Program)(No.2012AA020503).
Sponsored by Regenerative Medicine inInnovative Medical Subjects of
Zhejiang Province. Medical andhealth science and technology plan of
Department of Health ofZhejiang Province (2013RCA010). The
Postdoctoral Foundation ofChina (2014M561775, 2014M551759). The
Technology Develop-ment project (CXZZ20130320172336579) from the
Science Tech-nology and Innovation Committee of Shenzhen
Municipality. Weare grateful to The Core Facilities of Zhejiang
University School ofMedicine 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.
References
[1] Discher DE, Mooney DJ, Zandstra PW. Growth factors,
matrices, and forcescombine and control stem cells. Science
2009;324(5935):1673e7.
ffolds with or without exposure to Y-27632 for 7 days,
respectively. The middle columnare merged images of the left and
middle images. Scale bars, 100 mm.
-
[2] Chowdhury F, Na S, Li D, Poh YC, Tanaka TS, Wang F, et al.
Material propertiesof the cell dictate stress-induced spreading and
differentiation in embryonicstem cells. Nat Mater
2010;9(1):82e8.
[3] Gentleman E, Swain RJ, Evans ND, Boonrungsiman S, Jell G,
Ball MD, et al.Comparative materials differences revealed in
engineered bone as a functionof cell-specic differentiation. Nat
Mater 2009;8(9):763e70.
[4] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity
directs stem celllineage specication. Cell 2006;126(4):677e89.
[5] McMurray RJ, Gadegaard N, Tsimbouri PM, Burgess KV, McNamara
LE, Tare R,et al. Nanoscale surfaces for the long-term maintenance
of mesenchymal stemcellphenotype and multipotency. Nat Mater
2011;10(8):637e44.
[6] Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P,
et al. Thecontrol of human mesenchymal cell differentiation using
nanoscale symmetryand disorder. Nat Mater 2007;6(12):997e1003.
[7] Yin Z, Chen X, Chen JL, Shen WL, Hieu NTM, Gao L, et al. The
regulation oftendon stem cell differentiation by the alignment of
nanobers. Biomaterials2010;31(8):2163e75.
[8] Chen W, Villa-Diaz LG, Sun Y, Weng S, Kim JK, Lam RH, et al.
Nanotopographyinuences adhesion, spreading, and self-renewal of
human embryonic stemcells. ACS Nano 2012;6(5):4094e103.
[9] Cen L, Liu W, Cui L, Zhang W, Cao Y. Collagen tissue
engineering: developmentof novel biomaterials and applications.
Pediatr Res 2008;63(5):492e6.
[10] Liu Y, Ramanath HS, Wang DA. Tendon tissue engineering
using scaffoldenhancing strategies. Trends Biotechnol
2008;26(4):201e9.
[11] Shen W, Chen X, Chen J, Yin Z, Heng BC, Chen W, et al. The
effect of incor-poration of exogenous stromal cell-derived factor-1
alpha within a knittedsilk-collagen sponge scaffold on tendon
regeneration. Biomaterials2010;31(28):7239e49.
[20] O'Brien EJ, Frank CB, Shrive NGB, Hallgrimsson, Hart DA.
Heterotopic miner-alization (ossication or calcication) in
tendinopathy or following surgicaltendon trauma. Int J Exp Pathol
2012;93(5):319e31.
[21] Bi Y, Ehirchiou D, Kilts TM, Inkson CA, Embree MC, Sonoyama
W, et al.Identication of tendon stem/progenitor cells and the role
of the extracellularmatrix in their niche. Nat Med
2007;13(10):1219e27.
[22] Pietschmann MF, Frankewycz B, Schmitz P, Docheva D, Sievers
B, Jansson V,et al. Comparison of tenocytes and mesenchymal stem
cells seeded onbiodegradable scaffolds in a full-size tendon defect
model. J Mater Sci MaterMed 2013;24(1):211e20.
[23] Zhu J, Li J, Wang B, Zhang WJ, Zhou G, Cao Y, et al. The
regulation of phenotypeof cultured tenocytes by microgrooved
surface structure. Biomaterials2010;31(27):6952e8.
[24] Sharma RI, Snedeker JG. Biochemical and biomechanical
gradients for directedbone marrow stromal cell differentiation
toward tendon and bone. Bio-materials 2010;31(30):7695e704.
[25] Yin Z, Chen X, Zhu T, Hu JJ, Song HX, Shen WL, et al. The
effect of decellu-larized matrices on human tendon stem/progenitor
cell differentiation andtendon repair. Acta Biomater
2013;9(12):9317e29.
[26] Tong WY, Shen W, Yeung CW, Zhao Y, Cheng SH, Chu PK, et al.
Functionalreplication of the tendon tissue microenvironment by a
bioimprinted sub-strate and the support of tenocytic
differentiation of mesenchymal stem cells.Biomaterials
2012;33(31):7686e98.
[27] Tong HW, Wang M, Lu WW. Electrospinning and evaluation of
PHBV-basedtissue engineering scaffolds with different bre
diameters, surface topographyand compositions. J Biomater Sci Polym
Ed 2012;23(6):779e806.
[28] Bashur CA, Shaffer RD, Dahlgren LA, Guelcher SA, Goldstein
AS. Effect of berdiameter and alignment of electrospun polyurethane
meshes on mesen-chymal progenitor cells. Tissue Eng Part A
2009;15(9):2435e45.
[29] Hodgkinson T, Yuan XF, Bayat A. Electrospun silk broin ber
diameter in-uences in vitro dermal broblast behavior and promotes
healing of ex vivo
Z. Yin et al. / Biomaterials 44 (2015) 173e185 185[12] Neidert
MR, Lee ES, Oegema TR, Tranquillo RT. Enhanced brin remodelingin
vitro with TGF-beta1, insulin and plasmin for improved
tissue-equivalents.Biomaterials 2002;23(17):3717e31.
[13] Yoshizawa T, Takizawa F, Iizawa F, Ishibashi O, Kawashima
H, Matsuda A, et al.Homeobox protein MSX2 acts as a molecular
defense mechanism for pre-venting ossication in ligament broblasts.
Mol Cell Biol 2004;24(8):3460e72.
[14] Kishore V, Bullock W, Sun X, Van Dyke WS, Akkus O.
Tenogenic differentiationof human MSCs induced by the topography of
electrochemically alignedcollagen threads. Biomaterials
2012;33(7):2137e44.
[15] Czaplewski SK, Tsai TL, Duenwald-Kuehl SE, Vanderby Jr R,
Li WJ. Tenogenicdifferentiation of human induced pluripotent stem
cell-derived mesenchymalstem cells dictated by properties of
braided submicron brous scaffolds.Biomaterials
2014;35(25):6907e17.
[16] Teh TK, Toh SL, Goh JC. Aligned brous scaffolds for
enhanced mechanores-ponse and tenogenesis of mesenchymal stem
cells. Tissue Eng Part A2013;19(11e12):1360e72.
[17] Kolambkar YM, Bajin M, Wojtowicz A, Hutmacher DW, Garcia
AJ, Guldberg RE.Nanober orientation and surface functionalization
modulate humanmesenchymal stem cell behavior in vitro. Tissue Eng
Part A 2014;20(1e2):398e409.
[18] Lin L, Shen Q, Xue T, Yu C. Heterotopic ossication induced
by Achillestenotomy via endochondral bone formation: expression of
bone and cartilagerelated genes. Bone 2010;46(2):425e31.
[19] Yee LPP, Wong YM, Rui YF, Lee YW, Chan LS, Chan KM.
Expression of chondro-osteogenic BMPs in ossied failed tendon
healing model of tendinopathy.J Orthop Res 2011;29(6):816e21.wound
models. J Tissue Eng 2014;5. 2041731414551661.[30] McBeath R,
Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cyto-
skeletal tension, and RhoA regulate stem cell lineage
commitment. Dev Cell2004;6(4):483e95.
[31] Nikukar H, Reid S, Tsimbouri MP, Riehle MO, Curtis AS,
Dalby MJ. Osteogenesisof mesenchymal stem cells by nanoscale
mechanotransduction. ACS Nano2013;7(3):2758e67.
[32] Ker ED, Nain AS, Weiss LE, Wang J, Suhan J, Amon CH, et al.
Bioprinting ofgrowth factors onto aligned sub-micron brous
scaffolds for simultaneouscontrol of cell differentiation and
alignment. Biomaterials 2011;32(32):8097e107.
[33] Wang W, Deng D, Li J, Liu W. Elongated cell morphology and
uniaxial me-chanical stretch contribute to physical attributes of
niche environment forMSC tenogenic differentiation. Cell Biol Int
2013;37(7):755e60.
[34] Caliari SR, Harley BA. Composite growth factor
supplementation strategies toenhance tenocyte bioactivity in
aligned collagen-GAG scaffolds. Tissue EngPart A
2013;19(9e10):1100e12.
[35] Cheng X, Tsao C, Sylvia VL, Cornet D, Nicolella DP,
Bredbenner TL, et al.Platelet-derived growth-factor-releasing
aligned collagen-nanoparticle berspromote the proliferation and
tenogenic differentiation of adipose-derivedstem cells. Acta
Biomater 2014;10(3):1360e9.
Electrospun scaffolds for multiple tissues regeneration in vivo
through topography dependent induction of lineage specific ...1.
Introduction2. Materials and methods2.1. Fabrication of PLLA
scaffolds2.2. Morphology of PLLA scaffolds2.3. SEM imaging2.4.
Alkaline phosphatase (ALP) staining2.5. Quantitative PCR2.6. Animal
model2.7. Immunofluorescence2.8. Histological evaluation and
staining2.9. Immunohistochemistry2.10. Mechanical testing2.11.
Determination of collagen content2.12. Transmission electron
microscopy2.13. Radiographic evaluation2.14. Statistical
analysis
3. Results3.1. Fabrication and morphological characterization of
scaffolds3.2. The effects of aligned and randomly-oriented
scaffolds on neo-tissue formation3.2.1. Histology of repaired
tendons3.2.2. Histology of bone formation3.2.3. Mechanical
properties of repaired tendons
3.3. The effects of topographical cues on MSCs3.4.
Topography-induced lineage commitment of MSCs is dependent on
cytomyosin cytoskeleton
4. Discussion5. ConclusionAcknowledgmentAppendix A.
Supplementary dataReferences