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Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen
nanofibers
Lin Jia, Molamma P Prabhakaran, Xiaohong Qin and Seeram Ramakrishna
J Biomater Appl published online 28 March 2014
DOI: 10.1177/0885328214529002

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Guiding the orientation of smooth Reprints and permissions:
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muscle cells on random and aligned DOI: 10.1177/0885328214529002
jba.sagepub.com
polyurethane/collagen nanofibers

Lin Jia1,2,3, Molamma P Prabhakaran3, Xiaohong Qin1 and Seeram Ramakrishna3

Abstract
Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge
in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the
current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid
polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyureth-
ane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are
in the range of 174–453 nm and 145–419 for random and aligned fibers, respectively, where they closely mimic the
nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic
wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic
smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/
collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned
scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen
(3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen
(3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate
the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration.

Keywords
Polyurethane, collagen, aligned nanofibers, cell orientation, smooth muscle cells

Introduction
Scaffolds should have biological and structural func-
Atherosclerosis and coronary arterial restenosis are the tionality, such that they can support cell attachment,
two major diseases of the cardiovascular system, that growth and proliferation and provide biocompatible
cause morbidity and mortality in humans, which is template which allows for the in-growth of cells and
mainly attributed to the modern life style pursued by newly formed tissues. In order to satisfy these require-
people all over the world.1 Small diameter (<6 mm) ments, scaffolds should be porous and preferably bio-
vascular grafts are a potential requisite for patients resorbable, to allow for the gradual replacement of
waiting to replace the coronary blood vessel.2 The
main reason for the failure of small vascular substitute 1
Key Laboratory of Textile Science & Technology, Ministry of Education,
is owing to neointima formation and arterial occlusion, College of Textiles, Donghua University, Shanghai, China
which occurs from increased migration of smooth 2
Collagen of Textiles, Henan Institute of Engineering, Zhengzhou, Henan,
muscle cells (SMCs) from vascular media to the arterial China
3
lumen. In native vascular tissue, SMCs control the Center for Nanofibers and Nanotechnology, Nanoscience and
blood pressure and tissue homeostasis, which are very Nanotechnology Initiative, Faculty of Engineering, National University of
Singapore, Singapore
essential for arterial contraction and dilation.3,4 Hence,
developing functional scaffolds that have the ability to Corresponding author:
Molamma P Prabhakaran, Center for Nanofibers and Nanotechnology,
support and promote the function of native SMCs is E3-05-14, Nanoscience and Nanotechnology Initiative, Faculty of
critical towards the success of vascular tissue engineer- Engineering, National University of Singapore, 2 Engineering Drive 3,
ing (TE). Singapore 117576. Email: nnimpp@nus.edu.sg

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2 Journal of Biomaterials Applications 0(0)

new tissues.5 Electrospun nanofibrous scaffolds provide is useful for tissue regeneration applications.24–26
high surface area with interconnected pores; have con- Because of these reasons, collagen incorporated in PU
trollable biodegradation and mechanical properties; can produce scaffolds with better biocompatibility and
and are able to incorporate bioactive ingredients mechanical properties. In order to mimic the aligned
within synthetic polymers to enhance cell behaviors. fibrous structure of ECM in native arteries, aligned
Electrospun nanofibrous scaffolds are widely used in PU/Coll scaffolds were fabricated by electrospinning
various TE applications because of these advantages.6–8 and the phenotype, orientation, and functionality of
In addition, electrospinning can produce nanofibers SMCs on these aligned scaffolds were analyzed in our
with aligned morphology, whereby the aligned fibers study. The novelty of this work include fabrication of
can influence the alignment and function of cells random and aligned PU/Coll nanofibrous scaffolds to
seeded on them.9,10 In the human body, the extracellu- support and promote the alignment of SMCs, thus pos-
lar matrices (ECM) of tissues such as the nerve, muscle, itioning them highly as suitable scaffolds for vascular
tendons, and ligaments, have a regular and oriented tissue regeneration.
architecture, which co-relate much to the tissue func-
tion. In such cases, fabricating a scaffold with defined
fiber orientation is necessary to precisely mimic the Materials and methods
native ECM.11 Lee et al.12 produced aligned electro-
spun polyurethane (PU) nanofibers using a rotating
Materials
collecting target and found that the morphology of Polyurethane (PU) was generously provided by the
fibroblasts grown on these aligned scaffolds more clo- Lubrizol Corporation (Korea). Human aortic SMCs,
sely mimic the morphology of fibroblasts in vivo. In smooth muscle cell medium (SMCM), smooth muscle
another study, Xu et al.13 electrospun aligned poly(L- growth supplement (SMCGS), penicillin/streptomycin
lactide-co-caprolactone) (PLCL) nanofibers and found solution (P/S), and fetal bovine serum (FBS) were pur-
that the SMCs on aligned nanofibers expressed oriented chased from ScienceCell, USA. 4,6-diamidino-2-pheny-
morphology. Studies by Nivison-Smith et al. also lindole, dihydrochloride (DAPI) and Alexa Fluor 488
demonstrated that the SMCs grew longitudinally and Alexa Fluor 594 were all purchased from
along the orientation of aligned elastin nanofibers and Invitrogen Corporation, USA. Collagen type I was
expressed native a-smooth muscle protein actin.14 obtained from Koken, Japan, and 1,1,1,3,3,3-hexa-
The chemical composition of scaffolds is very fluor-2-propanol (HFP) was purchased from Sigma-
important for TE, since it can influence the adhesion Aldrich Pte Ltd., Singapore. Mouse anti-human alpha
and distribution of proteins and further control the smooth muscle actin (SMA) and anti-smooth muscle
attachment and migration of cells. Many biodegradable myosin heavy chain antibody (MHC) were obtained
and biocompatible polymers have been fabricated as from Abcam (Cambridge, UK).
nanofiber-based scaffolds through electrospinning. PU
have remarkable mechanical properties, possess bio-
Electrospinning of nanofibers
degradability, and electrospun PU nanofibers are pro-
mising substrates for cell culture.12,15,16 In this research, Pure PU was dissolved in HFP at a concentration of
we utilized an aliphatic, polyether-based PU mainly due 6 wt% and then stirred for 48 h at room temperature to
to its elastic behavior for application in vascular regen- obtain a uniform solution. Clear solutions of 6 wt%
eration. Pure PU lacks functional groups such as the PU/collagen with a weight ratio of 75:25, 50:50, and
carboxyl or amino groups, and hence they are not 25:75 were prepared by dissolving PU and collagen in
favorable as a sole matrix for tissue regeneration.16,17 HFP to obtain PU/Coll (3:1), PU/Coll (1:1), and PU/
However, the biocompatibility of PU can be improved Coll (1:3) solution, respectively. The PU and PU/Coll
by incorporating native proteins with PU, while preser- solution were separately fed into four 3-ml plastic syr-
ving its mechanical strength. Collagen (Coll) is a key inges attached to a blunted 27-G stainless steel needle
element of the extracellular matrix (ECM), is the most and a syringe pump (KDS 100, KD Scientific,
plentiful proteins in native vascular, and together the Hollistion, MA) was used to feed the polymer solution
collagen and elastin accounted for 58–75% of the dry into the needle tip at a flow rate of 1.0 ml/h. A high
defatted artery.18–20 In the adventitia and media layers voltage of 16 kV (Gamma High Voltage Research,
of arterial walls, collagen fibers are oriented along the USA) was applied to the polymer solution and
circumferential direction, and this unique microstruc- random nanofibers were collected on a flat collector
ture plays a key role towards the functionality of vas- wrapped with aluminum foil kept at a distance of
cular tissue.21–23 Due to the ease of isolation of collagen 12 cm from the needle tip. Aligned nanofibers were col-
from many sources, and due to its non-immunogeni- lected on a rotating drum with a rotational speed of
city, low antigenicity, and cell compatibility, collagen 2000 r/min. Nanofibrous scaffolds were dried under

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Jia et al. 3

vacuum and used for characterization and cell culture 37 C in a humidified atmosphere containing 5% CO2.
experiments, respectively. After every 3 days, SMCs were fed with fresh media
and trypsinized upon confluency using trypsin–EDTA
for cell sub-culture or cell seeding in vitro. SMCs of
Characterization evaluation of nanofibrous scaffolds
passage 5 were used in this study. The 15-mm coverslips
The morphology of the nanofibers was observed under with different samples were placed in 24-well plate and
SEM (FEI-QUANTA 200 F, the Netherlands) after pressed with a stainless steel ring to make sure of com-
gold coating (JEOL JFC-1200, Japan) of the nanofi- plete contact of the scaffolds with the wells. The sam-
bers. The average fiber diameters were measured ples were sterilized under UV light before they were
based on the SEM micrographs by measuring about washed three times with phosphate-buffered saline
100 random fibers using image analysis software (PBS) for 2 h. To enhance the cell adhesion, the speci-
(ImageJ, National Institutes of Health, Bethesda, mens were firstly incubated with SMCM 3 h before cell
MD). The degree of fiber alignment was evaluated seeding. SMCs were finally seeded on random and
using the ImageJ plugin OrientationJ software.27 aligned PU, PU/Coll (3:1;1:1,1:3) nanofibrous scaf-
From the SEM images, the orientation test software folds, and tissue culture plate (TCP as a positive con-
measured fiber number in different directions varying trol) at a density of 8000 cells per well. All the
from 0 to 180 . During this study, the dominant fiber nanofibrous scaffolds and TCP were used in triplicate,
direction and alignment degree were studied to evaluate and the experiments were repeated at least three times.
the direction and intensity of fiber alignment. The
alignment degree is the alignment index (AI) of the
Cell proliferation
nanofibers, which ranged from 0% to 100%, where
values 0% and 100% represent the complete random- The proliferation of SMCs on different nanofibrous
ness and complete alignment, respectively.27 The pore scaffolds and TCP was studied using 3-(4,5-
sizes of nanofibrous scaffolds were measured by a CFP- dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-
1200-A capillary flow porometer (PMI, New York, (4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric
NY). Nanofibrous scaffolds were peeled from alumi- assay. After 3, 6, 9, and 12 days of cell culture, the sam-
num foil and the mats had a surface area of 4 cm  4 cm ples were rinsed with PBS and incubated with 1 ml of
and a thickness of about 35 mm. The capillary flow DMEM containing 20% of MTS kit (CellTiter 96
porometry provides a simple technique to measure the AQueous One Solution reagent; Promega, Madison,
average pore size and distribution. Considering the WI). After incubating for a period of 3 h, the deeply
hydrophilic/hydrophobic property of the nanofibers, colored culture medium was aliquotted to a 96-well
water contact angle measurement was studied by plate (100 ml/well), and the absorbance of each well at
VCA Optima Surface Analysis System 213 (AST prod- 490 nm was measured using a microplate reader. The
ucts, Billerica, MA). Avatar 380 FTIR spectrometer background absorbance was also measured for cell-
(Thermo Nicolet, Waltham, MA) was used to analyze free DMEM containing MTS reagent.
the surface characterization of nanofibers over a range
of 400–4000 cm1 at a resolution of 2 cm1. A tabletop
Morphological evaluation of SMCs
tensile Tester (Instron5345, USA) was utilized to ana-
lyze the tensile properties of the electrospun nanofibers After 9 days of cell culture, morphologies of SMCs on
at a cross-head speed of 10 mm min1. The nanofibrous random and aligned electrospun nanofibers were stu-
membrane was cut into rectangular specimens with died by SEM. The samples were firstly washed two
10  30 mm for the tensile test. For each type of scaf- times with PBS and then incubated in 3% glutaralde-
folds, six samples were tested, and tensile strength and hyde for 3 h at room temperature to fix cells on scaf-
break elongation were calculated based on the stress– folds. The scaffolds were further washed using
strain curves. For studies such as the porosity measure- deionized water, followed by increasing concentrations
ment, FTIR, and tensile property measurement, the of aqueous ethanol (50%, 70%, 90%, and 100%) for
nanofibrous mat with a thickness of approximately 10 min each. The samples were finally treated with hex-
35 mm was peeled off from the aluminum foil after amethyldisilazane and air-dried in a fume hood. The
electrospinning. cell-scaffold construct was observed under SEM after
gold coating.
In vitro cell culture and seeding
Immunostaining of SMCs
SMCs were cultured in a 75 cm2 cell culture flask using
SMCM supplemented with 2% FBS, 1% SMCGS, and After 9 days of cell culture, SMCs grown on different
1% pencillin–streptomycinin. Cells were incubated at electrospun nanofibers and TCP were processed for

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4 Journal of Biomaterials Applications 0(0)

protein staining. The cells were fixed in formalin at Results

room temperature for 30 min, washed with PBS
three times for 15 min, and incubated in 0.1%
Characterization of nanofibrous scaffolds
TritonX 100 solution for 5 min followed by incuba- Electrospun PU and PU/Coll nanofibers with different
tion in 3% BSA for 90 min to permeabilize the cell fiber morphology (random and aligned) were fabricated
membrane and block the non-specific sites. The sam- in this study and Figure 1 shows the SEM images of the
ples were then incubated with either of the SMCs- nanofibers. Smooth and uniform fibers were observed
specific marker proteins SMA or MHC at a dilution for all the random and aligned nanofibers. The random
1:100 for 120 min at room temperature. Further the nanofibers expressed interconnected fiber morphology.
hybrid solution of secondary antibodies goat anti- The diameter of the hybrid nanofibers decreased with
mouse AF 488 (green) (1:400) for SMA, or AF increasing collagen content within the hybrid scaffolds.
594 (red) (1:400) for MHC with DAPI (1:500) One possible explanation for this is that the addition of
were added for 90 min. The samples were then collagen into PU solution enhanced the conductivity of
removed from 24-well plate and mounted on a hybrid PU/Coll solution and hence the composite PU/
glass slide using mounting medium and finally the Coll nanofibers had smaller fiber diameter compared to
protein expressions were observed under the fluores- pure PU nanofibers.28 However, due to the stretching
cent microscope (Olympus FV 1000). force produced by the rotating drum during the electro-
spinning process, the aligned nanofibers had smaller
diameter than their respective random nanofibers.29
The insets of Figure 1 shows the distribution of nano-
Statistical analysis
fibers by the direction of orientation, which ranged
All the data in this study were showed as mean  stand- from 0 to 180 . The random nanofibers expressed
ard deviation (SD) of the mean. The means of differ- random distributions between 0 and 180 and the
ent data sets were compared with single-factor analysis alignment index (AI) of random nanofibers were
of variance using Microsoft Excel data analysis, and about 0%, indicating complete randomness.
the value of p  0.05 was considered statistically Compared to the random nanofibers, the aligned
significant. nanofibers represented one-directional orientation of

90º
180º 0º
Figure 1. SEM images of electrospun nanofibers with in situ figures of their distributions from 0 to 180 directions. (A) R-PU (B) R-
PU/Coll (3:1) (C) R-PU/Coll (1:1) (D) R-PU/Coll (1:3) (E) A-PU (F) A-PU/Coll (3:1) (G) A-PU/Coll (1:1) (H) A-PU/Coll (1:3). The
arrow in F illustrates the directions of water contact angle measurement and tensile test: perpendicular (PP) and parallel (PL) to the
direction of the fiber orientation. R: random; A: aligned.

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Jia et al. 5

the nanofibers. The AI of aligned PU and PU/Coll scaf- incorporated collagen into PU to fabricate hybrid
folds are shown in Table 1, the AI of hybrid PU/Coll PU/Coll scaffolds to enhance the cell attachment and
nanofibers decreased with increasing collagen content. cell proliferation on the scaffolds. The contact angle
The addition of native protein, collagen into PU solu- values of electrospun random and aligned scaffolds
tion decreased the solution viscosity, and it also influ- are also shown in Table 1. For the nanofibers with
enced the fiber alignment. Schnell et al.30 electrospun aligned morphology, we surveyed the contact angle in
aligned poly-"-caprolactone (PCL) and PCL/collagen two different directions, i.e. along perpendicular (PP)
nanofibers and found that the addition of collagen and parallel (PL) direction with respect to the orienta-
into PCL solution reduced the alignment degree of tion of the fibers, as shown in Figure 1(F). Compared
PCL/Coll nanofibers. In a similar way, during this to the hydrophilicity of pure PU scaffolds (27.1  1.68
study, the alignment of PU/Coll nanofibers was com- for R-PU), the incorporation of collagen did not dras-
promised with respect to pure PU nanofibers. tically reduce the hydrophilicity of PU/Coll nanofibers,
Details of the fiber diameters and pore diameters of but might assist to maintain the integrity of the hybrid
the scaffolds are also given in Table 1. The mean pore scaffolds. In addition, an anisotropic wettability was
size of random PU, PU/Coll (3:1), PU/Coll (1:1), and observed for aligned nanofibers. Regardless of pure
PU/Coll (1:3) nanofibers was obtained as 0.64 mm, PU and PU/Coll scaffolds, the contact angle in PL dir-
0.36 mm, 0.31 mm, and 0.28 mm, respectively. The pore ection was significantly higher than in the PP direction
diameters of hybrid PU/Coll nanofibers were much (p  0.05) for aligned nanofibers.
smaller than the pore diameter of pure PU nanofibers, Surface characterization of the electrospun scaffolds
and this can be attributed to the reduced fiber diameter was studied by FTIR and Figure 2 shows the FTIR
of hybrid PU/Coll nanofibers. Compared to the pore spectra of PU and PU/Coll nanofibrous scaffolds,
diameter and the pore size distribution of random where PU scaffolds showed characteristic carbonyl
nanofibers, bigger pore diameter and wider range of peak at 1710 cm1, C-C absorption peak at
pore size distribution were observed for aligned nano- 1530 cm1, C-O stretching peak at 1110 cm1, –CH–
fibers, which can be accredited to the ‘one directional’ stretching vibrations at 777 cm1, thus representing
orientation of aligned fibers. the presence of substituted benzene.31 In addition to
The surface hydrophobic-hydrophilic properties of the characteristic absorption band of PU, the stretching
electrospun nanofibers have a major influence towards peak of N-H and C-H at 3310 cm1 and 3062 cm1 for
cell adhesion and proliferation behavior. Pure PU used amide A and amide B were observed on electrospun
in this study is hydrophilic, but PU nanofibers got PU/Coll scaffolds, indicating the existence of amino
wrinkled and started shrinking due to the extremely functional groups. Furthermore, the electrospun PU/
high elastic behavior of pure PU elastomer, which is Coll hybrid nanofibers also showed the C ¼ O stretch-
not a desirable aspect required of vascular graft, and ing and N-H deformation peaks at 1651 cm1 (amide I)
this would indirectly influence the cell growth and and 1535 cm1 (amide II). Because the u (C-C) absorp-
behaviors on pure PU scaffolds. Hence, we tion peak at 1530 cm1 of PU is very close to the N-H

Table 1. Properties of electrospun random (R) and aligned (A) nanofibers.

Alignment Water contact

Scaffolds Fiber diameter (nm) index (AI) Pore diameter (mm) angle ( )

R-PU 453  126 9% 0.64  0.28 27.1  1.21

A-PU 419  82 82% 0.73  0.46 PL:36.2  2.87
PP:22.3  1.34
R-PU/Coll (3:1) 273  76 7% 0.36  0.12 39.2  1.58
A-PU/Coll (3:1) 217  67 75% 0.42  0.24 PL:46.9  1.37
PP:34.6  1.19
R-PU/Coll (1:1) 206  78 8% 0.31  0.11 45.8  1.43
A-PU/Coll (1:1) 166  45 73% 0.33  0.21 PL:55.6  1.07
PP:41.2  1.69
R-PU/Coll (1:3) 174  61 6% 0.28  0.13 52.1  0.98
A-PU/Coll (1:3) 145  55 64% 0.31  0.20 PL:58.1  1.34
PP:47.4  1.57
PL: parallel direction with respect to the orientation of the fibers; PP: perpendicular direction with respect to the orientation of the fibers.

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6 Journal of Biomaterials Applications 0(0)

1535
3310 3062
PU/Coll (1:3) 1651

PU/Coll (1:1)

777
PU/Coll (3:1)

PU
1710 1220
2960 1110

3940 3440 2940 2440 1940 1440 940 440

Wavelength (cm-1)

Figure 2. FTIR spectra of electrospun PU and PU/Coll nanofibers. Coll: collagen; PU: polyurethane; PP: perpendicular; PL: parallel.

(A)12 (B) 32 (C) 8 A-PU-PP

R-PU A-PU-PL
7 A-PU/Coll(3:1)-PP
28 A-PU/Coll(3:1)-PL
10 R-PU/Coll(3:1) A-PU/Coll(1:1)-PP
R-PU/Coll(1:1) A-PU/Coll(1:1)-PL
24 6 A-PU/Coll(1:3)-PP
Tensile Stress (MPa)

Tensile Stress (MPa)

Tensile Stress (MPa)

R-PU/Coll(1:3) A-PU/Coll(1:3)-PL
8
20 5

6 16 4

12 3
4
8 2
2
4 1

0 0 0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 450 450 0 30 60 90 120 150 180 210 240 270 300
Tensile Strain (%) Tensile Strain (%)
Tensile Strain (%)

Figure 3. Stress–strain curves of electrospun nanofibers. (A) Random nanofibers. (B) Aligned nanofibers in PL direction. (C) Aligned
nanofibers in PP direction. PP: perpendicular; PL: parallel. R: random; A: aligned; Coll: collagen; PU: polyurethane; PP: perpendicular;
PL: parallel.

deformation peaks at 1535 cm1, the two peaks pro- Figure 3 shows the typical stress–strain curves of the
duced a bigger and more prominent peak at electrospun scaffolds for both the random and aligned
1535 cm1 for PU/Coll nanofibers. Comparing the electrospun scaffolds. Because of the random and inter-
spectra of hybrid PU/Coll nanofibers with different col- lacing arrangement of nanofibers, random PU and PU/
lagen contents (Figure 2), the difference was mainly Coll scaffolds expressed a linear segment first, followed
with respect to the intensity of the peaks. With increas- by a non-linear curve. Compared with the tensile prop-
ing content of collagen in the hybrid scaffolds, the erties of pure PU scaffolds, the incorporation of colla-
intensity of N-H stretching peak for amide A increased gen into PU led to a remarkable decrease in ultimate
and a corresponding decrease in the intensity of the strain of the hybrid PU/Coll fibers. The hybrid PU/Coll
characteristic absorption band of PU was observed. (3:1) and PU/Coll (1:1) retained high tensile strain due
Therefore, the intensity of the amide peaks for PU/ to the elasticity of PU and also had higher tensile
Coll (1:3) was higher compared to the amide peaks of strength. These results indicated that a moderate
PU/Coll (3:1) scaffolds. FTIR analysis showed that the amount of collagen (25%) incorporated in PU can
surface of electrospun PU/Coll nanofibers had amino enhance the tensile strength of PU/Coll (3:1) scaffolds
groups, thus improving the biocompatibility of scaf- and still retain its tensile strain. Aligned PU and PU/
folds by providing ligands to support the cell prolifer- Coll nanofibrous scaffolds expressed anisotropic mech-
ation on PU/Coll scaffolds better than the pure PU anical properties, and their tensile strengths and
scaffolds. Young’s modulus in PL direction were remarkably

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Jia et al. 7

Table 2. Tensile properties of electrospun random (R) and aligned (A) nanofibers.

Scaffold Ultimate tensile stress (MPa) Ultimate strain (%) Tensile modulus (MPa)

R-PU 4.83  0.54 345  45.70 1.41  0.07

A-PU-PL 14.07  1.41 423  56.40 4.05  0.85
A-PU-PP 4.21  1.22 264  50.10 1.38  0.09
R-PU/Coll (3:1) 11.49  1.31 128  11.38 16.67  2.17
A-PU/Coll (3:1)-PL 27.49  2.54 141  29.90 27.48  3.78
A-PU/Coll (3:1)-PP 7.38  0.78 127  27.80 6.54  1.09
R-PU/Coll (1:1) 5.91  0.34 102  12.50 7.79  2.07
A-PU/Coll (1:1)-PL 28.67  3.56 107  16.70 21.46  4.58
A-PU/Coll (1:1)-PP 5.81  0.69 94  18.10 4.26  0.76
R-PU/Coll (1:3) 2.87  0.68 59  10.80 9.16  1.24
A-PU/Coll (1:3)-PL 13.85  3.26 67  14.70 30.97  4.21
A-PU/Coll (1:3)-PP 2.45  0.37 37  15.40 7.96  1.56
Coronary arterya 1.4–11.14 45–99 –
a
See Xu et al.44

TCP R PU R PU /Coll (3:1)

1.1 R PU/Coll (1:1) R PU/Coll (1:3) APU
1 A PU/Coll (3:1) A PU/Coll (1:1) A PU/Coll (1:3)
0.9
0.8
Absorbance at 490nm

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Day 3 Day 6 Day 9 Day 12
Culture time (Days)

Figure 4. SMCs proliferation on electrospun nanofibers and TCP, as determined by MTS assay. *Significant compared with cell
proliferation on random PU at p  0.05. #Significant compared with cell proliferation on aligned PU scaffolds at p  0.05. Coll: collagen;
PU: polyurethane; PP: perpendicular; PL: parallel; TCP: tissue culture plate.

higher (p  0.05) than in PP direction (Table 2). Studies carried out by MTS assay (Figure 4). The proliferation
using electrospun aligned poly(3-hydroxybutyrateco-3- of SMCs on hybrid PU/Coll scaffolds was remarkably
hydroxyvalerate; PHBV) and PHBV/collagen nanofi- (p  0.05) higher than on pure PU nanofibers, which
bers also showed that the tensile strength of aligned can be explained due to the presence of collagen in
nanofibers in PL direction increased with increasing these scaffolds. Comparing the cell proliferation on dif-
degree of fiber alignment (than along the PP ferent PU/Coll scaffolds, the proliferation of SMCs on
direction).32 PU/Coll (3:1) was found slightly higher than on the
other two scaffolds. After 12 days of cell culture, the
cell proliferation on random PU/Coll (3:1) was 1.2%
SMCs proliferation on different nanofibrous scaffolds
and 2.3% higher than on random PU/Coll (1:1) and
After 3, 6, 9, and 12 days of cell culture, the prolifer- PU/Coll (1:3) scaffolds, respectively. A possible
ation of SMCs on different electrospun nanofibers were reason is that the PU/Coll (3:1) scaffolds had better

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8 Journal of Biomaterials Applications 0(0)

structural integrity and higher mechanical properties (Figure 5F–H) and random PU/Coll (3:1) (Figure 5B)
compared to PU/Coll (1:1) and PU/Coll (1:3) scaffolds. nanofibers had a highly aligned phenotype of cell
During our study, grown either on pure PU or PU/Coll behavior. However, the alignment of SMCs on
scaffolds, proliferation of SMCs on random nanofi- random PU/Coll (1:1) and PU/Coll (1:3) scaffolds
brous scaffolds was slightly higher than on their were not so pronounced like the SMCs on PU/Coll
respective aligned scaffolds. After culturing for 12 (3:1) scaffolds. Compared to TCP and random PU scaf-
days, the SMCs proliferation on random PU, PU/ folds, the addition of collagen into PU might have
Coll (3:1), PU/Coll (1:1), and PU/Coll (1:3) were induced cell orientation on hybrid PU/Coll scaffolds.
3.7%, 3.4%, 2.9%, and 1.8% higher than on their In addition, the PU/Coll (3:1) scaffolds had better
respective aligned scaffolds, demonstrating the import- mechanical properties than PU/Coll (1:1;1:3), while
ance of the chemical composition of the fibers than its the alignment of SMCs on PU/Coll (3:1) scaffolds
architecture. was higher than PU/Coll (1:1;1:3) scaffolds. These
results highlight the fact that the random nanofibrous
scaffolds with suitable composition and higher mechan-
Morphological evaluation of SMCs ical properties could induce the alignment of SMCs
After 9 days of cell culture, the morphologies of SMCs even on random structures.
on random and aligned nanofibers were evaluated by
SEM (Figure 5). Compared with PU nanofibers, more
cells were found attached on PU/Coll nanofibers, indi-
Expression of SMC markers
cating that the collagen-containing scaffolds improved To validate the functionality of SMCs, immunofluores-
the adhesion and attachment of SMCs. In addition, cent staining of SMA and MHC was carried out for
SMCs on aligned nanofibers (Figure 5E–H) were SMCs on different nanofibrous scaffolds. SMA is the
found elongated along the fiber direction, illustrating earliest marker of SMCs.4 Figure 7 shows the represen-
that the aligned nanofibers can promote the orientation tative images of SMA-stained SMCs on different sub-
of cells through contact guidance and the SMCs on strates. Regardless of the morphology of the fibers,
aligned nanofibrous scaffolds were of one-directional fewer SMCs were found to attach on PU nanofibers,
growth. However, SMCs on TCP (Figure 6A) and with less SMA expression (Figure 7A and E).
random PU nanofibers were observed in a non-direc- Conversely, on PU/Coll scaffolds (Figure 7B–D and
tional manner. The direction of SMCs orientation are F–H), more number of cells exhibited SMA markers
shown (black colored arrows) in Figure 5, which with microfilament bundles phenotype. SMCs cultured
also indicated the alignment of SMCs to some extent. on aligned PU/Coll nanofibers (Figure 7F–H)
It was observed that the SMCs on aligned PU/Coll expressed high density of SMA with aligned

Figure 5. SEM images of SMCs on electrospun (A) R-PU (B) R-PU/Coll (3:1) (C) R-PU/Coll (1:1) (D) R-PU/Coll (1:3) (E) A-PU (F)
A-PU/Coll (3:1) (G) A-PU/Coll (1:1) (H) A-PU/Coll (1:3) nanofibers after 9 days of cell culture.

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Jia et al. 9

microfilament bundles phenotype. SMCs on TCP also PU/Coll (3:1) and random PU/Coll (3:1) (Figure 8B)
(Figure 6B) showed higher SMA expression, but the nanofibers demonstrated a high density of MHC
orientation of the cells was non-directional. Similar to expressions, and it also demonstrated an aligned orien-
the results of SEM analysis, SMCs on random PU/Coll tation of cells compared to MHC expression on TCP
(1:1) and PU/Coll (1:3) (Figure 7C–D) showed good and random PU nanofibers (Figure 8A). These results
expression of SMA. However, SMCs on random PU/ indicated that the morphologies and protein staining of
Coll (3:1) (Figure 7B) exhibited aligned SMA expres- SMCs on random and aligned PU/Coll (3:1) were
sion with microfilament bundles phenotype. better than on all other scaffolds.
The functionality of SMCs on the electrospun nano-
fibers were further elucidated by expression of MHC
protein. MHC is considered as the most discriminating
Discussion
marker of SMCs.4 SMCs seeded on random and An important challenge in vascular TE involve the
aligned PU scaffolds (Figure 8A and E) showed development of a suitable vascular graft substitute
random and uneven expression of MHC in much that is ‘smart’ enough to instruct the in vivo environ-
lesser amounts. However, the SMCs seeded on aligned ment to form vascular tissue. PU had been applied in

Figure 6. SEM images and immunofluorescent staining of SMCs on TCP (A) SEM image; (B) SMA expression (C) MHC expression.

Figure 7. Immunofluorescent staining of SMA on (A) R-PU (B) R-PU/Coll (3:1) (C) R-PU/Coll (1:1) (D) R-PU/Coll (1:3) (E) A-PU
(F) A-PU/Coll (3:1) (G) A-PU/Coll (1:1) (H) A-PU/Coll (1:3) nanofibers.

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10 Journal of Biomaterials Applications 0(0)

Figure 8. Immunofluorescent staining of MHC on (A) R-PU (B) R-PU/Coll (3:1) (C) R-PU/Coll (1:1) (D) R-PU/Coll (1:3) (E) A-PU
(F) A-PU/Coll (3:1) (G) A-PU/Coll (1:1) (H) A-PU/Coll (1:3) nanofibers.

many fields of TE because of its excellent mechanical key role on the durable mechanical properties of
properties.15,16 However, it lacks chemical functional- arteries.
ities, such as carboxyl or amino groups, and poor cell Scaffolds with nanofibrous topography can attach
affinity become the bottleneck of PU when challenged and organize cells around them, and might promote
as vascular grafts. Collagen is the most plentiful protein cell attachment, proliferation, and differentiation of
found in native blood vessel and it has been used in a stem cells in vitro, especially because the diameter of
variety of tissue regeneration applications because of its the fibers are smaller than the diameter of the cells.35,36
resorbable, non-immunogenic, low antigenic, and good Furthermore, some researchers also found that nanofi-
cell compatibility effect.20–22 However, pure collagen bers with smaller diameter can enhance the cell prolif-
nanofibers cannot be utilized as a vascular graft due eration and differentiation compared to fibers with
to its poor mechanical properties. Hence, we incorpo- higher fiber diameter.37,38 In this study, aligned PU/
rated collagen with PU to fabricate hybrid scaffolds Coll nanofibers were produced with smaller fiber diam-
with excellent mechanical properties and good compati- eter and the oriented morphology of the fibers might
bility for vascular TE. assist to enhance the attachment and proliferation of
Electrospinning is the most popular method used SMCs, direct the cell orientation, and elongation of
for the fabrication of nanofibrous scaffolds, whereby SMCs. The porosity and pore size distribution of scaf-
these nanofibers mimic the native morphology of folds have a great impact on the nutrient flow and cell
ECM in tissues. In addition to fiber diameter, the migration, thus influencing the cells attachment behav-
orientation of the fibers is another crucial factor iour.39 During this study, pore size of the scaffolds
that is considered while choosing scaffolds for vascu- ranged from 0.2 to 1.2 mm and the mean pore size
lar TE. In tissues such as the nerve, muscle, tendon, was proportional to the fibers diameter and these
and ligament, the alignment of cells and surrounding results were also in accordance with the results reported
ECM impart their desired functionality by providing by other researchers.40,41 Due to the alignment of fibers
mechanical strength and cell communication. In TE, in one direction, there are less interconnected pores
aligned nanofibers can take on the role of native between the fibers, and the pore size distribution of
ECM chemically and architecturally, provide mechan- aligned nanofibrous scaffolds were less uniform com-
ical strength and biological cues for cell attachment.33 pared to the random fibers. Studies by Lee et al.12
In the native vascular tissue, the key feature of arter- also found that the pore size distribution of electrospun
ial micro-architecture is the alignment of SMCs elon- aligned PU nanofibers was much boarder than the
gating their axis towards the circumferential random PU nanofibers, similar to our results. At the
direction.34 The alignment of SMCs control the con- same time, Meng et al.34 reported that the random
striction or dilation of blood vessels, and along with nanofibers showed higher porosity than the aligned
their fibrous ECM, the alignment of SMCs play a ones, which was also similar to our observation.

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Jia et al. 11

With regard to the chemical composition and topo- minimizing neointima formation and arterial occlu-
graphic pattern of scaffolds, it is the contact angle that sion.3,33 Our cell proliferation results show the biocom-
reflects its hydrophilicity, which demonstrate the ability patibility of PU/Coll scaffolds for application in
of the scaffolds to absorb proteins and assist in cell vascular TE. These results also highlighted the import-
adherence. Anisotropic wetting was observed for the ance of choosing an optimized amount of protein while
electrospun aligned nanofibers. Similar results were incorporating it with a synthetic polymer, suggesting
observed by Kai et al.10 and Chung et al.42 during the potential of PU/Coll nanofibers as appropriate sub-
their studies for aligned PCL/gelatin and poly strates than pure PU nanofibers for vascular tissue
(dimethylsiloxane) (PDMS) scaffolds, respectively. regeneration. It was reported that the effect of nanofi-
One possible reason is that the grooves and ridges on ber orientation on cell proliferation varied greatly
nanofiber surfaces in PL direction limited the elong- between different studies. Some researchers found that
ation of water droplet, causing a higher water contact cell numbers on aligned nanofibers were remarkably
angle, while in PP direction, the droplet could elongate higher than those on random nanofibers scaffolds
easily and express longer footage and lower contact after 3–7 days of culture, the reason could be the
angle.42 The aligned topology and anisotropic wettabil- aligned nanofibers greatly enhanced cell migration
ity might guide the cell orientation on aligned nanofi- than the random nanofibers.45–47 In contrast, few
brous scaffolds. FTIR analysis showed that the other researchers reported that the cell proliferation
electrospun PU/Coll nanofibers have amino groups on aligned nanofibrous scaffolds was lower compared
on the surface of the scaffolds, thus improving the bio- with their random controls, and they explained that the
compatibility of scaffolds by providing ligands to sup- interconnected porosity of random nanofibrous scaf-
port the cell proliferation on PU/Coll scaffolds better folds promoted cell attachment and proliferation,
than on pure PU scaffolds. mainly because the aligned nanofibrous scaffolds had
The mechanical properties of the scaffolds play an poor porosity, which limit cell adhesion.12,48,49 In our
important role in tissue regeneration. Scaffolds suitable study, SMCs proliferation on random nanofibrous
for vascular TE should be soft and elastic enough to scaffolds was slightly higher than on their respective
reduce the local shear stresses. Compared to Dacron aligned scaffolds. The proliferation of SMCs indicated
(PET), the commonly used large-diameter vascular that the random and aligned PU/Coll (3:1) nanofibers
grafts with a tensile modulus of about 14,000 MPa,43 are more cell-compatible, less cytotoxic, and could be
the nanofibrous scaffolds fabricated in this study are more suitable as a vascular graft.
more compliant and they can be utilized as small diam- It was reported that the morphology and alignment
eter vessels such as the coronary arteries. In addition, of SMCs have a great influence towards the function of
the PU/Coll scaffolds are less stiffer (more compliant) arteries. In the native artery, the contractile forces pro-
than the PLCL nanofibers with a tensile modulus of duced by aligned SMCs adjust the constriction and
156 MPa utilized by Xu et al.44 as a vascular graft. dilation of blood vessels, while the alignment of
Hence, they are competent enough to endure the high SMCs and their ECM collectively regulate the move-
physiological pressure, and could be a better choice for ment and circumferential strength of the artery.14 The
constructing a completely biological and fully func- aligned nanofibrous scaffolds could promote the align-
tional coronary artery. Scaffolds suitable for vascular ment of SMCs through contact guidance, during this
TE should not only be soft and elastic enough to reduce study. However, the aligned PU/Coll scaffolds (Figure
the local shear stresses but also afford anisotropic 5F–H) supported greater cell alignment than aligned
mechanical properties to closely mimic the native PU scaffolds (Figure 5E), suggesting that the physical
artery. Aligned PU and PU/Coll nanofibrous scaffolds properties (morphologic patterns) and biochemical cues
showed anisotropic mechanical properties, and their (biological) of scaffolds can both influence the attach-
Young’s modulus and tensile strengths in PL direction ment and alignment of vascular cells. On the other
were significantly higher (p  0.05) than those of their hand, SMCs grown on random PU/Coll (3:1) expressed
corresponding values along the PP direction, thus a highly aligned phenotype. Previously, Blit et al.50
resembling the native artery. The tensile properties of reported that elastin-like polypeptide-4 cross-linked
native coronary artery and electrospun PU/Coll scaf- flat films helped to promote cell alignment, and they
folds are also summarized in Table 2. We found that explained that the biochemical composition could pro-
the electrospun random and aligned PU/Coll (3:1) scaf- mote the alignment of cells to a certain extent. Taken
folds had sufficient tensile strength and elastic modulus together, the aligned PU/Coll nanofibers not only pro-
to be utilized as a vascular graft with regard to the vided ligands for better cell proliferation but also facili-
tensile properties of native coronary artery. tated topographical cues for cell orientation and
Studies have suggested that SMCs seeded scaffolds elongation. Meanwhile, the random PU/Coll (3:1)
could improve the function of arteries through nanofibers provided mechanical support as well as

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12 Journal of Biomaterials Applications 0(0)

biological cues for SMCs alignment and elongation

(Figure 5B). Hence, the aligned morphological features
Conclusion
of SMCs on random and aligned PU/Coll (3:1) scaf- Alignment of SMCs is very important in controlling
folds appeared more similar to the phenotype and the constriction or dilation of blood vessels, while
behavior of SMCs in the native artery itself. scaffolds that promote the alignment of SMCs are
SMCs constitute an important component of blood more crucial for vascular regeneration. In this paper,
vessel, the medial layer of native artery is composed of electrospun PU and PU/Coll nanofibrous scaffolds in
several layers of SMCs in a matrix of collagen and two different morphologies (random and aligned) were
elastin.34 In this study, we chose two important mar- fabricated. Aligned nanofibrous scaffolds showed
kers (SMA and MHC) to determine the functionality anisotropic wetting performance and mechanical prop-
of SMCs grown on different scaffolds. SMCs on pure erties compared to their random counterparts. SMCs
PU scaffolds did not spread, and SMCs on hybrid cultured on aligned PU/Coll scaffolds displayed ori-
PU/Coll scaffolds attached and directed along the ented morphology with higher protein expression com-
orientation of nanofibers. In addition, electrospun pared to SMCs on aligned PU scaffolds. However, the
PU/Coll scaffolds appeared to have enhanced the random PU/Coll (3:1) nanofibers itself were able to
expression of SMA over pure PU scaffolds. Unlike induce SMC alignment, due to its superiority in mech-
the cells on TCP, SMCs on aligned and random anical properties and biochemical composition.
PU/Coll (3:1) expressed SMA with aligned microfila- Incorporation of 25% of collagen with PU was ade-
ment bundles of morphology. Similarly, SMCs on quate enough to fabricate a scaffold [PU/Coll (3:1)] to
random and aligned PU/Coll (3:1) nanofibers exhib- potentially serve as a substrate suitable for vascular
ited MHC expression with an aligned cell phenotype regeneration.
too. The positive expression of SMA and MHC on
PU/Coll nanofibers showed that the SMCs retained Acknowledgments
its functionality. Nivison-Smith et al.14 cultured The first author acknowledges the China Scholarship Council
SMCs on electrospun synthetic elastic fibers and for granting a scholarship that enabled her to pursue this
reported the expression of SMA filament. Similarly, work at NUS, Singapore
the expression of MHC on elastin-like polypeptide-
modified PU nanofibers were previously demonstrated Conflict of interest
by Blit et al.50 However, our studies showed strong, None declared.
uniform, aligned SMA and MHC expressions of
SMCs cultured on aligned and random PU/Coll Funding
(3:1) nanofibrous scaffolds. Taken together, our results This research is supported by the Singapore National
confirmed the biocompatibility of hybrid scaffolds, Research Foundation under CREATE programme: The
with especially the aligned and random PU/Coll Regenerative Medicine Initiative in Cardiac Restoration
(3:1) nanofibers, and had the ability to induce cell Therapy (NRF-Technion R-398-001-065-592) and
orientation and might serve as promising substrates Nanotechnology Initiative, Faculty of Engineering,
for vascular TE. National University of Singapore, Singapore. This work
The alignment of SMCs play a key role on the func- was also partly supported by grants (50973014 and
tionality of blood vessel, and an ideal scaffold for vas- 11172064) from the National Natural Science Foundation
cular TE should support the growth of SMCs and guide of China and from the Foundation for the Author of
National Excellent Doctoral Dissertation of P.R. China
the alignment of SMCs. During this study, we evalu-
(200961), as well as sponsored by Shanghai Rising-Star
ated the morphologies and functionalities of SMCs on Program in China (10QA1400100) and Fok Ying Tong
random and aligned PU/Coll nanofibrous scaffolds, Education Foundation (121071) to Prof. Xiaohong Qin. It
and observed that the SMCs cultured on random and was also supported by Program for New Century Excellent
aligned PU/Coll (3:1) possess aligned phenotype and Talents in University (NCET-10-0322) and the Fundamental
revealed oriented expression of proteins. These results Research Funds for the Central Universities as well as
indicated that apart from having aligned nanofibers, ‘ShuGuang’ (11SG33) project supported by Shanghai
the random fiber with appropriate biochemical com- Municipal Education Commission and Shanghai Education
position and suitable mechanical performance were Development Foundation.
capable enough to create SMC alignment. Two essen-
tial properties within hybrid PU/Coll scaffolds, namely
the ‘elasticity’ of PU combined with the ‘biocompati- References
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