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Cross-linking porcine peritoneum by oxidized konjac glucomannan: a novel method to improve the properties of cardiovascular substitute material

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The use of natural polysaccharide crosslinkers for decellularized matrices is an effective approach to prepare cardiovascular substitute materials. In this research, NaIO 4 was applied to oxidize konjac glucomannan to prepare the polysaccharide crosslinker oxidized konjac glucomannan (OKGM). The as-prepared crosslinker was then used to stabilize collagen-rich decellularized porcine peritoneum (DPP) to construct a cardiovascular substitute material (OKGM-fixed DPP). The results demonstrated that compared with GA-fixed DPP and GNP-fixed DPP, 3.75% OKGM [1:1.5 (KGM: NaIO 4 )]-fixed DPP demonstrated suitable mechanical properties, as well as good hemocompatibility, excellent anti-calcification capability, and anti-enzymolysis in vitro. Furthermore, 3.75% OKGM [1:1.5 (KGM: NaIO 4 )]-fixed DPP was suitable for vascular endothelial cell adhesion and rapid proliferation, and a single layer of endothelial cells was formed on the fifth day of culture. The in vivo experimental results also showed excellent histocompatibility. The current results demonstrted that OKGM was a novel polysaccharide cross-linking reagent for crosslinking natural tissues featured with rich collagen content, and 3.75% OKGM [1:1.5 (KGM: NaIO 4 )]-fixed DPP was a potential cardiovascular substitute material. Graphical Abstract
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Pengetal. Collagen and Leather (2023) 5:5
https://doi.org/10.1186/s42825-023-00114-w
RESEARCH
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Open Access
Collagen and Leather
Cross-linking porcine peritoneum
byoxidized konjac glucomannan: anovel
method toimprove theproperties
ofcardiovascular substitute material
Xu Peng1,2, Li Li3, Jiaqi Xing4, Can Cheng1, Mengyue Hu1, Yihao Luo1, Shubin Shi1, Yan Liu2, Zhihui Cui5 and
Xixun Yu1*
Abstract
The use of natural polysaccharide crosslinkers for decellularized matrices is an effective approach to prepare cardio-
vascular substitute materials. In this research, NaIO4 was applied to oxidize konjac glucomannan to prepare the poly-
saccharide crosslinker oxidized konjac glucomannan (OKGM). The as-prepared crosslinker was then used to stabilize
collagen-rich decellularized porcine peritoneum (DPP) to construct a cardiovascular substitute material (OKGM-fixed
DPP). The results demonstrated that compared with GA-fixed DPP and GNP-fixed DPP, 3.75% OKGM [1:1.5 (KGM:
NaIO4)]-fixed DPP demonstrated suitable mechanical properties, as well as good hemocompatibility, excellent
anti-calcification capability, and anti-enzymolysis in vitro. Furthermore, 3.75% OKGM [1:1.5 (KGM: NaIO4)]-fixed DPP
was suitable for vascular endothelial cell adhesion and rapid proliferation, and a single layer of endothelial cells was
formed on the fifth day of culture. The in vivo experimental results also showed excellent histocompatibility. The
current results demonstrted that OKGM was a novel polysaccharide cross-linking reagent for crosslinking natural
tissues featured with rich collagen content, and 3.75% OKGM [1:1.5 (KGM: NaIO4)]-fixed DPP was a potential cardiovas-
cular substitute material.
Keywords Sodium periodate, Oxidized KGM, Peritoneum, Collagen, Cardiovascular substitute material
*Correspondence:
Xixun Yu
yuxixun@163.com
Full list of author information is available at the end of the article
Page 2 of 17
Pengetal. Collagen and Leather (2023) 5:5
Graphical Abstract
1 Introduction
Heterogeneous decellularized tissues have been widely
used in the preparation of vascular grafts because their
construction is suitable for cell adhesion, prolifera-
tion, and migration. e products currently developed
include the Artegraft bovine common carotid artery
[1], ProCol bovine mesenteric vein [2, 3], and Syner-
graft bovine ureter [4]. However, some decellularized
tissues have not been used clinically, including the
human umbilical vein, porcine carotid artery, porcine
saphenous artery, and other acellular matrices. All the
decellularized scaffolds were rich in collagen and elas-
tin. Elastin is the main extracellular matrix component
of vascular tissues. Lack of elastin can lead to failure of
blood vessels, including aneurysms, thrombosis, and
intimal hyperplasia [58]. e peritoneum of the Bama
pig is also a tissue rich in elastin and is easier to obtain
than other tissues. e peritoneum, rich in blood cap-
illaries, lymphatic vessels, and nerves, exhibits flex-
ibility and a smooth surface. e peritoneum has the
same embryologic origin as blood vessels and a low risk
of thrombosis. Additionally, the Bama pig peritoneum
demonstrates desired permeability along with a certain
degree of regenerative properties, adequate mechanical
properties, and a thickness similar to that of the 2mm
diameter-natural vessels (300–500 μm). erefore, it
is suitable for use in preparing artificial vessels with
diameters of 2mm.
However, the direct application of porcine perito-
neum onto the construction of replacement grafts can
cause immune rejection. DPP can greatly reduce its
immunogenicity, but its poor mechanical properties
and stability in physiological environments prevent
its direct use or preservation. erefore, the pre-fix-
ation of these animal-derived decellularized tissues is
required to address these problems [9]. Glutaraldehyde
(GA), the most common crosslinking agent used for
pre-fixation, can enhance the biomechanical properties
of acellular tissues and improve their stability. However,
as many researchers have confirmed, the application of
Glutaric dialdehyde (GA) is limited by calcification and
cytotoxicity as well as the inability to crosslink elastin
Page 3 of 17
Pengetal. Collagen and Leather (2023) 5:5
[10]. Genipin (GNP) is an excellent, naturally occur-
ring crosslinking agent that has been developed in
recent years. Its cytotoxicity is lower than that of GA
alone. However, the color of tissues crosslinked by GNP
is dark blue, and the thickness of GNP-fixed tissues
increases excessively [11]. ese shortcomings restrict
thefurther application of GNP. ere is an urgent need
to find a suitable crosslinking agent to pre-fix acellular
tissues to improve their mechanical properties, further
reduce their antigenicity, and enhance their resistance
to enzymatic degradation while maintaining their origi-
nal ultrastructure.
Konjac glucomannan (KGM) is a high-molecular-
weight, water-soluble, non-ionic natural polysaccharide.
It exhibits good biocompatibility, biodegradability, and
excellent gel-forming properties and is used in cosmet-
ics, drug-controlled release materials, and plasma sub-
stitutes. KGM is linked by β-1,4 glycosidic bonds and
is rich in hydroxyl groups, providing opportunities for
this polysaccharide to be modified for other biomedi-
cal applications. e o-hydroxyl groups of KGM can be
selectively oxidized by sodium periodate (NaIO4) into
aldehyde groups, yielding oxidized KGM (OKGM) with
multiple functional aldehyde groups. OKGM can bridge
amine groups in heterogeneously derived acellular tissues
through reactive formyl-aldehyde groups to form a stable
cross-linking structure [1214].
is research evaluated various physical and chemical
properties of OKGM-fixed DPP, including its ultrastruc-
ture, mechanical properties, stability, cytocompatibil-
ity, hemocompatibility, invitro calcification, and invivo
inflammation. isresearch first provides an experimen-
tal basis for OKGM as a new cross-linking reagent for fix-
ing natural tissues featured with rich collagen content.
2 Materials andmethods
2.1 Preparation andcharacterization ofOKGM
After 5 g of KGM (K8170, Solarbio) was dissolved in
500mL distilled water, 7.5g of NaIO4 (S817518, Solar-
bio) was added to the KGM solution. e oxidation
reaction proceeded in the dark at room temperature for
48h. Subsequently, 20mL ethylene glycol was added to
the mixture to remove excess NaIO4. Crude OKGM was
obtained via precipitation with excess ethanol and suc-
tion filtration. Finally, the obtained OKGM was imme-
diately dialyzed (with a molecular weight cutoff of 3000)
with deionized water for 3 days and subsequently lyo-
philized using a vacuum freeze dryer to obtain purified
OKGM.
e aldehyde content of OKGM was determined by the
potentiometric titration of hydroxylamine hydrochloride.
e weight-average molecular weight (Mw), number-
average molecular weight (Mn), and polydispersity (Mw/
Mn) of OKGM were determined using aqueous gel per-
meation chromatography (Waters 1515). Zeta potential
(ζ) measurements of products were analyzed in a Mal-
vern Zeta sizer (Nano Model ZS90) at a constant tem-
perature of 25°C. For the FTIR measurement of OKGM,
4mg of dry samples and 40mg of KBr were pressed into
a disc. A Nicolet 560 infrared spectrometer was used to
measure the FTIR spectra at a resolution of 4 cm1 in the
wavenumber range of 400–4000 cm1.
2.2 Preparation ofDPP andxation index ofOKGM‑xed
DPP
efresh peritoneum was immersed in 1% Triton X-100/
deionized water and 0.5% SDS/deionized water and
shaken (60rpm) for 3h. Subsequently, the samples were
soaked while undergoing continuous magnetic mechani-
cal stirring for 3h. Finally, the samples were washed sev-
eral times with PBS to remove cellular remnants. DNase
I (200 U/mL) was used to treat samples at 37°C for 4h.
DPP was obtained using the decellularization process
described above.
DPPs were immersed in a series of OKGM/PBS solu-
tions with different OKGM concentrations (3.75, 7.5, and
15% m/v) to fix the samples. eDPPs were all fixed at
37°C for 24h in a constant temperature oscillator.
At predetermined times, all the OKGM-fixed DPPs
were lyophilized for at least 12h to a constant weight.
After heating the NHN solution to boiling for 20 min,
lyophilized tissues were removed, and the optical absorb-
ance of the solution was measured using an enzyme-
labeled instrument (Multiskan FC, ermo Fisher) at
570 nm. Glycine at various known concentrations was
used as the standard, and the fixation index (FI) was cal-
culated using the following formula:
2.3 Surface characterization ofsamples
Field-emission scanning electron microscopy (FE-SEM,
Oberkochen) was used to evaluate the surface topogra-
phies of the samples. e hydrophilicity of the surfaces
of the samples (lyophilized 2.5 cm × 2.5 cm) was char-
acterized by a water contact angle goniometer (WCA,
SL200KS).
2.4 Cytocompatibility andinvitro endothelialization
ofOKGM‑xed DPP
Human vascular endothelial cells (HVEC, purchased
from the Lab of Transplant Immunology West China
Hospital) were inoculated with 1 × 105 cells/mL on
Co-60 irradiated sterilized samples (2 cm × 2 cm) in
FI
%=
(NHNreactive amine)
fresh
(NHNreactive amine)
fixed
(NHNreactive amine)
fresh
Page 4 of 17
Pengetal. Collagen and Leather (2023) 5:5
6-well culture plates. DMEM (2000μL with 10% FBS)
was added after 30 min. e culture medium was
changed every two days. CCK8 (Cell Counting Kit-8,
Beyotime) assays were performed on days 1, 3, and 5.
Cell live/dead fluorescent staining (Calcein AM, Beyo-
time) was performed on day 2 to observe cell adhesion
and activity on the surface of the sample. SEM was used
to observe HVEC attachment morphology on sterilized
samples after incubation for 3days. After the fifth day
of culturing, the cell sample constructs were washed
with PBS, fixed with paraformaldehyde, and stained
with HE to observe the endothelialization of OKGM-
fixed DPP invitro.
2.5 Hemocompatibility measurement
2.5.1 Hemolytic test
Red blood cell (RBCs) separation 10mL of rabbit blood
anticoagulated with sodium citrate were added to 20mL
DPBS to ensure uniform dispersion. e RBCs were sep-
arated by centrifugation at 500 × g for 10min. ereaf-
ter, the RBCs were rinsed with DPBS several times and
re-suspended in 100mL DPBS solution. RBCs dispersed
in DPBS were used as negative controls, while those dis-
persed in distilled water were used as positive controls.
Gradient concentration hemolysis test All OKGM-fixed
DPP samples were ground to powders and dispersed in
PBS at gradient concentrations to form a suspension.
0.4mL of RBC solution was then added to 1.6mL sus-
pensions at each concentration soon afterward, and all
mixtures were incubated for 3h at 37°C.
Direct contact hemolysis test e sample was cut into
2cm × 2cm pieces and placed flat in a 6-well plate. en,
2mL of RBC suspension was added to each well, and the
samples were incubated for 3h at 37°C.
e suspension was centrifuged, and the absorbance of
the supernatant was measured at 540nm. e hemolysis
rate (%) was calculated as follows:
2.5.2 Static platelet adhesion test
Whole blood was centrifuged (1000 r/min for 10min) to
separate platelet-rich plasma (PRP). 400 μL of PRP was
dropwise added on the surface of DPP-samples, which
have been pre-immersed and equilibrated in PBS at 37°C
for at least 1h. en, they were incubated at 37 °C for
1h. ese DPP-samples were mildly washed with PBS to
Hemolysis rate
(
%
)
=(experimental groupnegative control group)
/(positive control groupnegative control group
)
×100
remove non-adherent platelets before being fixed with a
2.5% GA/PBS solution for 8h at 4°C. Finally, the DPP-
samples were dehydrated using gradient ethanol (50, 70,
85, 90, 95, and 100%) for 10min per concentration, and
the morphology and aggregation of the attached platelets
were observed using SEM.
2.5.3 Ex vivo blood contact test
e DPP-samples were cut into square patches
(4mm × 9 mm) and pre-immersed in PBS for 1h. e
patches were then attached to the internal walls of sili-
cone tubes (ID = 3.1mm) pre-soaked with heparin. Gen-
eral anesthesia of experimental rabbits weighing 3 kg
each was induced through the intravenous administration
of 10mg/kg pentobarbital sodium salt (Sigma, 57-33-0).
40μg/kg of the sedative dexmedetomidine hydrochloride
(Zoetis, 1852761) was also administered intravenously.
0.3mg/kg Butorphanol (MSD, A141A04) for preemptive
analgesia was administered intramuscularly.
After opening the abdominal cavity from the midline,
2cm of the abdominal aorta was separated and exposed.
e vessel was clamped using arterial clamps at the dis-
tal and proximal ends. Next, the artery was cut from the
middle, and the vascular stump was flushed with heparin-
ized saline. A cannula (0.8mm × 1.6mm) was inserted
into the vascular stump and bound using sutures to pre-
vent cannula dislodgement. Blood was collected from the
proximal arterial drainage tube and infused into degassed
sample tubes to interact with the materials using peristal-
tic pumps (YZ1515x, Longer). Blood was then injected
into the distal arterial drainage tube to complete circu-
lation. e infusion lasted for 2h. After completing the
perfusion procedure, the DPP-samples were removed,
fixed with 2.5% GA solution, and observed using SEM.
2.6 Enzymatic hydrolytic resistance
After being lyophilized to constant weight, all sam-
ples were immersed into 1.5 mL collagenase I/PBS
(S10053, Yuanye Shanghai) (25 U/mL), collagenase IV/
PBS (S10056, Yuanye Shanghai) solutions (25 U/mL),
Elastase/PBS (S10165, Yuanye Shanghai) solutions (25 U/
mL), respectively, and incubated at 37°C with constant
shaking. e samples were then transferred to a 10mM
EDTA solution to terminate the enzymatic hydrolysis
reaction at predetermined time points (0.5, 1, 3, 6, 12,
24, 48, and 72h). e enzymatically hydrolyzed samples
were lyophilized and weighed. e percentage of weight
loss (ΔW%) was calculated using the following formula:
W%=
W
0
W
t
W0
×
100%
Page 5 of 17
Pengetal. Collagen and Leather (2023) 5:5
W0 represents the initial weight before degradation, and
Wt represents the weight of the corresponding sample
after degradation.
2.7 In vitro calcication
e lyophilized and sterilized 1cm × 1cm samples were
immersed in 2 mL of simulated body fluid (SBF, JISS-
KANG) and incubated in a constant temperature incuba-
tor at 37°C. e SBF was changed weekly and the entire
process remained aseptic. e samples were collected
and washed with distilled water at pre-determined times
(after 30, 60, and 90 d). After dehydration by gradient
alcohol and drying at the critical point of CO2, the sur-
faces of the samples were observed by SEM, and the sur-
face calcium content of the samples was detected using
EDS.
2.8 Mechanical properties
2.8.1 Uniaxial tensile test
e samples were investigated before and after fixation
to determine if any improvement in the biomechanical
properties could be detected. e samples were cut into
rectangular strips of 40 mm × 10mm, and their thick-
nesses were measured by a micrometer. Uniaxial tensile
tests were performed using an Instron material test-
ing machine (Instron Co., USA) at an extension rate of
7mm/min. e ultimate tensile strain and the ultimate
tensile stress were recorded when the sample was torn.
e ultimate elastic modulus was determined from the
ultimate tensile stress–strain curve.
2.8.2 Suture retention strength
e suture retention test was performed according to the
American National Standard Institute /Association for
the Advancement of Medical Instruments (ANSI/AAMI)
VP20 standard. Each sample was cut into rectangular
strips (40mm × 10mm). A single 5–0 absorbable suture
loop was created 2mm away from the sample edge and
secured to a hook connected to the clamp of the testing
device. e suture retention strength was defined as the
maximum force recorded at an extension rate of 7mm/
min when the sample was torn. Suture retention ten-
sion was obtained by normalizing the suture retention
strength to the thickness of each sample.
2.9 Subcutaneous implantation
Subcutaneous implantation in rats is the standard FDA-
accepted technique for evaluating cardiovascular mate-
rials. SD rats were purchased from Chengdu Dossy
Experimental Animals CO, Ltd. Rats were inhalation
anesthetized with of 1–2% isoflurane gas, and sterilized
DPP-samples were implanted subcutaneously on both
sides of their backs. After recovery from anesthesia, all
rats were fed a standard diet. Rats were euthanized with
excess isoflurane gas after 90 days, and each collected
implantation sample was fixed in 4% formaldehyde for
HE staining analysis.
2.9.1 Statistical analysis
Data are expressed as mean ± standard deviation and
were analyzed by one-way ANOVA using SPSS (version
20.0), p < 0.05.
3 Results andanalysis
3.1 Preparation andcharacterization ofOKGM
3.1.1 Oxidation degree, molecular weight, andpolydispersity
ofOKGMs prepared withdierent feeding ratios
ofKGM/NaIO4
With the increase in NaIO4, the Mn and Mw of OKGM
showed a downward trend, and the aldehyde group con-
tent within OKGM increased because of the breaking
of the polysaccharide chain (Table 1). e molecular
weights and physiological activities of the crosslinking
agents are different. OKGM with a large molecular
weight has both low cytotoxicity and low crosslinking
properties owing to its better diffusion and ability to
reach more reaction sites to react with the amino groups
within the tissue. In addition, the molecular weight of the
crosslinking agent has an important relationship with its
metabolic time invivo. e larger the molecular weight
of OKGM, the more complex its structure and the more
difficult it is for OKGM invivo [15, 16]. erefore, appro-
priate NaIO4 consumption is key to the preparation of
OKGM. According to the pre-experiment, we selected
three OKGM samples prepared with different feeding
ratios of KGM/NaIO4 (mass ratios of KGM/NaIO4 were
1:1, 1:1.5, and 1:2) to further investigate their crosslink-
ing properties and the physicochemical and biological
properties of OKGM-fixed samples to ascertain the best
Table 1 Oxidation degree, molecular weight, and polydispersity of various OKGMs
KGM/NaIO4 mass ratios Oxidation degree Number‑average Molecular
Weight Mass average molar mass Mw/Mn
1:1 21% ± 2.3% 3623 15,310 4.23
1:1.5 32% ± 1.8% 3001 7500 2.49
1:2 47% ± 1.7% 2534 7475 2.95
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Pengetal. Collagen and Leather (2023) 5:5
matching of KGM and NaIO4 for preparing OKGM used
as a crosslinking agent.
3.1.2 FTIR analysis ofOKGM
OKGM was prepared by the NaIO4 oxidation method
(Fig.1a). e presence of dialdehyde groups was verified
by FTIR analysis. As shown in Fig.1b, the spectrum of
OKGM shows a new characteristic peak at 1727.5 cm1,
which is the most characteristic band of the C=O vibra-
tions of the aldehyde group. is peak clearly indicates
the formation of active aldehyde groups in the OKGM
molecular chain. A new infrared band at 892 cm1 sug-
gests the formation of hemiacetals between the aldehyde
groups and non-oxidized hydroxyl groups. erefore,
the aldehyde group was successfully introduced through
periodate oxidation.
3.1.3 Zeta potential ofOKGM
e OKGMs with different degrees of oxidation pre-
sented different zeta potential values. is may be owing
to the different types and numbers of functional groups
on the surface of OKGMs. e higher the feeding ratio
of NaIO4, the higher thetable degree of oxidation of an
OKGM (Table2), and more aldehyde groups are formed
in the OKGM molecular chain. Owing to the elec-
tronegativity of oxygen atoms in aldehyde groups, the
electron cloud of the carbon–oxygen double bond will
shift toward the oxygen atoms, eventually leading to an
increase in the electronegativity of the overall system.
is increase in polarity is manifested as an increase in
the absolute value of the potential.
3.2 Decellularization ofperitoneum andcharacterization
ofDPP
e fresh porcine peritoneum (FPP) is a semi-transpar-
ent film with a thickness of approximately 0.2–0.5mm.
After decellularization, the tissue thickness significantly
increased to about 0.8 mm (Additional file 1: Fig. S1).
Lyophilized DPP appeared as a white sheet with good
flexibility, as shown in Fig.2a.
3.2.1 Residual DNA ofDPP
Residual cellular components in ECM can cause adverse
host reactions in vivo and increase the risk of hetero-
geneous antigen transmission. Double-stranded DNA
(dsDNA) can be quantitatively assessed as an indicator of
cell residue in the ECM. Previous studies have suggested
that the minimal standard concentration of dsDNA in the
ECM is < 50ng/mg. Our research demonstrates that the
total DNA content of DPP is significantly decreased by
91.5% (44 ± 12ng/mg) after decellularization when com-
pared to that of FPP (519 ± 80 ng/mg) (Fig. 2c), which
meets the standard of clinical application of ECM. DAPI
staining of fresh and decellularized porcine peritoneum
was also performed to determine the extent of DNA
removal. As shown in Fig.2b, the number of intact nuclei
in the FPP is much higher than that in the DPP. Almost
no nuclei are observed in the DPP. HE staining is a simple
qualitative indicator used to observe whether tissues con-
tain nuclei to confirm the removal of nucleic acids from
tissues [17]. In this experiment, there are no residual cells
with intact structures in the DPP fiber voids after decel-
lularization, based on the HE staining results (Fig.2e).
Fig. 1 Characterization of OKGM. a Schematic diagram of preparing OKGM. b FTIR analysis of OKGM
Table 2 Zeta potential of OKGM
Material Zeta
potential
(mV)
KGM 17.40
1:1 OKGM 20.76
1:1.5 OKGM 23.90
1:2 OKGM 25.97
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Pengetal. Collagen and Leather (2023) 5:5
Fig. 2 Characterization of decellularized peritoneum. a Lyophilized DPP. b DAPI staining of FPP and DPP. c Residual DNA of DPP. d Microstructure of
fiber in samples under SEM. e HE, Masson, EVG staining of collagen fibers, muscle fibers and elastic fibers in samples. f Expression of α-Gal, α-SMA,
collagen I, collagen IV in samples
Page 8 of 17
Pengetal. Collagen and Leather (2023) 5:5
3.2.2 Ultrastructure ofDPP andFPP
e SEM results (Fig. 2d) show that after decellulari-
zation, the fibers in the peritoneum became loose but
less broken, and the directions of the fibers are similar.
e results of HE, Masson, and EVG staining (Fig.2f)
show that collagen and elastic fibers remain intact after
decellularization.
3.2.3 The expression level ofα‑Gal, α‑SMA, collagen I
andcollagen IV inDPP
α-Gal is widely expressed in most mammalian tissues
(including humans and higher primates), and α-Gal
remaining in the cytomembrane can cause hyperacute
immune rejection reactions after xenotransplantation
[18, 19]. e absence of α-Gal could be considered a
sign of successful decellularization. α-SMA is a marker
protein of vascular smooth muscle, and its expression
level can be used as an indicator of the degree of clear-
ance of smooth muscle cells [20]. e weak expression
of α-Gal and α-SMA (Fig.2f) in DPP indicatesthe suc-
cessful decellularization of FPP and the safety of this
animal-derived substitutional material.
Collagen accounts for approximately one-third of
the total proteins in all vertebrates. It is biodegradable
with weak antigenicity and good biocompatibility [21].
However, after decellularization, its natural assembly
structure is destroyed, resulting in decreased mechani-
cal properties and stability. e results of collagen
immunohistochemical staining (Fig.2f) shows that the
expression of type I and type IV collagen both in FPP or
DPP is abundant and mainly concentrated in the basal
layer [22, 23].
3.3 Preparation andcharacterization ofOKGM‑xed DPP
After crosslinking with OKGM, GNP, and GA, it was
found that GNP-fixed DPP becomes dark bluish, while
the color of both GA-fixed and OKGM-fixed DPP turn
yellow. e freeze-dried DPP-samples are shown in
Fig.3b.
3.3.1 The xation ofDPP byOKGM
e FI was used to quantitatively evaluate the degree
of crosslinking. e resultsdemonstrated that the FI of
OKGM-fixed DPP was correlated with the feed ratio of
KGM/NaIO4 for preparing OKGM and the concentra-
tion of OKGM. As shown in Fig.3a, the fixation of sam-
ples by 7.5 and 3.75% OKGM prepared with different
feeding ratios of KGM/NaIO4 (the mass ratio of KGM/
NaIO4 is 1:1, 1:1.5, 1:2) is stable, and after 24h of fixa-
tion, their FI reaches a maximum (> 60%). ere is no
significant difference in FI during the entire course of
the test between the 3.75 and 7.5% OKGM-fixed DPP.
Studies have reported that the degradation rate of
decellularized tissue affects tissue regeneration after
implantation [24]. e invivo degradation rate of animal-
derived scaffolds could be controlled by their FI. e 95%-
FI bovine pericardium was more resistant to enzymatic
degradation than its 60%-FI counterpart in research of
GNP-fixed bovine pericardium [25]. Consequently, tissue
regeneration was limited in the 95%-FI acellular tissue
throughout the entire research (1year post-operatively),
whereas tissue regeneration was observed in the 60%-
FI acellular tissue [26]. In conclusion, FI determines the
degradation rate of acellular tissue and its regeneration
pattern. erefore an appropriate FI ( 60%) should be
takeninto account the ability to resist enzymolysis and
tissue regeneration.
Based on the principles of easy dissolution, short fixa-
tion time, and stable FI, the following three conditions:
(1) 1:1, 3.75%, 24h; (2) 1:1.5, 3.75%, 24h; and (3) 1:2,
3.75%, 24 h, were initially selected to prepare OKGM,
and the prepared OKGM was further used for the follow-
ing experiment.
3.3.2 Water contact angle
Appropriate surface hydrophilicity is a prerequisite and
a basis for good cytocompatibility of biomaterials. Supe-
rhydrophilic or superhydrophobic surfaces are not con-
ducive to cell spreading and adhesion. e water contact
angle (WCA) of the DPP was approximately 77° (Fig.3c).
After crosslinking with GA and OKGM, the WCA of the
fixed samples increased to 87.6° and 91.1°, respectively.
is may be because of the massive consumption of
amino groups on the surface of DPP by GA or OKGM,
leading to a weakened interaction force between the fixed
sample and the water droplet. erefore, the surfaces of
DPP fixed with GA or OKGM exhibited slight hydropho-
bicity compared to that of DPP alone. After crosslinking
with GNP, the WCA of the fixed samples decreased sig-
nificantly to 14.1°. is might be because a large number
of amino groups remained on the DPP surface due to the
different crosslinking mechanisms of GNP. Moreover, the
carboxyl and hydroxyl groups on the GNP were exposed.
All these events resulted in a reduction in WCA. e sur-
faces of GA-fixed DPP and OKGM-fixed DPP are neither
too hydrophilic nor too hydrophobic, which could effec-
tively promote cell adhesion and spreading.
3.3.3 The ber structure ofxed DPP
After crosslinking with GA and GNP, the DPP fibers
exhibited anisotropic characteristics (Fig.3d). e fibers
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Pengetal. Collagen and Leather (2023) 5:5
in the GA-fixed DPP were tighter than those in the GNP-
fixed DPP. For OKGM-fixed DPP, with the increase in
NaIO4 concentration, the aldehyde group content within
OKGM (the oxidation of OKGM) increased, the struc-
tures of the fibers were more compact, and the fiber
orientation was basically the same. is indicated that
the cross-linking by OKGM did not destroy the original
structure of the fiber but rather made the fiber structure
denser.
3.4 Cytocompatibility andinvitro endothelialization
ofOKGM‑xed DPP
e lumen surface of a vascular graft can trigger throm-
bosis owing to the deposition of platelets. e absence or
Fig. 3 Characterization of fixation and water contact angle. a Influence of different oxidation degree, concentration, and crosslinking time of OKGM
on the fixation of samples. b Preparation of OKGM-fixed DPP (from left to right is DPP, GA-fixed DPP, GNP-fixed DPP and DPPs crosslinked with
various OKGM prepared with different feeding ratios of KGM/NaIO4 (the mass ratio of KGM/NaIO4 is 1: 1, 1:1.5, 1:2). c Water contact angle. d Fiber
structure of fixed DPP
Page 10 of 17
Pengetal. Collagen and Leather (2023) 5:5
delay of endothelialization and the migration and pro-
liferation of smooth muscle cells (SMC) on the lumen
surface of vascular grafts can lead to intimal hyperplasia
[27]. Re-endothelialization is the first key point in inhib-
iting intimal hyperplasia and thrombosis. A completely
spread monolayer of endothelial cells can ensure normal
blood flow and the functional expression of EC [28, 29].
After seeding HUEVCs on fixed DPP and culturing
for 1, 3, and 5days, the proliferation of HUEVCs was
detected by CCK8 (Fig. 4a). e adhesion of HUEVCs
on the second and third days was detected by live/dead
cell fluorescence staining (Fig. 4b) and SEM (Fig. 4c),
respectively. e proliferation and endothelialization of
HUEVCs on samples on the fifth day tested by HE stain-
ing (Fig.4d). e results showed that the samples in the
1:1 OKGM-fixed and 1:1.5 OKGM-fixed groups had
similar relative generation rates (RGR) of HUEVCs, basi-
cally maintaining around 70–90% within 5days. is was
significantly higher than that of the GA-, GNP-, and 1:2
OKGM-fixed DPP. Live/Dead cell fluorescence stain-
ing and SEM results showed that HUEVCs had good
adhesion on the surface of 1:1 OKGM-fixed and 1:1.5
OKGM-fixed DPP, spreading fully with their characteris-
tic fusiform morphology on sample surfaces. In contrast,
the GA- and 1:2 OKGM-fixed samples showed a certain
degree of cytotoxicity, with no obvious normal adhe-
sion of HUEVCs on 1:2 OKGM-fixed DPP. As shown in
Fig.5d, compared with counterparts in other groups, sig-
nificantly confluent monolayers are found on the surfaces
of the 1:1 OKGM- and 1:1.5 OKGM-fixed DPP. e above
results indicated that 1:1 OKGM- and 1:1.5 OKGM-fixed
DPP presented low cytotoxicity and high HEVC cyto-
compatibility. is was partly because of the good bio-
compatibility of OKGM. Furthermore, OKGM could also
crosslink collagen and elastin, which enhances the abil-
ity of OKGM-fixed DPP to resist enzymatic hydrolysis
invitro, avoiding the cytotoxicity resulting from the small
molecule toxic substances produced by rapid degradation
of samples in vitro. Conversely, the excessive aldehyde
groups in the 1:2 OKGM-fixed DPP could result in some
cytotoxicity.
3.5 Hemocompatibility ofOKGM‑xed DPP
Hemocompatibility is the most important criterion for
the successful application of cardiovascular substitute
materials invivo [30]. Figure5a shows the hemolysis rate
of various DPP-samples as a function of suspension con-
centration of the samples after incubation with RBCs at
37°C for 3h. As shown in the figure, the hemolysis rate
of OKGM-fixed samples is lower than that of GA-fixed
and GNP-fixed samples. In addition, the hemolysis rate
is less than 5% for 1:1 and 1:1.5 OKGM-fixed samples at a
concentration of 25μg/mL, which was much higher than
the concentration value that was possibly reached invivo.
Fig. 4 Cytocompatibility and in vitro endothelialization of OKGM crosslinked DPP. a HEVCs-cytocompatibility of fixed DPP (# means significant
difference compared with DPP group). b Live/dead cell fluorescent staining image of HUEVCs on samples on the second day. c HUEVCs adhered on
fixed DPP on the third day. d Proliferation and endothelialization of HUEVCs on samples on the fifth day
Page 11 of 17
Pengetal. Collagen and Leather (2023) 5:5
e complete DPP-sample direct contact hemolysis
test results (Fig.5b) show that the hemolysis rate of the
OKGM-fixed samples is significantly lower than that of
the GA-fixed and GNP-fixed samples, and the hemolysis
rates of all samples are less than 5%. e hemolysis rate
tested by the two methods met the clinical requirements
for implants according to the international standard ISO
10993–4:2017(E).
Anticoagulation is an essential factor for the long-
term patency of blood vessels [31]. Platelet adhesion
and deposition are the leading causes of thrombosis
and intimal hyperplasia. erefore, inhibition of platelet
adhesion on cardiovascular substitute materials is very
important for small-diameter vascular grafts. Owing to
the participation of GP lb, vWF, and integrin αIIβ3d, the
adhesion of platelets onto the surface of cardiovascular
substitute materials undergoes five processes (flowing-
disc-shaped platelets, rolling ball-shaped platelets, hem-
isphere-shaped platelets, firm reversible adhesion, and
spreading irreversible adhesion), and the platelet mor-
phology undergoes corresponding changes during these
processes. Among these processes, only the last step
(spreading irreversible adhesion) is irreversible, which
is conducive to capturing more proteins and platelets
to adhere to the surface of the cardiovascular substitute
materials, leading to platelet aggregation and thrombus
formation of thrombi [32]. As observed by SEM (Fig.5c),
more flat platelets are spread irreversibly and aggregated
on the DPP surface. ere were many hemispherical
platelets on the GA-fixed DPP, which were basically in
the rolling ball-shape stage, on the GNP-fixed DPP, the
platelets were slightly flat and their pseudopodia were
more stretched, and most of them were in the hemi-
sphere shape stage. e number of platelets adhering to
OKGM-fixed DPP was significantly lower than that of the
GA-fixed and GNP-fixed samples. Among these OKGM-
fixed DPP, no platelets spread irreversibly on the surface
of the 1:1.5 OKGM-fixed DPP. e results showed that
OKGM-fixed DPP, especially 1:1.5 OKGM-fixed DPP,
could significantly inhibit platelet adhesion.
e carotid artery in rabbits is thin and prone to
thrombosis after puncturing owing to hemodynamic
changes at the site of the puncture needle and cannula.
In view of this, a semi-ex vivo circulation (Fig.6a, b) was
Fig. 5 Hemocompatibility and static platelet adhesion of OKGM-fixed DPP. a Hemolysis rate of various DPP-samples as a function of suspensions
concentration of samples. b Complete DPP-samples direct contact hemolysis test. c Static platelet adhesion of samples (the box represents the
typical platelet shape; the arrow represents the fibrin)
Page 12 of 17
Pengetal. Collagen and Leather (2023) 5:5
constructedby ligating the abdominal aorta and cannu-
lating the upper and lower ends of the vessel, and using
a peristaltic pump to set the blood flow rate invivo. e
sheet sample was fixed to the inner surface of the blood
circulation line, and arterial blood was extracted from
rabbits for semi-ex vivo circulation to assess the anticoag-
ulation properties and leukocyte adhesion of the sample.
After circulation for 2h, no thrombus is observed on the
surface of the samples in each group (Fig.6c). As shown
in the SEM image (Fig.6d), there are variable amounts
Fig. 6 Ex vivo blood contact test. a Abdominal aortic blood flow pathway (red arrows indicate the direction of arterial blood flow). b Sample sheet
fixed to the inner wall of circulation pipe (black box indicates the location of sample). c Samples exposed to the flow of blood without heparin for
2 h. d SEM of sample surface. (White boxes indicate the typical platelets; blue arrow and blue boxes indicate the leukocyte)
Page 13 of 17
Pengetal. Collagen and Leather (2023) 5:5
of adherent platelets without a fibrin network on the
sample surface. Among them, more platelets areon the
GA- and GNP-fixed DPP. In addition, a certain amount
of leukocytes is also observed to adhere to their surfaces.
e number of platelets adhering to the OKGM-fixed
DPP was lower than that of the DPP, GA-, and GNP-fixed
samples, and no leukocytes were found on their surfaces.
is indicated a lower risk of thrombosis for OKGM-
fixed DPP and also that GA- and GNP-fixed DPP might
trigger a more severe inflammatory response. An impor-
tant feature of the inflammatory response process in ves-
sels is that leukocytes adhere to and infiltrate the vascular
endothelium and engulf pathogens or tissue debris from
exudation. erefore, the amount of leukocyte adhesion
and exudation could reflect the degree of inflammation
[33]. At the site of vascular injury, excessive leukocyte
adhesion needs to be avoided as excessive leukocyte
adhesion and accumulation could lead to atherosclero-
sis [34]. Conversely, excessive leukocyte adhesion and
infiltration is an important manifestations of vascular
endothelial dysfunction [35]. Based on these results, the
OKGM-fixed DPP, especially the 1:1.5 OKGM-fixed DPP,
had better hemocompatibility.
3.6 In vitro anti‑enzymatic hydrolysis ofOKGM‑xed DPP
e main structural proteins in mammals are collagen
and elastin, which provide mechanical support, strength,
and elasticity in various tissues and organs. Collagen
and elastin are readily available, biodegradable, bio-
compatible, and can stimulate cell growth [36]. In the
anti-enzymatic hydrolysis experiments (collagenase I
and collagenase IV) of fixed samples invitro, the ability
of GNP-fixed DPP to resist collagenase digestion is bet-
ter than that of GA-fixed DPP at 72h, and OKGM-fixed
DPP is equivalent to GA-fixed DPP (Fig.7). is indicates
that GNP presents a better crosslinking effect on colla-
gen than OKGM and GA, while OKGM and GA present
the same crosslinking effect on collagen. Masson stain-
ing results (Additional file1: Figs. S2–S3) show that the
structure of collagen fiber in GNP-fixed DPP is well pre-
served. e same result was also observed for GA-fixed
and 1:1.5 OKGM-fixed DPP.
Elastin keeps ECM in its original shape under stress-
ful conditions. Elastin is mainly composed of hydropho-
bic amino acids [37]. Due to the lack of lysine residues
as reactive groups, it is difficult to fix and stabilize natu-
ral elastin through covalent interactions. erefore, the
commercial crosslinker GA cannot effectively crosslink
elastin. is conclusion was confirmed in the present
research. e ability of GNP-fixed and OKGM-fixed DPP
to resist elastase digestion was better than that of GA-
fixed DPP and DPP alone. EVG staining results showed
no obvious elastic fibers were observed in the GA-fixed
DPP after 72h of elastase-enzymolysis in vitro, and its
microstructure was seriously damaged. e elastic fiber
microstructure (Additional file1: Fig. S4) in the 1:1.5 and
1:2 OKGM-fixed DPP samples was preserved as well as
that of the GNP-fixed DPP. e results indicated that GA
had a poor crosslinking effect on elastin and was unsuit-
able for crosslinking DPP. However, GNP had a good
crosslinking effect on both collagen and elastin, and
their resistance to enzymatic hydrolysis (collagenase and
elastase) invitro was the best. e crosslinking effect of
OKGM on collagen and elastin was second only to that
of GNP. It also demonstrated a good crosslinking effect
on DPP.
3.7 In vitro calcication ofOKGM‑xed DPP
e calcification process of the vessel and valve is simi-
lar to bone mineralization, and there are two main stages:
calcium phosphate nucleation and further growth of
crystals [3840]. e initiation and progression of calci-
fication depend on the microstructure of the biomateri-
als (including collagen fibers and elastic fibers). e low
crosslinked elastin protein has large open spaces between
molecules because of the lack of functional groups that
will react with aldehydes and epoxy compounds. It has
many calcium-binding sites that provide a central site for
Fig. 7 Weight loss rate of fixed samples during enzymatic hydrolysis (*indicates significant difference compared to the GA-DPP group)
Page 14 of 17
Pengetal. Collagen and Leather (2023) 5:5
crystal nucleus formation. erefore, the more elastin the
biomaterials contain, the faster their calcification rates.
As representative small-molecule crosslinking agents,
GA and GNP are usually used to crosslink bovine peri-
cardium to construct artificial heart valves. However, this
artificial heart valve presents a serious risk of long-term
calcification, which limits its long-term implantation.
Grafts fixed by other crosslinking agents (including EDC,
ethylene oxide, proanthocyanidins, sodium alginate,
etc.) also demonstrated serious instances of calcification
either in vitro or in vivo, causing implantation failure
[41]. We developed a novel OKGM to crosslink DPP and
detected the calcification of OKGM-fixed DPP invitro
through EDS (Fig.8a) and SEM (Fig.8b). DPP exhibited
the maximum degree of calcification, and its calcium
content reached more than 30% after incubation for 60
and 90days. e calcium content of GA-fixed and GNP-
fixed DPP was approximately 1% after incubation for 60
and 90days. e OKGM-fixed DPP presented the low-
est degree of calcification because of its smooth surface
with almost no calcium deposits. e results suggested
that OKGM-fixed DPP had significantly better anti-calci-
fication capability invitro than GA-fixed and GNP-fixed
DPP. Although OKGM does not wholly prevent the first
stage of calcification in the peritoneum, it can delay the
mineralization of elastin and reduce the second stage
of calcification. However, the underlying mechanism
remains unclear. We speculate that one of the reasons for
this is that OKGM can simultaneously crosslink collagen
and elastin.
3.8 The mechanical properties ofOKGM‑xed DPP
Adequate mechanical properties are essential for car-
diovascular materials, particularly for artificial heart
valves and small-diameter vascular grafts [42]. Accord-
ing to the literature, the axial E-modulus of a 6mm aorta
is 9.13MPa, and the ultimate tensile stress is 4.03MPa
[4345]. Uniaxial tensile testing (Fig.9) is one of the most
commonly used methods for measuring the mechanical
properties of cardiovascular materials. e ultimate tensile
stress (Fig.9a, b) of OKGM-fixed DPP is 3.43MPa, which
is slightly lower than that of the artery, GA-fixed DPP and
GNP-fixed DPP, but there is no significant difference in
the ultimate tensile stress among them. Conversely, the
E-modulus of OKGM-fixed DPP (Fig.9c) is 12MPa, which
is slightly higher than that of the artery, GA-fixed DPP and
GNP-fixed DPP. However, there was still no significant dif-
ference in the E modulus among them.
Fig. 8 In vitro calcification detected by a EDS and b SEM (OKGM-DPP indicate the 1:1.5 OKGM-fixed DPP)
Fig. 9 The mechanical properties of crosslinked DPP. a Stress-strain curves. b Tensile stress at maxiun load(MPa). c E-modulus(MPa). d Suture
retention strength(N)
Page 15 of 17
Pengetal. Collagen and Leather (2023) 5:5
Vascular grafts require a certain suture retention
strength to ensure clinical use, and the demand for suture
retention strength of vascular grafts is at least 1N. e
suture retention strength results for various samples are
shown in Fig.9d. e suture retention strength of GA-
fixed DPP and GNP-fixed DPP was significantly higher
than that of the 6mm artery and OKGM-fixed DPP. Fur-
thermore, OKGM-fixed DPP had an equivalent suture
retention strength to that of the 6 mm artery, which
was approximately 7.9–9.2N. In fact, suture retention
strength that is too high is not conducive to anastomosis.
e above results confirm that the mechanical proper-
ties of the OKGM-fixed DPP is the nearest to the human
artery, meeting the mechanical performance require-
ments for vascular grafts.
3.9 Degradation andinammation ofOKGM‑xed DPP
invivo
A subcutaneous implantation model of the SD rat sub-
jects for 90days was used to investigate the biocompat-
ibility of the samples. As shown in Fig.10a, compared
with OKGM-fixed DPP, there are more blood vessels
around the DPP and GA-fixed DPP/GNP-fixed DPP
12weeks after subcutaneous implantation. is dem-
onstrated that the inflammation caused by DPP, GA-
fixed DPP, and GNP-fixed DPP was still obvious, and
the sample morphologies showed noticeable changes
due to degradation in vivo. e morphology of the
GNP-fixed DPP and OKGM-fixed DPP remained rela-
tively complete. No apparent morphological changes
due to degradation and no obvious inflammation were
observed in the OKGM-fixed samples, indicating their
good histocompatibility. Samples were retrieved from
the subdermal sites for histological analysis. HE stain-
ing results showed (Fig.10b) that DPP presented more
fiber breakage invivo and more inflammatory cell infil-
tration compared with samples in other groups. GA-
fixed DPP and GNP-fixed DPP maintained better fiber
morphology because of their ability to resist enzymatic
digestion, but inflammatory cell infiltration in these
samples was also high, indicating serious inflamma-
tion. OKGM-fixed DPP presented fewer broken fibers,
Fig. 10 Sample morphology (a) and inflammatory cell infiltration (b) after 12 weeks of subcutaneous implantation (black curve indicates sample
edge; black circles indicate neovascularization)
Page 16 of 17
Pengetal. Collagen and Leather (2023) 5:5
and its shape retention was second only to GNP-fixed
DPP. It is to be noted that there was no inflammatory
cell infiltration observed in the sample, while massive
neovascularization around the sample was found. is
indicated that OKGM-fixed DPP had good histocom-
patibility invivo, which was beneficial to the migration
of endothelial cells, and thus could promote angiogen-
esis in implants invivo.
4 Conclusion
e peritoneum is a thin elastic membrane. After decel-
lularization, DPP is a promising animal-derived bioma-
terial that remains intact ECM and suitable mechanical
properties. In this research, NaIO4 was appliedto oxidize
KGM to prepare a polysaccharide crosslinker, OKGM,
and then used OKGM to stablizeDPP to prepare a novel
cardiovascular substitute material. For the preparation
of OKGM, the results showed that OKGM obtained
using a feeding ratio of 1:1.5 (KGM:NaIO4) possessed an
appropriate molecular weight and degree of oxidation,
with almost no cytotoxicity. Furthermore, 3.75% OKGM
[1:1.5 (KGM:NaIO4)] was used as a cross-linking agent to
fix DPP to prepare a cardiovascular substitute material
and the prepared material exhibited good cross-linking
characteristics. Compared to GA-fixed DPP and GNP-
fixed DPP, this novel 3.75% OKGM-fixed DPP exhibited
suitable mechanical strength, excellent HEVC-cytocom-
patibility, hemocompatibility, anti-calcification capabil-
ity, and resistance to enzymatic degradation in vitro,
as a cardiovascular substitute material. It also exhibits
good histocompatibility in vivo. is research provides
an experimental basis for OKGM as a new cross-linking
reagent for fixing natural tissues featured withrich col-
lagencontent and 3.75% OKGM-fixed DPP as a potential
cardiovascular substitute material.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s42825- 023- 00114-w.
Additional le1. Fig.S1. Morphology of peritoneum. Fig.S2. Masson
staining of samples hydrolyzed by collagenase I. Fig.S3. Masson staining
of samples hydrolyzed by collagenase IV. Fig.S4. EVG staining of samples
hydrolyzed by elastase.
Acknowledgements
We would be grateful to the help of Xiaomei Zhang from Experimental and
Research Animal Institute, Sichuan University, for providing the fresh porcine
peritoneum and raising animals.
Author contributions
XP: experiment, analysis, and writing. LL and CC: experiment, editing. JX: analy-
sis and editing. MH, YL, SS: experiment, editing. YL and ZC: Preparation and
staining of pathological tissues. XY: funding acquisition, review, and editing. All
authors read and approved the final manuscript.
Funding
This work was financially supported by National Key Research and Devel-
opment Program of China [Nos. 2016YFC1100900, 2016YFC1100901,
2016YFC1100903 and 2016YFC1100904]. “From 0 to 1” innovative research
projects of Sichuan University (2022SCUH0045). The Key Research and Devel-
opment Program of Sichuan Province (2020YFS0278).
Availability of data and materials
All data from this research are presented in the paper and the supplementary
material.
Declarations
Ethics approval and consent to participate
All animal experiments were conducted in compliance with the guidelines of
the Administration Committee of Experimental Animals in Sichuan Province
and the Animal Care Committee of Sichuan University (No. K2021036). Male
SD Rat (220 ± 10 g) and New Zealand rabbit (3 kg) were purchased from
Experimental and Research Animal Institute, Sichuan University.
Competing interests
The authors declare no conflict of interest.
Author details
1 College of Polymer Science and Engineering, Sichuan University,
Chengdu 610065, People’s Republic of China. 2 Experimental and Research Ani-
mal Institute, Sichuan University, Chengdu 610065, People’s Republic of China.
3 Department of Oncology Hematology, Western Theater Command Air Force
Hospital, Chengdu 610021, People’s Republic of China. 4 Max Planck Institute
for Polymer Research, 55128 Mainz, Germany. 5 Department of Growth
and Reproduction, Copenhagen University, Copenhagen, Denmark.
Received: 9 October 2022 Revised: 31 December 2022 Accepted: 5 Janu-
ary 2023
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