Cell Tissue Bank (2021) 22:277–286

https://doi.org/10.1007/s10561-020-09876-7 (0123456789().,-volV)
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FULL LENGTH PAPER

Porcine carotid arteries decellularized

with a suitable concentration combination of Triton X-100
and sodium dodecyl sulfate for tissue engineering vascular
grafts
Zhiwen Cai . Yongquan Gu . Yonghao Xiao . Cong Wang . Zhonggao Wang

Received: 8 January 2020 / Revised: 14 October 2020 / Accepted: 16 October 2020 / Published online: 29 October 2020
Ó Springer Nature B.V. 2020

Abstract Tissue engineering vascular grafts
(TEVGs) constructed by decellularized arteries have
the potential to replace autologous blood vessels in
bypass surgery for patients with cardiovascular dis-
ease. There are various methods of decellularization
without a standard protocol. Detergents approaches
are simple, and easy control of experimental condi-
tions. Non-ionic detergent Triton X-100 and ionic
detergent sodium dodecyl sulfate (SDS) are the most
commonly used detergents. In this study, we used
Triton X-100 and SDS with different concentrations to
decellularize porcine carotid arteries. After that, we
investigated the acellular effect and mechanical prop-
erties of decellularized arteries to find a promising
concentration combination for decellularization.
Results showed that any detergents’ combination
would damage the inherent structure of extracellular
matrix, and the destruction increased with the increase
of detergents’ concentration. We concluded that the
decellularization approach of 0.5% Triton X-100 for
Z. Cai
Y. Gu (&)
C. Wang
Z. Wang (&)
Department of Vascular Surgery, Xuan Wu Hospital,
Capital Medical University, No. 45, Changchun Street,
Xicheng District, Beijing 100053, China
e-mail: yongquangu@126.com
Z. Wang
e-mail: Zhonggaowang37@163.com
Y. Xiao
School of Materials Science and Engineering, Beijing
Institute of Technology, Beijing 100081, China
24 h combined with 0.25% SDS for 72 h could help to
obtain decellularized arteries with minimum destruc-
tion. This protocol may be able to prepare a clinically
suitable vascular scaffold for TEVGs.
Keywords TEVGs
Decellularization
Triton
X-100
SDS
Introduction
Cardiovascular diseases are the leading cause of death
worldwide (Michele and Paolo 2018). Bypass grafting
is the most important treatment to reconstruct blood
flow. It was estimated that more than 1 million
vascular grafts are needed in the United States every
year (Schneider et al. 2016). Autologous blood vessels
are the most successful vascular conduits and the first
choice for grafts in bypass surgery. Unfortunately, up
to 40% of patients do not have feasible autologous
grafts due to previous diseases, operation history, and
inappropriate size (Salacinski et al. 2001). In order to
solve this problem, Weinberg and Bell (1986) first
created tissue engineering vascular grafts (TEVGs) for
bypass grafting. In recent years, many studies have
been done on materials for TEVGs (Mcclure et al.
2011; Fukunishi et al. 2017; Wang et al. 2017a, b). The
two main materials are synthetic polymers such as
poly-e-caprolactone (PCL) (Wang et al. 2014) and

123

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278
natural materials represented by decellularized arter-
ies (Porzionato et al. 2017). TEVGs made from
biodegradable polymer synthetic materials have short-
comings such as thrombosis, intimal hyperplasia, and
calcification (Gong et al. 2016). Decellularized
xenografts have their unique advantages because they
retain the original three-dimensional structures of the
blood vessels and maintain well-preserved mechanical
properties. Even more exciting, they possess molec-
ular cues such as growth factors that support the
migration of host cells and vascular remodeling
(Schneider et al. 2018). Nagaoka et al. (2014) created
decellularized vascular scaffolds from rat abdominal
arteries and implanted them in rat carotid arteries, and
the patency rate was 83.33% after 4 weeks. Amensag
et al. (2017) also demonstrated the possibility of small
artery reconstruction using decellularized scaffolds.
However, decellularized xenografts are still facing
some problems such as thrombosis, aneurysms, and
intimal hyperplasia. Rapid endothelialization and
vascular remodeling are the keys to solve these
problems. Many studies have applied surface modifi-
cation to decellularized arteries to promote endothe-
lialization (Zeng et al. 2012; Koobatian et al. 2016;
Kong et al. 2018).
Decellularization is aimed at the complete removal
of immunogenic cells from tissues while maintaining
the integrity of extracellular matrix (ECM). Decellu-
larization mainly includes physical, chemical, enzy-
matic methods, and their combinations, while there is
no standard protocol so far (Crapo et al. 2011). The
detergent treatments are simple, and easy control of
experimental conditions. Non-ionic detergent Triton
X-100 and ionic detergent sodium dodecyl sulfate
(SDS) are the most commonly used detergents. Triton
X-100 is relatively moderate, and its mechanism is to
destroy the lipid-lipid and lipid-protein interactions,
but not protein-protein (Seddon et al. 2004). It usually
needs to be combined with other methods of decellu-
larization to remove the cellular components com-
pletely. SDS could remove the cellular elements
effectively by dissolving cytoplasm and nucleus, but
it can also damage the cell matrix (Deeken et al. 2011).
Our previous studies combined SDS with Trixon-100
to prepare acellular tissue matrix and obtained
promising results (Cheng et al. 2019; Cai et al. 2019).
This study was designed to explore a promising
combination of Trixon-100 and SDS with a suit-
able concentration to prepare decellularized vascular
123
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Cell Tissue Bank (2021) 22:277–286
scaffolds. We used Triton X-100 and SDS with
different concentrations to decellularize porcine
carotid arteries. After that, we investigated the acel-
lular effect and mechanical properties of decellular-
ized arteries to find a promising concentration
combination for decellularization.
Materials and methods
Vascular harvesting
Fresh porcine carotid arteries about 8 cm long (inner
diameter of 2–5 mm) were obtained from a nearby
slaughterhouse (Beijing no. 5 meat factory). The
vessels were immersed in ice-cold phosphate buffered
saline (PBS) containing 100 U/ml penicillin and
100 g/l streptomycin and transferred to the laboratory
immediately. In the laboratory, the adherent loose
connective tissue was removed completely using
scalpel and scissors, and PBS was used repeatedly to
remove blood clots. After that, the arteries were stored
in PBS at - 20 °C for further use.
Decellularization
Native arteries were firstly washed with distilled water
on an orbital shaker (TS-100, Qilinbeier Instrument
Equipment Manufacturing Co. Ltd, Haimen, China)
(100 r/min) for 12 h to lyse the blood cells. The vessels
were then randomly divided into four groups (A, B, C,
D) for subsequent decellularization.
Group A was immersed into 1% Triton X-100
solution (Labest Biological Technology Co. Ltd,
Beijing, China) and kept oscillating for 24 h (100
r/min). After washing with PBS for 2 h, then
immersed into 1% SDS solution (Hanran Biological
Technology Co. Ltd, Shanghai, China) and continuous
shaking for 48 h. Group B was incubated in 1% Triton
X-100 solution for 24 h on the orbital shaker (100r/
min). After washing with PBS for 2 h, decellularized
in 0.5% SDS solution and oscillated for 60 h. Group C
was immersed into 1% Tritonx-100 solution and kept
oscillating for 24 h. After washing with PBS, then
immersed into 0.25% SDS solution and continuous
shaking for 72 h. Group D was treated with 0.5%
Tritonx-100 solution for 24 h and 0.25% SDS solution
for 72 h in sequence. After decellularization, all the
vessels were washed with PBS under orbital shaking
Cell Tissue Bank (2021) 22:277–286
for 72 h to remove the residual detergents and stored
at - 20 °C for future use.
Histological analysis
Samples were fixed in 4% Paraformaldehyde, embed-
ded them in paraffin, and cut into 5-lm sections. After
dewaxing and re-hydration, H&E and DAPI staining
were performed to confirm the absence of cellular
elements. Elastica Van Gieson (EVG) staining and
Masson’s trichrome staining were applied to visualize
elastin and collagen, respectively.
DNA quantification
To further determine whether the cellular components
were completely removed, the total amount of DNA in
native and decellularized arteries were quantified
using a DNeasy Blood & Tissue Kit (Qiagen, Venlo,
The Netherlands). According to the instructions pro-
vided, 20 mg dry weight samples of native and
decellularized arteries were digested using Proteinase
K at 56 °C until completely decomposed (n = 5 for
each group). Then, we added a protein precipitation
solution and centrifuged to remove the protein frac-
tion. After that, added isopropanol and ethanol to the
supernatant and then centrifuged and rehydrated to
isolate the DNA. Total DNA was finally quantified
using a spectrophotometer at 260 nm and expressed as
ng of DNA per mg of sample dry weight.
Scanning electron microscopy (SEM)
Native and decellularized arteries were cut into 5 mm
lengths and fixed in 2.5% glutaraldehyde for 2 h. After
washed with PBS three times for 10 min each, arteries
were immersed into a 1% osmium tetroxide solution
for 2 h and dehydrated in 50%, 70%, 90%, 100%
alcohol gradient for 10 min each. After that, put them
into hexamethyldisilazane (BASF SE, Ludwigshafen,
Germany) for 10 min and air-dried overnight. At last,
these samples were sputter-coated with gold and
examined utilizing the SU-8010 scanning electron
microscope (Hitachi, Tokyo, Japan).
Biomechanical properties
The stress–strain, suture strength, and burst pressure
tests were used to investigate the biomechanical
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279
properties of native and decellularized arteries. The
uniaxial tests including the longitudinal strength and
the circumferential strength were performed to mea-
sure the stress–strain. The longitudinal strength test
was carried out in the following steps: the arteries were
cut into 20 mm in length and 4 mm in width by a
custom-made die. Then use a digital micrometer to
measure the thickness of each sample. After clamping
at both ends with a uniaxial mechanical testing
machine (DLL-5000, Shanghai, China), the samples
were stretched at a rate of 20 mm/min until fracture.
The maximum strength, breaking strength, and the
elongation at break in the longitudinal were recorded.
When test the circumferential strength,these samples
were cut into 4 mm wide rings and each thickness was
measured. Then pass two triangular metal bars through
the circular specimen with the bottom side parallel,
and fix them on the upper and lower clamps of the
uniaxial mechanical testing machine respectively.
After that, the samples were stretched at a rate of
20 mm/min until fracture and recorded the circumfer-
ential strength.
When test the suture strength, the sample was cut
into 4mm 9 10 mm. One end was fixed on a cloth strip
with 2 stitches of 7–0 suture (Eethilon, ethicon, Inc.,
USA), the needle distance was 2 mm. The cloth strip
was fixed on the upper clamp of the testing machine.
Then clipped the other end on the lower clamp of the
testing machine. Stretched at a speed of 5 mm/min,
and the tension when the suture tore out the vessel was
the suture strength.
For the burst pressure test, a balloon connected to a
three-limb tube was inserted from one end of the
sample (4 cm long), and the other end was ligated and
sealed. The other two ends of the three-limb tube were
connected with a manometer and a small air com-
pressor respectively. Air was injected into the balloon
at a rate of 5 ml per minute. When the sample ruptured
and the balloon remained intact, the pressure on the
manometer was recorded as the burst strength.
Thrombosis test in vitro
Active partial thrombin time (APTT) was used to
reflect the samples’ thrombogenic characteristics.
Each sample was cut into 3 9 5 mm and put into a
four-hole colorimetric cup. Then added 50 ul standard
plasma with citric acid and 50 ll Pathromtin SL into
the cup, and preheated for 3 min at 37 °C. Finally,
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280
added 50 ll calcium chloride solution and the APTT
was measured through an automatic coagulation
analyzer (TEChrom IV plus).
Statistical analysis
Statistical analysis was performed by means of SPSS
(version 19; IBM, Armonk, NY, USA). Numeric data
were expressed as mean ± standard deviation. Single
comparisons between two groups were carried out
using the Student t test. One-way ANOVA analysis
was used to analyze multiple comparisons. The results
were considered statistically significant at P \ 0.05.
The bar chart was drawn by GraphPad Prism
(GraphPad Software, Inc., USA).
Results
Histology
The cellular components and nuclei of the native
arteries are clearly visible in H&E staining (Fig. 1a)
and DAPI staining (Fig. 1f), respectively. After
decellularization, H&E staining (Fig. 1b–e) showed
no residual cells in all groups, confirmed by DAPI
staining (Fig. 1g–j) which demonstrated the absence
of nuclei in the vascular matrix. EVG staining and
Masson’s trichrome staining showed elastic fiber
(black) and collagen (blue) (Fig. 1k–t) were well
preserved without severe disruption. However, the
density of collagen and elastic fibers in group A
(Fig. 1l, q) was lower than that in the other three
groups (Fig. 1m–o, r–t) which indicated that the
concentration combination of detergents in group A
was more disruptive than other groups.
DNA quantification results
As shown in Fig. 2, DNA content of acellular vessels
in all groups was significantly lower than that in native
arteries (Native:693.10 ± 19.78, A:28.61 ± 1.98,
B:32.26 ± 1.82, C:35.96 ± 1.14, D:39.38 ± 1.38 ng
DNA per mg dry weight, P \ 0.001).
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Cell Tissue Bank (2021) 22:277–286
SEM
SEM was used to observe the microstructure of the
samples. The lumen surface of native arteries was
covered with an intact layer of endothelial cells
(Fig. 3a), and the fibers in the vessel walls were tightly
connected (Fig. 3f). After decellularization, no resid-
ual cellular components were visible on the lumen
surface or in the vessel wall (Fig. 3b–e, g–j). However,
the basement membrane was more or less damaged
and the subintimal collagen fiber network was
exposed. Group A (Fig. 3b) showed the most obvious
collagen fiber exposure and the most pores, followed
by group B (Fig. 3c) and group C (Fig. 3d), and the
basement membrane of group D (Fig. 3e) was well
preserved. The vascular wall of decellularized arteries
became porous and loose, and it was the most obvious
in group A (Fig. 3g).
Biomechanical properties
The longitudinal maximum strength and breaking
strength of all decellularized vessels were weaker than
that of native arteries, among which, group A and
group B showed the most significant decrease, while
group D showed the least (Fig. 4a, b). The longitudinal
elongation at break of decellularized arteries in group
A and group B were significantly shorter than that of
native arteries, while group C and group D were
similar to native arteries, with the smallest difference
in group D (Fig. 4c). There was no significant
difference in the circumferential strength between
native and decellularized arteries in each group,
including the circumferential maximum strength
(Fig. 4d), the circumferential breaking strength
(Fig. 4e), and the circumferential elongation at break
(Fig. 4f).
There was a significant decrease in the suture
strength of decellularized arteries in group A and B,
while that of acellular arteries in group C and D was
little lower than native arteries with no significant
differences (Fig. 4g).
The burst pressure of acellular vessels decreased to
a certain degree in all groups, but there was no
statistical difference compared with that of native
arteries (Fig. 4h).
Cell Tissue Bank (2021) 22:277–286 281

H&E DAPI EVG Masson’s trichrome

Native
1%Triton X-100
Group A

1%SDS
1%Triton X-100
0.5%SDS
Group B
1%Triton X-100
0.25%SDS
Group C
0.5%Triton X-100
0.25%SDS
Group D

Fig. 1 H&E (a–e), DAPI (f, g, h, i, j), EVG (k–o) and Masson’s trichrome (p–t) of native (a, f, k, p), group A (b, g, l, q), group B (c, h,
m, r), group C (d, i, n, s) and group D arteries (e, j, o, t). (Color figure online)

APTT assay mammary artery is the best method at present, but

APTT is an important indicator of thrombosis in vitro.
As shown in Fig. 5, the APTT value of acellular
arteries in these four groups showed no significant
difference and was similar to that of native arteries.
Discussion
Cardiovascular diseases are still the most dangerous
killer threatening human health (Matsuzawa et al.
2015). Bypass grafting using a saphenous vein or
autologous blood vessels are not always feasible
(Pellegata et al. 2013). The continuous development of
TEVGs provides new approaches to solve this prob-
lem. Using decellularized arteries to prepare vascular
scaffolds for cell attachment, migration, and prolifer-
ation has become an attractive option in TEVGs
thanks to the specific ECM components and structure
(Ishino and Fujisato 2015).
Xenogeneic vascular grafts can cause an immune
response that leads to failure. It is necessary to remove
antigenic epitopes associated with cell membranes and
intracellular components of tissues effectively (Bady-
lak et al. 2009). Decellularization aims to remove cells

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282
Fig. 2 DNA content of the native, group A (1% Triton X-100
and 1% SDS), group B (1% Triton X-100 and 0.5% SDS), group
C (1% Triton X-100 and 0.25% SDS) and group D (0.5% Triton
X-100 and 0.25% SDS) arteries. ***P \ 0.001, significantly
different from the native arteries
and nuclear material while retaining three-dimen-
sional structure and protein components, reducing
damage to the matrix. There is still no consensus on
the method of decellularization. ECM can be destruc-
ted by physical methods, including freezing, pressure,
sonication, and agitation (Tebyanian et al. 2017).
Enzymatic approaches cannot be easy to control such
as proteases, nucleases, and calcium chelators.
Besides, enzymatic methods can also remove fibro-
nectin, laminin, and elastin, while these compositions
are important. But chemical approaches are simple
and cost-effective such as detergents, alkaline or
acidic solutions, and chelators. Detergents are com-
monly used for decellularization because they dissolve
cells and nuclear membranes and dissociate DNA
from proteins (Tuan-Mu et al. 2014). In this study,
non-ionic detergent Triton X-100 and ionic detergent
SDS were chosen to prepare acellular vascular grafts
because they are efficient and easy to obtain. The
reported concentrations of detergents vary widely
from 0.1 to 2% (Schneider et al. 2018). Mancuso et al.
(2014) used 1% SDS to remove nuclei and cellular
components completely, but the samples became
stiffer with a significant decrease in compliance. A
successful decellularization process can be guaranteed
by concentrations of 1% Triton X-100 and 0.5% SDS,
but the basement membrane on the lumen surface
suffered damage and the collagen fibers were exposed
(Crapo et al. 2011). Any acellular steps designed to
remove cells will damage or alter the inherent
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Cell Tissue Bank (2021) 22:277–286
structure of the ECM, thereby affecting the biome-
chanical properties of acellular blood vessels. (Fitz-
patrick et al. 2010). The balance between the complete
removal of cellular components and the best possible
preservation of the ECM remains studied. We inves-
tigated a comparison of Triton X-100 and SDS with
different concentrations after shaking for appropriate
time on decellularized arteries. 1% Triton X-100
followed by 1%, 0.5%, 0.25% SDS respectively and
0.5% Triton X-100 with 0.25% SDS were used to
prepare decellularized arteries. These concentrations
were commonly used and easy to prepare. We
conducted a series of pre-experiments to ensure that
the cells were completely removed within the appro-
priate time, and the timing of each step was easy to
manipulate.
The results of histology showed that cells and
nuclear materials could be completely removed in all
groups. Besides, the DNA content was significantly
reduced in all groups, less than 50 ng per mg dry
weight, which reached the decellularization standard
proposed by Crapo et al. (2011). According to EVG
staining and Masson’s trichrome staining, 1% SDS
had more significant destruction on the ECM than
0.25% SDS, despite the shorter treatment time. Under
SEM, we found that the endothelial cells on the inner
surface of blood vessels disappeared after decellular-
ization. The basement membrane damage and colla-
gen fiber network exposure were most obvious in
group A and least in group D. The basement
membrane plays an essential role in endothelial cell
adhesion and antithrombosis (Uzarski et al. 2013).
Therefore, the decellularized arteries in group A were
likely to lead to thrombosis and were not suitable for
TEVGs. Although there was no significant difference
in APTT between native and decellularized arteries in
each group, further study on thrombosis in vivo is
needed. Compared with native arteries, the vascular
walls of acellular arteries became loose and porous
which resist the arterial pressure in the body to prevent
aneurysms formation. The collagen and elastin in
group D suffered the least damage, and the pores
between the fibers were the smallest. Therefore, the
decellularized vascular scaffolds in group D may be
more suitable for TEVGs, and the combination of
Triton-X and SDS with lower concentration had
smaller damage to ECM.
Collagen provides strength and the collagen fibers
were mainly oriented circumferentially in the outer
Cell Tissue Bank (2021) 22:277–286 283

Fig. 3 SEM results of the Inner lumen Vascular wall

inner lumen of native (a),
group A (b), group B (c),
group C (d) and group D
arteries (e) and the vascular

Native
wall of native (f), group A
(g), group B (h), group C
(i) and group D arteries (j)

1%Triton X-100
Group A

1%SDS
1%Triton X-100
0.5%SDS
Group B
1%Triton X-100
0.25%SDS
Group C
0.5%Triton X-100
0.25%SDS
Group D

adventitia and media of arteries while the collagen strength of decellularized arteries was similar to native
fibers that distributed near the regions of internal and arteries in our study; this indicated that the collagen
external elastic lamina were mainly longitudinally arranged circumferentially remained intact. Good
oriented (Ghazanfari et al. 2012). The circumferential biomechanical properties of decellularized arteries

123

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284
Fig. 4 Biomechanical properties of the native, group A (1%
Triton X-100 and 1% SDS), group B (1% Triton X-100 and
0.5% SDS), group C (1% Triton X-100 and 0.25% SDS) and
are important to avoid aneurysms formation after
implantation. The biomechanical properties of the
samples in all groups were consistent with the results
of histology and SEM. By comparing the results of
group A, B and C, it can be found that despite the
longer treatment time, the lower the concentration of
detergent SDS, the greater the longitudinal maximum
strength and breaking strength. Compare with group C
and D, it predicted that when the concentration of
Triton-X decreased, the longitudinal tensile strength
of the samples also increased. Results of the longitu-
dinal elongation at break indicated the arteries in
group A and B became stiffer after SDS treatment with
a high concentration. The suture strength test was to
ensure that the grafts can be anastomosed without
123
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Cell Tissue Bank (2021) 22:277–286
group D (0.5% Triton X-100 and 0.25% SDS) arteries. *P \
0.05, significantly different from the native arteries
rupture. There was a significant decrease in the suture
strength of samples in group A and group B, resulting
in the grafts being easily torn during bypass surgery.
The burst pressure of decellularized arteries in all
groups was slightly lower than that of native arteries,
indicating that they can withstand blood flow (Wang
et al. 2017a, b). This finding was consistent with
several studies showing that the burst pressure does
not change significantly after decellularization (Gui
et al. 2009; Pellegata et al. 2013). In our study, the
arteries were stored at - 20 °C before and after
decellularization. This freezing step may damage the
vessels. In order to minimize its impact, repeated
freezing and thawing should be avoided, and the
arteries should not be frozen for a long time.
Cell Tissue Bank (2021) 22:277–286
Fig. 5 APTT of the native, group A (1% Triton X-100 and 1%
SDS), group B (1% Triton X-100 and 0.5% SDS), group C (1%
Triton X-100 and 0.25% SDS) and group D (0.5% Triton X-100
and 0.25% SDS) arteries. There was no significant difference
among the groups
Conclusions
The combined treatment of Triton X-100 and SDS can
decellularize porcine carotid arteries successfully
without residual cell components. Any combination
of detergent concentrations will damage or alter the
inherent structure of the ECM. The destruction of
collagen and elastin increased with the increase of
detergents’ concentration. The decellularization
approach of 0.5% Triton X-100 for 24 h combined
with 0.25% SDS for 72 h can help to obtain vascular
extracellular matrix and three-dimensional structure
with minimum destruction. This protocol may be able
to prepare a clinically suitable vascular scaffold for
TEVGs.
Acknowledgements This work was supported by the National
Key Research and Development Program of China (Grant No.
2017YFC1104100) and the Capital Health Research and
Development of Special, Beijing, China (Grant No. 2016-1-
2012).
Compliance with ethical standards
Conflict of interest All of the authors declare that they have
no conflicts of interest.
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