Three-dimensional electrospun poly(lactide-co-ɛ-caprolactone) for small-diameter vascular grafts.

Abstract

Nanofibers have been applied to tissue engineering scaffolds because fiber diameters are of the same scale as the physical structure of protein fibrils in the native extracellular matrix. In this study, we utilized cell matrix engineering combined with cell sheet matrix and electrospinning technologies. We studied small-diameter vascular grafts in vitro by seeding smooth muscle cells onto electrospun poly(lactide-co-ɛ-caprolactone) (PLCL) scaffolds, culturing and constructing a three-dimensional network. The vascular grafts constructed using cell matrix engineering were similar to the native vessels in their mechanical properties, such as tensile strength, tensile strain, and e-modulus. Also, they had a self-sealing property more improved than GORE-TEX because PLCL has compatible elasticity. Small-diameter vascular grafts constructed using matrix engineering have the potential to be suitable for vascular grafts.

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Three-Dimensional Electrospun
Poly(Lactide-Co-e-Caprolactone)
for Small-Diameter Vascular Grafts
Cho Hay Mun, M.S.,1,2Youngmee Jung, Ph.D.,1Sang-Heon Kim, Ph.D.,1Sun-Hee Lee, M.S.,1
Hee Chan Kim, Ph.D.,2Il Keun Kwon, Ph.D.,3and Soo Hyun Kim, Ph.D.1
Nanofibers have been applied to tissue engineering scaffolds because fiber diameters are of the same scale as the
physical structure of protein fibrils in the native extracellular matrix. In this study, we utilized cell matrix
engineering combined with cell sheet matrix and electrospinning technologies. We studied small-diameter
vascular grafts in vitro by seeding smooth muscle cells onto electrospun poly(lactide-co-e-caprolactone) (PLCL)
scaffolds, culturing and constructing a three-dimensional network. The vascular grafts constructed using cell
matrix engineering were similar to the native vessels in their mechanical properties, such as tensile strength,
tensile strain, and e-modulus. Also, they had a self-sealing property more improved than GORE-TEX because
PLCL has compatible elasticity. Small-diameter vascular grafts constructed using matrix engineering have the
potential to be suitable for vascular grafts.
Introduction
C
dedicated to improve the understanding of coronary artery
disease, the shortage of donors and the high cost of coronary
artery bypass procedures limit effective treatment.5–7Al-
though there have been some developments in large-
diameter artificial vascular grafts, much less success has been
reported using synthetic vascular grafts as replacement for
small-diameter blood vessels (inner diameter <5mm).8–11
Also, noncardiac bypass vascular procedures, such as arte-
riovenous fistulae for hemodialysis access, have not been
accomplished using tissue engineering.
Nanostructured materials offer a great potential for tissue
engineering, such as controlling the nanofiber diameter, ge-
ometry, and mechanical properties of biomaterials. Nanofi-
bers have been recently applied to tissue engineering
scaffolds because nanosized fiber diameters are of the same
scale as the physical structure of protein fibrils in the native
extracellular matrix (ECM).12,13Cells can attach and organize
easily around fibers that have smaller diameters.14In addi-
tion, the nanofibrous structure has a high surface area-
to-volume ratio and this may provide more surface area for
cell attachment and protein absorption. Recent studies have
shown that electrospun nanofibers of polymers and matrix
ardiovascular disease is the leading cause of death in
the United States.1–4Although many efforts have been
proteins allow adhesion, proliferation, and organized as-
sembly of cells in vitro.14–17
Native blood vessels consist of three distinct layers: in-
tima, media, and adventitia layers. The intima is composed
of a simple epithelium called endothelial cells. The media
contains numerous smooth muscle cells (SMCs) and the
adventitia layer is made up of several connective tissues. The
media layer in blood vessels plays an important role to
maintain the vessel structure and to withstand, regulate, and
endure blood pressure. Also, blood vessels are a dynamic
tissue with high elasticity and strength suited for the pres-
sure and flow of blood.9,11,18Having the right mechanical
properties, conferred mainly by the media layer of blood
vessels, is essential in the development of a functional tissue-
engineered blood vascular graft.
The nature of native blood vessels requires the artificial
blood vascular grafts to possess the right mechanical prop-
erties, such as sufficient elasticity and strength. Recently,
poly(lactide-co-e-caprolactone)
been applied as biomaterials for vascular grafts because they
have high elastic and biodegradable properties.10,19–22
In this study, we fabricated vascular scaffolds that pos-
sessed similar mechanical properties as native vessels and
combined several techniques, such as electrospinning (ELSP)
and cell matrix. We seeded SMCs on the two-dimensional
(2D) thin electrospun scaffold. The seeded SMCs grew and
(PLCL) copolymershave
1Division of Life and Health Sciences, Biomaterials Research Center, Korea Institute of Science and Technology, Seoul, Korea.
2Department of Biomedical Engineering, Seoul National University, Seoul, Korea.
3Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul,
Korea.
TISSUE ENGINEERING: Part A
Volume 18, Numbers 15 and 16, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2011.0695
1608

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formed a cell layer on the electrospun scaffold. After this
process, the firmly attached SMC layer on the electrospun
scaffold was rolled around, creating a tubular-shaped vas-
cular graft. The electrospun scaffold supported the SMC
layer as a frame to exert appropriately their mechanical
properties such as burst of strength. This approach was
designated as cell matrix engineering. We generated small-
diameter vascular grafts using cell matrix engineering in vitro
by seeding SMCs onto the electrospun scaffolds, culturing
them, and constructing a tubular-shaped vascular graft. We
investigated the feasibility of using an electrospun scaffold
with SMCs as a vascular graft by testing the biomechanical
properties and cell compatibility in vitro.
Materials and Methods
Scaffold preparation
Copolymerization of PLCL was prepared as previously
described. Briefly, PLCL (50:50) was polymerized in a
100mL glass ampoule containing L-lactide (100mmol) and e-
caprolactone (100mmol) at 150?C for 24h in the presence of
1,6-hexanediol (0.5mmol) and stannous octoate (1mmol) as
catalysts. The ampoule was sealed under vacuum after
purging three times with nitrogen at 90?C and heated to
170?C for 24h with stirring in an oil bath. After the reaction,
the obtained polymer was dissolved in chloroform and fil-
tered through a 4.5-mm-pore membrane filter. The polymer
was precipitated into an excess of methanol, filtered, and
dried under vacuum.
PLCL nanofibrous membranes were fabricated by ELSP.
The custom-designed ELSP machine consisted of a KD-200
syringe pump (KD Scientific), a high-voltage power supply
(Nano NC), and a rotating mandrel to collect the fibers. The
ELSP solution was prepared with 9wt% in 1,1,1,3,3,3-hexa-
fluoro-2-propanol (99+ %) (HIFP; Sigma-Aldrich). A positive
voltage (15kV) was applied to the polymer solution by the
high-voltage power supply. The polymer solution was de-
livered through a 22-gauge blunt-tip syringe needle at a
constant flow rate of 1mL/h using a syringe pump. The air
gap between the syringe tip and the mandrel was 18cm, and
the rotation rate was 500rpm.
Cell isolation and culture
SMCs were isolated from the descending aortas of male, 3-
week-old New Zealand white rabbits. After removing en-
dothelium, adventitia, fat, and connective tissue, the vascular
smooth muscle tissues were cut into pieces and incubated in
an enzymatic dissociation buffer under agitation for 90min
at 37?C. This buffer consisted of 0.125mg/mL elastase (Sigma-
Aldrich), 1.0mg/mL collagenase (CLS type I, 204units/mg;
Worthington Biochemical), 0.250mg/mL soybean trypsin in-
hibitor, and 2.0mg/mL crystallized bovine serum albumin
(Sigma-Aldrich). Following the complete dissolution of the
matrix, the resultant cell suspension was filtered through a
100-mm Nitex filter (Tetko) and centrifuged at 200 g for
5min. The pellet was resuspended in growth medium con-
sisting of Medium 199 (M199; Welgene) supplemented with
10% v/v fetal bovine serum (Gibco) and 100units/mL pen-
icillin and 0.1mg/mL streptomycin (Welgene). Isolated
SMCs were cultured in tissue culture flasks under a hu-
midified atmosphere of 5% CO2and 95% air at 37?C. The
culture medium was changed three times a week. SMCs
were used at passage 4.
Cell seeding and in vitro culture
Before SMCs were seeded on the electrospun PLCL scaf-
fold, 0.1% collagen type I was coated on the scaffold. SMCs
at passage 4 were seeded on the scaffold for 1 day for at-
tachment to occur and then the same process was repeated
for the other side of the scaffold. The density of cell seeding
was 104SMCs per 1cm2of scaffold (Fig. 1a). After the
seeding process, a 2D-shaped scaffold was rolled (Fig. 1b),
and then the flask was cultured. The nanofibrous sheet was
wrapped around a 4-mm-diameter mandrel and bound by
fibrin gel (Green Cross) to maintain the tubular shape (Fig.
1c). The medium was changed three times a week for 4
weeks.
Scanning electron microscopy
The morphology and surface topography of scaffolds and
tissue-engineered grafts were observed by scanning electron
microscopy (SEM; Hitachi) operated at 15kV. For the SEM
examination, cell-seeded scaffolds were fixed in 4% (v/v)
formaldehyde for 24h and then dehydrated using a graded
ethanol series and dried. The specimens were coated with
gold using a model IB3 sputter-coater (Eiko).
Tensile properties
A cell-matrix-engineered vascular graft cut from each graft
(5mm·10mm) was tested for tensile properties. The me-
chanical properties of the electrospun PLCL fiber sheets were
measured by a tensiometry using a model 5567 of instron
(TestResources) with a 10-N load cell number at a cross-head
speed of 10mm/min (n=3). Young’s modulus, tensile
strength, and elongation at break were obtained from the
stress–strain curves.
Needle-hole leakage comparison of the vascular grafts
A Gore-TEX vascular graft (W.L. Gore & Associates), an
ELSP-PLCL scaffold graft, and 8-week-old rabbit aorta were
compared for their self-sealing property. Grafts were tested
by an identical system that provided 100mmHg static water
pressure at ambient temperature. A needle puncture was
FIG. 1.
gram of the manufacture of
small-diameter vascular
grafts using cell matrix engi-
neering.
A schematic dia-
ELECTROSPUN PLCL SCAFFOLD FOR VASCULAR GRAFTS1609

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accomplished with a RENAX A.V. Fistula needle set (Sunder
Biomedical Tech). Two test cases were chosen: a single
puncture and a multiple puncture case. For a multiple
puncture case, two punctures were chosen, as it is the min-
imum puncture. After removal of the needle, leakage
through the resultant needle hole was measured for 1min.
Burst pressure strength
Grafts were mounted in a system custom designed for
pressurizing individual grafts to failure. They were pres-
surized with water, and the pressure was increased by 5kPa
to a maximum of 30kPa for 5min and maintained at 30kPa
for 30min. Then the pressure was gradually increased until
the grafts failed.
Cell proliferation and DNA quantification
For the in vitro cell adhesion and proliferation study, cell
suspensions (1·104SMCs/200mL) were seeded onto ELSP
PLCL sheet scaffolds, which were coated with type I col-
lagen (Sigma-Aldrich). Cell proliferation was measured at
days 1, 4, and 7 using the WST test (Dojindo Molecular
Technologies), which is based on the ability of living cells to
reduce a tetrazolium salt into a soluble colored formazan
product.
Viable cells in vascular grafts were determined by an
AccuPrep genomic DNA extraction kit (Bioneer). Briefly, the
scaffolds were freeze-dried for 3 days and 25mg DNA was
collected according to the manufacturer’s protocol for DNA
quantification. The DNA content was determined by a Na-
nodrop ND-1000 apparatus (Thermo Fisher Scientific).
Histology
Cell-matrix-engineered vascular grafts were harvested
after 1, 2, 3, and 4 weeks of incubation. The grafts were fixed
in 4% formalin for 24h for histologic analysis. The scaffolds
were processed in a tissue processor, embedded in paraffin,
and sectioned at a thickness of 5mm. Sections were depar-
affinized and stained with hematoxylin and eosin (H&E) to
stain the nucleus and cytoplasm. For the immunohisto-
chemical staining, the antibody against smooth muscle a-
actin (SMA, 1A4; DAKO) was used to identify SMCs. Also,
adhered cells were observed using a model TE2000-U fluo-
rescence microscope (Nikon) by staining with 4,6-diamino-2-
phenylindole-dihydrochloride (DAPI; Vector Laboratories).
Statistical analyses
Where applicable, all data are expressed as mean–standard
deviation. Student’s t-test and single-factor analysis of vari-
ance were used for parameter estimation and hypothesis
testing, with p<0.05 and p<0.01 considered as being statisti-
cally significant (*) and extremely significant (**), respectively.
Results
Scanning electron microscopy
Hexafluoro-2-propanol was used as a solvent to dissolve
PLCL. The 9wt% dissolved solution was electrospun with a
pumping rate of 1mL/h at 15kV. SEM of representative
electrospun PLCL fibers obtained by the cell matrix engi-
neering revealed a small-diameter vascular graft (Fig. 2a).
Fiber diameter determined by the image analysis of SEM
FIG. 2.
presentative image of a cell-matrix-engineered vascular graft. (b) Morphology of electrospinning sheet (·5000). (c) Cross-
section of a cell-matrix-engineered vascular graft (·150). (d) SMCs covering an outer surface of cell-matrix-engineered
vascular graft after 4 weeks (·1000). (e) SMCs covering an inner surface of cell-matrix-engineered vascular graft after 4
weeks (·3000). (f) SMCs covering an inner surface of cell-matrix-engineered vascular graft after 4 weeks (·5000). SMCs,
smooth muscle cells.
Scanning electron microscopic findings of a small-diameter vascular graft using cell matrix engineering. (a) Re-
1610MUN ET AL.

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images (Fig. 2b, c) was 1.05–0.23mm. Figure 2c shows a
cross-sectional SEM image of the vascular graft and the sheet
of ELSP with a thickness of 500–40 and 46–4mm, respec-
tively. SMCs (7.2·105) were seeded on both sides of a scaf-
fold coated by collagen type I. Attachment of SMCs on the
scaffolds was confirmed by SEM after 4 weeks (Fig. 2d–f).
Tensile properties
Tensile properties were analyzed by three values (i.e.,
stress at break, strain at break, and Young’s modulus; E-
modulus). These factors are important for assessing the
mechanical properties in the native environment in which
blood pressure exists. Small-diameter vascular grafts con-
structed using cell matrix engineering had similar tensile
properties as a rabbit aorta as a native vessel (Fig. 3). The
tensile strength values of small-diameter vascular grafts
were 1.91–0.56MPa (0 week), 2.09–0.2MPa (1 week),
2.38–0.53MPa (2 weeks), 3.16–0.4MPa (3 weeks), and
3.23–0.57MPa (4 weeks) at a strain 135%, 244%, 254%,
263%, and 270%, respectively (Fig. 3a, b). Also, the rabbit
aorta and GORE-TEX grafts were analyzed. The tensile
strength of the rabbit aorta was 2.61–0.4MPa at a strain of
86.7% and that of GORE-TEX was 14.03–0.72MPa at a strain
27.8% (Fig. 3a, b). The E-modulus of PLCL small-diameter
vascular grafts was lower than GORE-TEX: 0.85–0.14MPa
(0 week), 0.86–0.2MPa (1 week), 0.90–0.1MPa (2 weeks),
0.98–0.2MPa (3 weeks), and 1.2–0.3MPa (4 weeks) versus
31.61–4.76MPa (GORE-TEX), while it was higher than the
rabbit aorta, which had an E-modulus value of 0.72–0.1MPa
(Fig. 3c).
Needle-hole leakage comparison of the vascular grafts
The cell-matrix-engineered vascular graft that was made
using the ELSP technology, a GORE-TEX vascular graft, and
rabbit aorta were punctured by an A.V. fistula needle at
100mmHg static water pressure. Leakage of the rabbit aorta
was minimal for both puncture conditions: 0.48–0.05mL/
min for one-puncture condition and 1.0–0.03mL/min
for two-puncture condition (Fig. 4). The leakage of the con-
ventional GORE-TEX graft was 32.45–1.8mL/min for
one-puncture condition and 63–9.9mL/min for the two-
puncture condition, while the leakage of the engineered
vascular graft was 0.65–0.1mL/min and 1.18–0.1mL/min
for the one- and two-puncture conditions, respectively.
FIG. 3.
(a) Comparison of tensile strength; (b) tensile strain; (c) E-modulus.
Comparison of tensile properties of small-diameter vascular grafts, rabbit aorta, and GORE-TEX (RA: rabbit aorta).
ELECTROSPUN PLCL SCAFFOLD FOR VASCULAR GRAFTS1611

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Burst pressure strength
The burst pressure strength test was performed to identify
the maximum pressure that the scaffolds could endure be-
fore failure, and to determine whether the vascular graft
possessed adequate strength to endure the physiologic for-
ces. The burst pressure strength was enhanced with in-
creasing SMC culture time (Fig. 5). The burst pressure
strength at weeks 0, 1, 2, 3, and 4 was 604–4, 765–44,
809–44, and 933–22mmHg, respectively. When the burst
strength with a cell-matrix-engineered vascular graft using
cell matrix engineering was compared with the GORE-TEX
graft, the latter displayed a higher burst strength value at 4
weeks ofa small-diameter
933–22mmHg). The burst strength of the rabbit aorta was
the highest value among others at 1647–201mmHg.
vascular (1323–383 vs.
Cell proliferation and DNA quantification
The cell proliferation for up to 7 days for SMCs cultured
on PLCL ELSP scaffolds and tissue cell culture plates (TCPs)
is plotted in Figure 6. ELSP scaffolds supported better cell
proliferation than TCPs after 3 days. At 7 days, the viability
of SMCs on PLCL ELSP scaffold was significantly higher
than TCPs. As also seen in Figure 6, the number of viable
cells cultured on PLCL ELSP scaffolds was comparable to
that cultured on TCPs at 3 and 7 days. The DNA content was
measured to determine the growth quantification of SMCs
on small-diameter vascular grafts using PLCL cell matrix
engineering for up to 4 weeks. As seen from Figure 7, the
DNA content increased rapidly at week 1, while only a slight
increase was observed after 2 weeks. When the DNA con-
tents were compared with 0 week (1.4–0.1mg/mL), there
were significant differences after 1 week (4.2–0.6, 4.8–0.4,
5.1–0.2, and 5.6–0.3mg/mL, respectively, at 1, 2, 3, and 4
weeks) (p<0.01).
Histology
SMC nuclei and cytoplasm within the cell-matrix-
engineered vascular grafts were visualized by staining with
H&E. SMCs were present on each graft layer (Fig. 8a, b).
Immunochemical analysis showed that the formed tissue
stained positively for SMA, which indicates SMCs (Fig. 8c,
d). As shown in Figure 8c and 8d, SMCs were evident on
each layer of the scaffold, indicating SMC regeneration. To
examine the distribution of SMCs that adhered to small-
diameter,cell-matrix-engineered
staining was performed. DAPI-stained cells appeared as
bright blue spots on both sides of each layer of the vascular
grafts (Fig. 8e).
vasculargrafts,DAPI
Discussions
Electrospun nanofibrous scaffolds have several advan-
tages, such as an extremely high surface-to-volume ratio,
tunable porosity, and malleability to conform to a wide
range of sizes and shapes.8,23–25However, ELSP scaffolds
display some limitations as a means of culturing cells. Cells
have difficulty infiltrating into the electrospun scaffolds be-
cause they usually have nanosized pores.26–29Also, the cell
matrix usually does not have enough mechanical properties
to endure blood pressure as the artificial vascular grafts
FIG. 4.
ter vascular graft using cell matrix engineering and GORE-
TEX (p<0.01). PLCL, poly(lactide-co-e-caprolactone).
Needle-hole leakage comparison of a small-diame-
FIG. 5.
lar graft using cell matrix engineering and GORE-TEX
(*p<0.01).
Burst pressure strength of a small-diameter vascu-
FIG. 6.
scaffold and on TCPS. TCPS, tissue culture polystyrene.
Viability of SMCs cultured on PLCL electrospun
1612MUN ET AL.

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do.30–32To overcome difficulties associated with the nanos-
tructured scaffold and cell sheet matrix in this study, we
investigated the potential of adapting cell matrix engineering
based on ELSP scaffold as a fabrication method for vascular
scaffolds. In this approach, cell matrix engineering was
combined with cell matrix and electrospun scaffold. The 2D
electrospun PLCL scaffold could be seeded with more SMCs
than the tubular-shaped electrospun vascular grafts, because
there are different methods of scaffold preparation. Cells
were seeded on both sides of the 2D electrospun scaffold,
whose surface area is 144cm2, compared with the surface
area of 15.1cm2in cell-seeded area of three-dimensional (3D)
electrospun scaffold (common tubular scaffold: inner dia-
meter=4mm and length=60mm).
Cell-matrix-engineeredsmall-diameter
have been suitably fabricated. The scaffolds have several
favorable characteristics for the potential applications for
small-diameter vascular grafts, such as 3D-shaped blood-
vessel-like structure (Fig. 2a), biomechanical properties, and
in vitro biocompatibility. In this study, the small-diameter
vascular grafts were manufactured with a length of 6cm and
a thickness of 500–40mm (Fig. 2c). The surface of a small-
vasculargrafts
FIG. 7.
vascular graft using cell matrix engineering; SMCs were
seeded at a density of 1·104cells/cm2and cultured for short
term (1, 3, and 7 days) (*p<0.01).
DNA content of SMCs cultured on a small-diameter
FIG. 8.
tion of a small-diameter vas-
cular graft using cell matrix
engineering by H&E staining,
SMA, and DAPI staining. (a)
H&E staining of scaffold
cross-section cultured for 4
weeks (·200). (b) H&E stain-
ing of scaffold cross-section
cultured for 4 weeks (·400).
(c) SMA staining of scaffold
cross-section cultured for 4
weeks (·200). (d) SMA stain-
ing of scaffold cross-section
cultured for 4 weeks (·400).
(e) DAPI staining of scaffold
cross-section cultured for 4
weeks (·200). (f) DAPI
staining of scaffold cross-
section cultured for 4 weeks
(·400). H&E, hematoxylin
and eosin; SMA, smooth
muscle a-actin; DAPI, 4,6-
diamino-2-phenylindole-
dihydrochloride. Color
images available online at
www.liebertonline.com/tea
Histological evalua-
ELECTROSPUN PLCL SCAFFOLD FOR VASCULAR GRAFTS1613

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diameter vascular graft displayed an ECM-like matrix (Fig.
2b). This could provide a suitable environment to attach and
grow SMCs. After 4 weeks, we observed a confluent growth
of SMCs on the surface of vascular grafts by SEM (Fig. 2e, f).
The mechanical properties of PLCL cell-matrix-engineered
small-diameter vascular grafts are depicted in Figure 3.
Comparison of tensile properties between small-diameter
vascular grafts, rabbit aorta, and GORE-TEX included tensile
strength, tensile strain, and E-modulus. The tensile strength
and tensile strain were enhanced with increasing culture
time from 0 to 4 weeks in the small-diameter vascular grafts
(Fig. 3a, b).
The tensile strength of GORE-TEX was not comparable to
the rabbit aorta as a native vessel because the value of GORE-
TEX was seven times higher than that of the rabbit aorta.
Rather, the tensile strength of small-diameter vascular grafts
was comparable to that of the rabbit aorta (Fig. 3a). The tensile
strain results echoed those of the tensile strength. GORE-TEX
had the lowest tensile strain, indicating that it was stiff in
comparison to the rabbit aorta (Fig. 3b). The E-modulus value
was closely related to compliance because the inverse of
modulus is compliance in elastic materials like polymer.33,34
The E-modulus of small-diameter vascular grafts showed
a similar value to that of the rabbit aorta (Fig. 3c). We could
expect that the cell-matrix-engineered small-diameter vas-
cular grafts would have similar tensile properties to the
rabbit aorta, and this would cause less problems related to a
mechanical mismatch to native vessel in vivo.35–37
For some artificial vascular grafts, such as the A.V. Fistula,
which is used for dialysis, having a self-sealing property that
minimizes blood leakage following piercing is crucial.38–40
Self-sealing property of vascular grafts seems to depend on
the graft material elasticity. As seen in Figure 4, the small-
diameter vascular grafts showed a superior self-sealing
property than the GORE-TEX grafts. This was expected be-
cause of the elasticity of PLCL materials.19–22As shown in
Figure 3b, PLCL small-diameter vascular grafts were more
elastic than GORE-TEX grafts.
Figure 5 shows that the burst pressure strength increased
with SMC culture time. The burst pressure strength at 4
weeks in the PLCL small-diameter vascular scaffold rose to
933–22mmHg. Although it was less than the burst pressure
strength of the GORE-TEX grafts, it could be enough to en-
dure the physiological blood pressure.41–44The significant
improvement of the burst pressure strength of the PLCL
small-diameter vascular scaffolds confirmed the analysis of
cell proliferation and DNA quantification (Figs. 6 and 7). The
burst pressure strength increased about 35%, from 604–4 to
933–22mmHg, and DNA quantification was risen almost
400%, from 1.4–0.1 to 5.6–0.3mg/mL, from 0 to 4 weeks
(Figs. 5 and 7). The collective data indicate that SMCs on the
small-diameter vascular grafts grew and might have con-
ferred glue-like properties between each layer as the culture
period increased.
At less than 1 week of culture, the viability of SMCs on the
electrospun PLCL scaffolds was better than TCPs (Fig. 6).
There are several explanations. In this study, the vascular
scaffold was coated by type I collagen to improve cell-
seeding efficiency and growth on the scaffold. Collagen, one
of the main classes of structural ECM proteins, has excellent
cell-binding properties and cell compatibility, which makes it
a widely used biomaterial in the tissue engineering field.
Previous studies assessed the role of type I collagen, which is
the most important component of ECM in terms of amounts,
in cell growth, and function. The cell-seeding efficiency was
approximately twofold higher in collagen/SMC–incorpo-
rated scaffolds than in SMC-seeded scaffolds.19,44,45Electro-
spun nanofibers have a structure that is similar to the native
ECM, which is composed of nonoscaled protein fibrils, pro-
viding SMCs with a more familiar environment to attach and
grow on the scaffold.23,24,45–47H&E staining, SMA, actin, and
DAPI staining showed that the SMCs attached and grew on
the layers of the small-diameter vascular graft (Fig. 8). This
indicates that the electrospun cell matrix scaffold can support
long-term cell growth and proliferation.
Conclusion
Nanostructured materials have a tremendous potential for
tissue engineering. ELSP technology can be used to generate
nanofibrous scaffolds made of synthetic polymers or native
matrix molecules.
In this study, we pioneered an approach named cell ma-
trix engineering that combined the electrospun scaffold and
the cell matrix. We expected that the viable cell numbers on
the ELSP scaffold and mechanical strength properties of the
cell matrix could improve by cooperating. Usually, cells have
a difficulty infiltrating into the tubular-shaped electrospun
vascular grafts because of their nanosized pores. The cell-
matrix-engineered blood vascular grafts showed similar
mechanical properties as native vessels, such as tensile
strength, tensile strain, and E-modulus. Also, they had better
self-sealing property than the GORE-TEX grafts because
PLCL scaffold has higher elasticity.
We investigated the small-diameter vascular grafts in vitro
by seeding SMCs onto the electrospun scaffolds, cultured
them, and constructed a 3D vascular structure. We found
that the small-diameter vascular grafts constructed using the
cell matrix engineering have the potential to be used as ar-
tificial vascular grafts in tissue engineering.
Acknowledgment
This study was supported by a grant from the Korea
Healthcare Technology R&D Project, Ministry of Health &
Welfare (MOHW), Republic of Korea (A110962).
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Soo Hyun Kim, Ph.D.
Division of Life and Health Sciences
Biomaterial Research Center
Korea Institute of Science and Technology
39-1 Hawolgok-dong, Seongbook-Ku
Seoul 136-791
Korea
E-mail: soohkim@kist.re.kr
Received: December 08, 2011
Accepted: March 28, 2012
Online Publication Date: June 13, 2012
1616MUN ET AL.