Heparin-modified small-diameter nanofibrous vascular grafts.
ABSTRACT Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter vascular grafts. Here we produced 1-mm diameter vascular grafts with nanofibrous structure via electrospinning, and successfully modified the nanofibers by the conjugation of heparin using di-amino-poly(ethylene glycol) (PEG) as a linker. Antithrombogenic activity of these heparin-modified scaffolds was confirmed in vitro. After 1 month implantation using a rat common carotid artery bypass model, heparin-modified grafts exhibited 85.7% patency, versus 57.1% patency of PEGylated grafts and 42.9% patency of untreated grafts. Post-explant analysis of patent grafts showed complete endothelialization of the lumen and neovascularization around the graft. Smooth muscle cells were found in the surrounding neo-tissue. In addition, greater cell infiltration was observed in heparin-modified grafts. These findings suggest heparin modification may play multiple roles in the function and remodeling of nanofibrous vascular grafts, by preventing thrombosis and maintaining patency, and by promoting cell infiltration into the three-dimensional nanofibrous structure for remodeling.
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ABSTRACT: Tissue-engineered vascular grafts (TEVGs) offer an alternative to synthetic grafts for the surgical treatment of atherosclerosis and congenital heart defects, and may improve graft patency and patient outcomes after implantation. Electrospinning is a versatile manufacturing process for the production of fibrous scaffolds. This review aims to investigate novel approaches undertaken to improve the design of electrospun scaffolds for TEVG development. The review describes how electrospinning can be adapted to produce aligned nanofibrous scaffolds used in vascular tissue engineering, while novel processes for improved performance of such scaffolds are examined and compared to evaluate their effectiveness and potential. By highlighting new drug delivery techniques and porogenic technologies, in addition to analyzing in vitro and in vivo testing of electrospun TEVGs, it is hoped that this review will provide guidance on how the next generation of electrospun vascular graft scaffolds will be designed and tested for the potential improvement of cardiovascular therapies.Expert Review of Cardiovascular Therapy 06/2014;
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ABSTRACT: Hemocompatibility of tissue-engineered vascular grafts remains a major hurdle to clinical utility for small-diameter grafts. Here we assessed the feasibility of using autologous blood outgrowth endothelial cells to create an endothelium via lumenal seeding on completely biological, decellularized engineered allografts prior to implantation in the sheep femoral artery. The 4-mm-diameter, 2- to 3-cm-long grafts were fabricated from fibrin gel remodeled into an aligned tissue tube in vitro by ovine dermal fibroblasts prior to decellularization. Decellularized grafts pre-seeded with blood outgrowth endothelial cells (n = 3) retained unprecedented (>95 %) monolayer coverage 1 h post-implantation and had greater endothelial coverage, smaller wall thickness, and more basement membrane after 9-week implantation, including a final week without anti-coagulation therapy, compared with contralateral non-seeded controls. These results support the use of autologous blood outgrowth endothelial cells as a viable source of endothelial cells for creating an endothelium with biological function on decellularized engineered allografts made from fibroblast-remodeled fibrin.Journal of Cardiovascular Translational Research 01/2014; · 3.06 Impact Factor
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ABSTRACT: Silk fibroin (SF) scaffolds have been designed and fabricated for multiple organ engineering owing to the remarkable mechanical property, excellent biocompatibility and biodegradability, as well as its low immunogenicity. In this study, an easy-to-adopt and mild approach based on modified freeze-drying method was developed to fabricate a highly interconnected porous SF scaffold. Physical properties of the SF scaffold, including pore morphology, pore size, porosity and compressive modulus could be adjusted by the amount of added ethanol, freezing temperature and the concentration of SF. Fourier transform infrared (FTIR) illustrated that treatment of the lyophilized scaffolds with 90% methanol led to a structure transition of SF from silk I (random coil) to silk II (beta-sheet) which stabilized the SF scaffolds in water. We also incorporated heparin during fabrication to obtain a heparin-loaded scaffold which possessed excellent anticoagulant property. Heparin which was incorporated in SF scaffolds could be released in a sustain manner for approximately 7 days, inhibiting in vitro human smooth muscle cells (hSMCs) proliferation within the scaffold while promoting neovascularization in vivo. We therefore propose that the SF porous scaffold fabricated here may be an attractive candidate to be used as potential vascular graft for implantation based on its high porosity, excellent blood compatibility and mild fabrication process.Acta biomaterialia 01/2014; · 5.68 Impact Factor
22IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 11, NO. 1, MARCH 2012
Heparin-Modified Small-Diameter Nanofibrous
Randall Raphael R. Janairo, Jeffrey J. D. Henry, Benjamin Li-Ping Lee, Craig K. Hashi, Nikita Derugin,
Randall Lee, and Song Li*
Abstract—Due to high incidence of vascular bypass procedures,
an unmet need for suitable vessel replacements exists, especially
vascular grafts. Here we produced
1-mm diameter vascular grafts with nanofibrous structure via
electrospinning, and successfully modified the nanofibers by the
as a linker. Antithrombogenic activity of these heparin-modified
scaffolds was confirmed in vitro. After 1 month implantation using
a rat common carotid artery bypass model, heparin-modified
grafts exhibited 85.7% patency, versus 57.1% patency of PEGy-
lated grafts and 42.9% patency of untreated grafts. Post-explant
analysis of patent grafts showed complete endothelialization
of the lumen and neovascularization around the graft. Smooth
muscle cells were found in the surrounding neo-tissue. In addition,
greater cell infiltration was observed in heparin-modified grafts.
These findings suggest heparin modification may play multiple
roles in the function and remodeling of nanofibrous vascular
grafts, by preventing thrombosis and maintaining patency, and by
promoting cell infiltration into the three-dimensional nanofibrous
structure for remodeling.
Index Terms—Biomaterials, heparin, nanofibers, small diam-
eter, vascular graft.
unmet need is the suitable small-diameter vascular graft.
Small-diameter synthetic vascular grafts made of poly(ethylene
terephthalate), expanded poly(tetrafluoroethylene) (ePTFE),
URRENTLY, over half a million of bypass procedures
are performed annually to treat vascular diseases. An
Manuscript received December 04, 2010; revised June 29, 2011; accepted
November 09, 2011. Date of current version March 13, 2012. This work was
supported in part by National Institutes of Health Grants HL 078534 (to S.
Li), HL 083900 (to S. Li), HL 941622 (to J. J. D.Henry), and Training Grant
GM56847 from National Institute of General Medicine Sciences—Initiative for
Maximizing Student Development (NIGMS-IMSD) (to R. R. R. Janairo). As-
terisk indicates corresponding author.
R. R. R. Janairo, J. J. D. Henry, B. L.-P. Lee, and C. K. Hashi are with the
Department of Bioengineering, University of California, Berkeley, CA 94720
USA, and also with the UC Berkeley and UCSF Bioengineering Graduate Pro-
N. Derugin is with the Department of Neurological Surgery, University of
California, San Francisco, CA 94143.
R. Lee is with the UC Berkeley and UCSF Bioengineering Graduate Pro-
gram, and also with the University of California, Department of Medicine, San
Francisco, CA 94143 USA.
*S. Li is with the Department of Bioengineering, University of California,
Graduate Program and Department of Bioengineering, University of California,
Berkeley, CA 94720-1762 USA (e-mail: email@example.com).
Color versions of one or more of the figures in this paper are available online
Digital Object Identifier 10.1109/TNB.2012.2188926
and polyurethane have high clogging rate due to thrombus for-
mation; autologous grafts have lower failure rate, but suitable
autologousveins or arteriesarenot alwaysavailable for patients
, . Thus, much research has been focused on developing
small-diameter vascular grafts with patency that can match or
outperform that of transplanted native vessels.
An ideal vascular graft should have characteristics including
compliance similar to native vessels, and capability of self-re-
modeling. Researchers have attempted to address these ideal
properties by developing tissue-engineered cellular vessels
using vascular cells – as well as bone marrow cells
–. Cellular grafts, albeit successful in exhibiting many of
the same properties as autologous grafts, need a long period to
fabricate and require significant amounts of effort for storage,
shipping, and handling. To address this shortcoming, research
in the past 20 years has employed decellularized native matrix
as well as synthetic materials to develop vascular replace-
ments –. Recently, many research groups have utilized
electrospinning to produce fibrous scaffolds for vascular regen-
eration , , –. These scaffolds consist of fibers
that mimic the porous micro- and nanostructure of collagen
and elastin fibers, the major native extracellular matrix (ECM)
components in the blood vessel wall.
Matrix mimicry is not sufficient to prevent graft failure;
the luminal surface must be made nonthrombogenic, either by
cell seeding or surface modification. Heparin has been used
extensively in vascular therapies because of its strong antico-
agulant ability. In addition, heparin has been shown to prevent
thrombosis on synthetic surfaces, such as ePTFE, Gore-Tex,
polyurethane, as well as decellularized xenogenic tissue ,
–. However, the effects of immobilized heparin on
electrospun nanofibers are not well understood. In this study,
we investigate the effects of heparin-modified electrospun
nanofibers on graft patency and cell infiltration.
A. Electrospinning of Nanofibrous Vascular Graft Scaffold
Poly(L-lactide) (PLLA) (Lactel Absorbable Polymers,
Pelham, AL, 1.09 dL/g inherent viscosity) was dissolved in
1,1,1,3,3-hexafluoro-2-propanol (HFIP) at 20% (w/v) concen-
tration. Electrospinning was done as previously described with
minor modification . Briefly, a voltage of 4.5 kilovolts (kV)
was applied via high voltage generator (Gamma High Voltage,
Ormond Beach, FL) to a rotating stainless steel mandrel (1-mm
1536-1241/$31.00 © 2012 IEEE
JANAIRO et al.: HEPARIN-MODIFIED SMALL-DIAMETER NANOFIBROUS VASCULAR GRAFTS23
Fig. 1. Structure of nanofibrous vascular grafts. (A) Scanning electron micro-
graphofthe electrospun,nanofibrous vasculargraft.
Scanning electron micrograph of the highly porous surface of the electrospun
diameter; 75 revolutions per minute) and a positive voltage
of 4 kV was applied to a spinneret. Electrospinning continued
until scaffold wall thickness reached approximately 200
The resulting scaffold was then removed from the mandrel and
placed into a vacuum desiccator for 24 h to remove any residual
HFIP. Bulk scaffold and nanofiber quality and dimensions were
inspected using a Hitachi S-5000 scanning electron micro-
scope. The bulk scaffold was cut into 7 mm length segments,
sterilized in 70% isopropanol under germicidal ultraviolet light
for 30 min, and washed three times with phosphate buffer
B. Chemical Modification of Nanofibrous Vascular Graft
Heparin modification of nanofibers was accomplished
by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(sulfo-NHS) (Pierce Biotechnology, Rockford, IL) with
di-amino-poly(ethylene glycol) as a linker . A group of
grafts were also PEGylated, using EDC and sulfo-NHS to
conjugate only di-amino-poly(ethylene glycol) (PEG) to the
nanofibers. The presence of heparin on the nanofibrous scaffold
was verified and measured by using toluidine blue (Sigma
Aldrich, St. Louis, MO) as described previously . Briefly,
untreated, PEGylated, and heparin-modified nanofibrous scaf-
folds were placed in 0.0005% (w/v) Toluidine Blue solution
and vortexed for 10 min. Standard heparin solutions ranging
to 250 were also prepared in 0.0005%
(w/v) toluidine blue solution and vortexed for 10 min. After
vortexing, 3 ml of n-hexane was added to all samples and
standard heparin solutions to extract the remaining unbound
toluidine blue. To determine the concentration of remaining
toluidine blue, the absorbance was measured at 631 nm using
a spectrophotometer (BioRad, Model 550). The concentration
of immobilized heparin on each nanofibrous scaffold sample
was thus determined by comparing their absorbance values
of unbound Toluidine Blue to those obtained for the standard
Fig. 2. Antithrombogenic activity of immobilized heparin (with PEG linker).
(A) Heparin was conjugated to the ester groups on PLLA via PEG linker. (B)
Heparin conjugation significantly increased antithrombogenic activity of the
nanofibrous material. The inhibition of thrombin activity in the presence of an-
tithrombin-III was quantified (
significant difference (
) compared to other groups.
for all groups). * indicates statistically
C. Antithrombogenic Activity of Heparin-Conjugated
The antithrombogenic activity of heparin-conjugated grafts
was determined by measuring the level of thrombin inhibition
in the presence of antithrombin-III. As described previously
, the chromogenic substrate for thrombin, S-2238 (Di-
apharma, West Chester, OH), was used to detect thrombin
activity. Briefly, untreated, PEGylated, and heparin-modified
grafts were placed in a 50 mM Tris buffer containing 0.08
NIH units of human antithrombin-III (Sigma Aldrich, St.
Louis, MO), and shaken for 5 min at 37
containing 0 to 400 units of heparin were similarly prepared
in Tris buffer also containing 0.08 NIH units of human an-
tithrombin-III. Afterwards, 0.08 NIH units of human thrombin
(Sigma Aldrich, St. Louis, MO) were added to each sample
and standard solution, which were subsequently shaken for 30
s at 37
. Following thrombin addition, 5 mM S-2238 was
added to each sample/solution, which was then shaken for 10
min at 37
. All reactions were stopped by the addition of
40% acetic acid. Remaining thrombin activity in the solutions
was assessed by measuring the absorbance of the sample
supernatants and standard solutions at 405 nm (BioRad, Model
550). The heparin activity for all nanofibrous samples was
determined by comparing absorbance values to those obtained
for the standard heparin solutions.
. Standard solutions
D. Animal Studies
All procedures were approved by the Institutional Re-
view Board Service and Institutional Animal Care and Use
24IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 11, NO. 1, MARCH 2012
Fig. 3. Cross sections of the explanted untreated grafts [(A), (B), (C)], PEGylated grafts [(D), (E), (F)], and heparin-modified grafts [(G), (H), (I)] at 1 month
post-implantation. Hematoxylin staining was performed.
in A; in C.
Committee at the University of California, San Francisco,
and University of California, Berkeley. Animal studies were
performed as previously described . Untreated
, and Heparin-modified grafts
implanted into rats. Briefly, Sprague Dawley rats (200–240
grams) (Charles River) were anesthetized with 2.0% isoflu-
rane, placed in the supine position, and had a vascular graft
sutured end-to-end to the ligated left common carotid artery.
No additional heparin or other anti-coagulant was used at any
time during these animal studies. After 1 month post-operative
procedure, the rats were euthanized via
followed by bilateral thoracotomy, and the vascular grafts
were explanted and washed with heparinized saline to remove
E. Histological Analysis
Samples were cryopreserved at
Temperature compound (Sigma Aldrich) and subsequently
cryosectioned at 10-
thicknesses in the cross-sectional plane
of the grafts. Immunohistochemical staining was performed to
analyze the presence of CD31 (BD Biosciences, San Jose, CA)
and smooth muscle-myosin heavy chain (SM-MHC) (Santa
Cruz Biotechnology) in the tissue sections. Hematoxylin and
DAPI stains were also performed to visualize cell presence
within the graft sections. Images of all aforementioned stains
were captured with a Zeiss Axioskop 2 MOT microscope.
in Optimal Cutting
F. Statistical Analysis
The data are represented as
data were compared by using one-way ANOVA tests. Holm’s t
test was then performed to evaluate significant differences be-
tween pairs. A p-value less than 0.05 was considered statisti-
A. Electrospinning of Nanofibrous Grafts That Mimic Native
Matrix Fiber Structure
We successfully made nonwoven, nanofibrous conduits that
were suitable for use as vascular graft scaffolds [Fig. 1(A)]. The
electrospun PLLA nanofibrous scaffolds exhibited a structure
similar to native matrix, marked by the fiber morphology and
porous structure [Fig. 1(B)]. The range of fiber diameter was
200 nm to 3
, and the average diameter of the fibers was
approximately 700 nm.
B. Heparin Modification of Nanofibrous Vascular Graft
The conjugation scheme is shown in Fig. 2(A). According to
the assays performed with toluidine blue, heparin modification
heparin (with PEG linker) conjugated to the
C. Heparin-Conjugated Nanofibrous Scaffolds Exhibit
To detect the antithrombogenic activity of immobilized hep-
arin, the inhibition of thrombin activity in the presence of an-
tithrombin-III was quantified. Untreated, PEGylated and hep-
arin-modified grafts exhibited activity levels of 0.24, 0.9, and
, respectively [Fig. 2(B)], confirming that
heparin was successfully immobilized and retained significant
D. Heparin-Modified Grafts Significantly Improved Patency
Rates In Vivo
Patency for untreated, PEGylated, and heparin-modified
grafts were determined after 1 month post-operative procedure
by confirming unobstructed blood flow through the graft into
JANAIRO et al.: HEPARIN-MODIFIED SMALL-DIAMETER NANOFIBROUS VASCULAR GRAFTS 25
Fig. 4. Cross sections of the explanted untreated grafts (A), PEGylated grafts (B), and heparin-modified grafts (C) at 1 month post-implantation were stained with
DAPI dye to label the cells, and cell number was quantified (D). The dotted lines in (A)–(C) indicate the boundary between neo-tissue (left) and the graft (right).
. indicates statistically significant differencecompared to other groups.
the distally attached native carotid artery prior to explantation.
After 1 month, 3 out of 7 (42.9%) untreated grafts were patent,
4 of 7 (57.1%) PEGylated grafts were patent, and 6 of the 7
(85.7%) heparin-modified grafts were patent. Fig. 3 shows
the examples of patent and unpatent grafts in each group. The
histological results of the patent grafts of the three groups
(untreated, PEGylated, and heparin-modified) showed very
interesting similarities and trends. For the patent grafts, the
minimal thrombosis and/or intimal hyperplasia was consis-
tent throughout each group after 1 month. The hematoxylin
stain indicated that neo-tissues form around the vascular
grafts, and there were some cell infiltration into the scaffolds.
[Fig. 3(C), (F), and (I)].
E. The Effects of Heparin Modification on Cell Infiltration
stained by DAPI (Fig. 4). Cell infiltration had occurred by 1
month, but to different extents in each treatment group. Hep-
arin-modified grafts significantly promoted cell infiltration into
the grafts, suggesting the additional beneficial effects of heparin
F. Endothelialization and Smooth Muscle Cell (SMC)
After one month, complete endothelialization was observed
on the luminal surface of the patent grafts from all three groups
[Fig. 5(A), (C), and (E)]. In the neo-tissue, angiogenesis was
observed, as indicated by the endothelialized microvasculature
in the outer partof each graft [Fig.5(A),(C),and (E)].Thisneo-
microvasculature suggested the integration of the grafts. SMC
staining showed that SMCs were the major cell type in neo-
tissues around the grafts [Fig. 4(B), (D), and (F)].
In this study, we developed an electrospun, nanofibrous,
small-diameter vascular graft scaffold that exhibited excellent
performance when chemically modified with heparin. These
modified acellular grafts showed superior patency rate as well
as significantly enhanced cell infiltration after 1 month in vivo.
As expected, heparin showed obvious prevention of throm-
bosis and occlusion when conjugated to nanofibrous scaffolds.
In vitro assessment of the heparin-modified scaffold confirmed
that conjugated heparin maintained its antithrombogenic ac-
tivity. What is more revealing was the additional advantage
heparin provided in significantly promoting cell infiltration
into the graft. Although further characterization is needed
to identify the type of infiltrated cells, we speculate that the
cells could primarily be vascular stem cells, progenitor cells,
fibroblasts, or macrophages. In the case of macrophages, some
populations have been reported to promote a tissue remodeling
response . Thus, an increase in cellular penetration into the
scaffold might in turn promote quicker remodeling and faster
Heparin modification prevented thrombosis and occlusion
that would have delayed or prevented endothelialization of the
graft lumen. Furthermore, since heparin has a high binding
affinity to many growth factors in the circulating blood, e.g.,
vascular endothelial growth factor (VEGF), it may serve as a
26IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 11, NO. 1, MARCH 2012
Fig. 5. Endothelialization and SMCs in implanted vascular grafts after
1 month. ECs were stained by using CD31 antibody [(A), (C), (E)]
and SMCs were stained by using SM-MHC antibody [(B), (D), (F)].
substrate for VEGF binding and thus promote endothelial cell
(EC) proliferation and migration.
These results suggest that heparin in conjunction with nanofi-
brous scaffolds may result in superior vascular grafts to other
previously reported chemically modified scaffolds. Previous re-
when modified by the potent thrombin inhibitor hirudin .
However, the cell infiltration exhibited after 1 month and even 6
and cell infiltration in long-term studies, as well as their cell in-
filtration rates in vitro. Additionally, although heparin coating
has already been shown to improve patency of small-diameter
grafts , –, we have been able to amplify its benefit
by using it in combination with a nanofibrous scaffold, in which
heparin is immobilized onto nanofibers on the surface and in the
wall of the grafts. Electrospinning of materials like PLLA in-
creases surface-to-area ratio and porosity of scaffolds, allowing
for an increase in cellular infiltration and remodeling in vivo.
Heparin magnifies this advantage of electrospun scaffolds by
providing some cellular cues or a more favorable environment
for cells within the graft, perhaps via its immense potential to
immobilize anticoagulants, growth factors, and matrix proteins.
Electrospun PLLA also supplies a slowly degrading scaffold
that can provide sufficient mechanical stability to allow for re-
generation of ultimately self-supporting native vessels. How-
ever, such a slowly degrading material may not be necessary,
since heparin may promote faster native matrix deposition and
remodeling within the graft. Thus, it will be beneficial to opti-
mize the degradation rate of the polymer to achieve a balanced
polymer degradation and matrix synthesis during blood vessel
It will also be beneficial to explore how the organization of
the nanofibers, e.g., alignment of electrospun nanofibers, can
contribute to endothelialization, cell infiltration, vascular graft
patency, and native blood vessel regeneration, since it has al-
ready been shown to improve dermal wound healing in con-
junction with heparin coating .
The use of synthetic, biocompatible, bioabsorbable materials
like PLLA to produce matrix-mimicking nanofibrous scaffolds
not only addresses mechanical stability and graft remodeling
issues, but also the immunogenic and ethical concerns asso-
ciated with decellularized xenogeneic tissue-based grafts. Fur-
thermore, the electrospinning process has the potential to repro-
ducibly scale up the fabrication of high quality vascular grafts.
Chemical modification with heparin can be performed quickly
and easily to provide a stable antithrombogenic surface. Taken
together, heparin-modified, small-diameter vascular grafts may
provide a superior off-the-shelf option for bypass procedures.
Theauthorswould liketothank HenryLiuand DavidSchultz
for their technical assistance.
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