Materials Science & Engineering C 128 (2021) 112329

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Zwitterionic hydrogel-coated heart valves with improved

endothelialization and anti-calcification properties
Yu Luo a, Shenyu Huang b, Lie Ma a, *
a
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027,
China
b
Department of Ophthalmology, the Second Affiliated Hospital of Zhejiang University, College of Medicine, Hangzhou, Zhejiang, China

A R T I C L E I N F O A B S T R A C T

Keywords: Valve replacement surgery is the golden standard for end-stage valvular disease due to the lack of self-repair
Zwitterionic hydrogel ability. Currently, bioprosthetic heart valves (BHVs) crosslinked by glutaraldehyde (GA) have been the most
REDV popular choice in clinic, especially after the emerge of transcatheter aortic valve replacement (TAVR). Never­
Bioprosthetic heart valves
theless, the lifespan of BHVs is limited due to severe calcification and deterioration. In this study, to improve the
Endothelialization
Anti-calcification
anti-calcification property of BHVs, decellularized heart valves were modified by methacrylic anhydride to
introduce double bonds (MADHVs), and a hybrid hydrogel made of sulfobetaine methacrylate (SBMA) and
methacrylated hyaluronic acid (MAHA) was then coated onto the surface of MADHVs. Followed by grafting of
Arg-Glu-Asp-Val (REDV), an endothelial cell-affinity peptide, the BHVs with improved affinity to endothelial cell
(SMHVs-REDV) was obtained. SMHVs-REDV exhibited excellent collagen stability, reliable mechanical property
and superior hemocompatibility. Moreover, enhanced biocompatibility and endothelialization potential
compared with GA-crosslinked BHVs were achieved. After subcutaneous implantation for 30 days, SMHVs-REDV
showed significantly reduced immune response and calcification compared with GA-crosslinked BHVs. Overall,
simultaneous endothelialization and anti-calcification can be realized by this strategy, which was supposed to be
benefit for improving the main drawbacks for available commercial BHVs products.

1. Introduction such as cytotoxicity, hemocompatibility, immune response and calcifi­

Heart valve disease has become one of the major life-threaten
problems in the last few decades, especially for the elder people [1].
For now, valve replacement surgery is still the golden standard for end-
stage valvular disease due to inability of self-repair [2]. The first suc­
cessful implantation surgery was performed in 1952 [3]. Since then,
nearly 300,000 patients received valve replacement surgery each year,
and the number is estimated to be more than triple in 2050 [4].
Currently, there are two major types of commercial valve substitutes:
mechanical heart valves (MHVs) and bioprosthetic heart valves (BHVs).
BHVs exhibit better hemodynamic performance compared with MHVs,
which makes BHVs as a more attractive choice than MHVs. Since the
first successful performance of transcatheter aortic valve replacement
(TAVR) in 2002, BHVs can be implanted into human body in a micro-
invasive safer method [1,5].
Commercial BHVs are mainly made from glutaraldehyde (GA)
crosslinked xenograft heart valves or pericardium. Several drawbacks,
cation, shorten the lifespan of GA-crosslinked BHVs, despite GA cross­
linking can stabilize collagen via amidation reaction [6,7]. Due to the
cytotoxicity of remnant aldehyde groups, endothelial cells are pro­
hibited adhering and proliferating, thus impeding endothelialization
[6,8,9], which is crucial for the implanted cardiovascular devices [10].
Severe immune response elicited by GA crosslinking has been proved to
be responsible for degradation of elastin fibers, which may contribute to
mineral nucleation, subsequently causing calcification [11], leading to
the structural valve deterioration (SVD).
During the last few years, several methods have been developed to
conquer the above drawbacks, such as decellularization and the usage of
non-GA crosslinker. Decellularization, one way to remove cells from the
target tissues or organs, has been proved to be effective in improving the
anti-calcification ability of BHVs [12]. However, the stability of decel­
lularized heart valves is limited, which requires for further crosslinking
treatments. Till now, non-GA crosslinkers, including tannic acid [13],
quercetin [14] and carbodiimide [15], have been proved to be effective

* Corresponding author.
E-mail address: liema@zju.edu.cn (L. Ma).

https://doi.org/10.1016/j.msec.2021.112329
Received 24 April 2021; Received in revised form 4 July 2021; Accepted 18 July 2021
Available online 28 July 2021
0928-4931/© 2021 Elsevier B.V. All rights reserved.
Y. Luo et al.
in improving the anti-calcification property of BHVs. However, none of
the above crosslinkers has been utilized in clinic.
Recently, hydrophilic hydrogel films have been conjugated on the
surface of porcine pericardium via radical polymerization, which
endowed the hybrid pericardium with superior anti-fouling property.
Excellent protein and platelet resistance performance and mitigated
immune response were achieved simultaneously, and most importantly,
improved anti-calcification capacity was proved by in vivo subcutane­
ously implantation animal experiments [16]. Anti-fouling treatment is
considered as one novel method to improve the anti-calcification
property of BHVs. SBMA ([2-(Methacryloyloxy)ethyl]dimethyl-(3-sul­
fopropyl) ammonium hydroxide) is one typical zwitterions monomer
with both anionic and cationic groups. SBMA monomer can be poly­
merized into zwitterions polymer, which can induce the formation of
hydration layer, thus resist non-specific protein absorption [17]. SBMA
polymer based materials, such as hydrogel, have been widely utilized in
biomedical field during the last few years [18–22]. SBMA can be
introduced on the surface of biomaterials via ATRP [23] or covalent
coupling [24] according to the composition of target surface. There are
abundant amino groups inside the heart valves, which can be meth­
acrylated easily, thus introducing vinyl groups according to previous
research [6].
Endothelialization has been proved to be crucial for improving the
long-term stability of artificial vascular grafts [10]. However, only weak
endothelialization can be achieved after implantation of current com­
mercial BHVs due to the severe cytotoxicity of GA crosslinking. Despite
reduced calcification can be achieved via anti-fouling hydrogel films
coating, endothelialization cannot be achieved due to the lack of cell
affinity molecules. REDV peptide is one kind of endothelial cell-affinity
peptide, which have been applied widely to improve the endotheliali­
zation potential of an implant [25,26].
Herein, SBMA and MAHA (methacrylic anhydride modified hyal­
uronic acid) hybrid hydrogel film was copolymerized onto the surface of
methacrylic anhydride modified decellularized heart valves (MADHVs),
following by grafting of REDV peptide to fabricate SMHVs-REDV. As
shown in Scheme 1, freshly harvested heart valves were decellularized
to remove cells, after then, DHVs was treated with methacrylic anhy­
dride to obtain methacrylated DHV (MADHVs), in which vinyl groups
were introduced. MAHA was utilized as an extra double bonds supplier.
The hybrid hydrogel film was coated onto the surface of MADHVs via UV
light triggered free radical polymerization, after which REDV peptide
was grated onto the surface of SMHVs via “click” reaction. The obtained
SMHVs-REDV exhibited excellent collagen stability, hemocompatibility
and in vitro biocompatibility. In vivo subcutaneous implantation
confirmed that reduced calcification and mitigated immune response
can be achieved in SMHVs-REDV compared with GA group.
Scheme 1. Schematic illustration of SMHVs-REDV fabrication procedure.
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2. Materials and methods
2.1. Materials
Hyaluronic acid sodium salt (HA, Mw = 110 kDa) was purchased
from Zhenjiang Dong Yuan Biotech (Zhenjiang, China) Co., Ltd. N-
Lauroylsarcosine sodium salt, Deoxyribonuclease I, ribonuclease,
methacrylic anhydride (MA), [2-(Methacryloyloxy)ethyl]dimethyl-(3-
sulfopropyl) ammonium hydroxide (SBMA) and 3- (trimethoxysilyl)
propyl methacrylate (TMS) were purchased from Aladdin Co. (Shanghai,
China). Irgacure 2959 was purchased from BASF (Shanghai, China).
Collagenase I was purchased from Guangzhou saiguo biotech Co.
(Guangzhou, China).
Cys-Arg-Glu-Asp-Val (REDV) was purchased from GL Biochem Co.,
Ltd. (Shanghai, China). Methacrylic anhydride modified hyaluronic acid
(MAHA) was synthesized according to our previous work with slight
change [27]. Details were described in the Supplementary material.
LDH Cytotoxicity Assay Kit was purchased from Beyotime Biotech­
nology (Shanghai, China). All other reagents (analytical grade) were
purchased from Sinopharm Chemical Reagents Co. (Shanghai, China).
Fresh porcine aortic heart valves were obtained from the local
slaughterhouse and transferred into the sterilized cold PBS (containing
100 U mL− 1 penicillin and 100 μg mL− 1 streptomycin) for storage.
Valves were divided into different groups randomly (five heart valves for
each group). Native heart valves without any treatment (Native) were
set as the control.
2.2. Decellularization
Heart valves were decellularized according to our previous report
[28]. Briefly, the heart valves were treated with 1% N-Lauroylsarcosine
sodium salt (SLS) (w/v, 0.05 M Tris-HCl buffer, pH = 8) for 48 h at 37 ◦ C,
and transferred into DNase (100 mg mL− 1) and RNase (20 mg mL− 1) for
2 h at 37 ◦ C immediately. All the above procedures were conducted
under vacuum atmosphere (<100 Pa). After then, the valves were
washed thoroughly by sterilized PBS for 30 min with continuous shaking
under normal atmosphere. Finally, the obtained samples were perse­
vered in sterilized PBS (containing 100 U mL− 1 penicillin and 100 μg
mL− 1 streptomycin) at 4 ◦ C for further experiments. The obtained
decellularized heart valves were named as DHVs.
2.3. Preparation and characterization of methacrylic anhydride modified
decellularized heart valves
Methacrylic anhydride modified decellularized heart valves
(MADHVs) were obtained according to the previous research [6].
Briefly, DHVs were blotted dry and weighed, following by being
immersed into deionized water at a concentration of 0.1 g DHVs per mL
deionized water. Methacrylic anhydride was added dropwise into so­
lution (ice bath). The pH value was maintained at 7 by the addition of
NaOH (2 M) aqueous solution, and the reaction was carried out at room
temperature for another 24 h. The obtained MADHVs were washed
thoroughly by 50% (v/v) ethanol aqueous solution and deionized water
for at least three times. The feed ratio (MA: DHVs, v/w) varied and the
obtained MADHVs were namely as MADHVs 0.1, 0.5, 1.0 and 5.0 ac­
cording to the feed ratio, respectively.
The remnant amino groups of MADHVs were quantified by ninhydrin
assay according the previous reports with slight change [6,29]. Briefly,
the heart valves were cut into 5 mm × 5 mm pieces. Each piece of heart
valve was placed into a small centrifuge tube, following by adding 2 mL
ninhydrin solution (1% w/v, dissolved in 0.1 M sodium citrate, pH = 5).
The tube was kept into water bath at 80 ◦ C for 15 min. After then, the OD
values at 567 nm of supernatant were detected by Infinite M200 PRO
(TECAN, Switzerland). A gradient concentration of glycine solution was
utilized as the standard. The heart valves were lyophilized and weighed.
The remnant amino groups content can be calculated according to the
Y. Luo et al.
standard curve of glycine.
MADHVs were cut into 1 cm × 1 cm small pieces and washed thor­
oughly by deionized water for three times. After then, the heart valves
were transferred into centrifuge tube containing 0.5 mL concentrated
HCl for 24 h at 37 ◦ C for hydrolysis. The supernatant was dried by
vacuum oven and the obtained dried pellet was dissolved in D2O for 1H
NMR.
2.4. Preparation of hydrogel-coated hybrid heart valves
SMHVs: MADHVs were immersed into initiator solution (0.05% I
2959, w/v, dissolved in PBS) for 1 h at 37 ◦ C. After then, the heart valves
were blotted and flattened, followed by adding 30 μL hydrogel precursor
solution (3% MAHA and 10% SBMA, w/v, dissolved in PBS containing
0.05% I 2959) on the surface. The curing process were completed by UV
light (365 nm, 100% intensity, IntelliRay 400, Uvitron, USA) for 2 min.
The heart valves were flipped over, and the above procedures were
repeated.
SMHVs-REDV: SMHVs were immersed into Na2CO3/NaHCO3 buffer
(pH = 9, containing 2 mg mL− 1 REDV peptide) for 2 h at 37 ◦ C, followed
by washing with PBS for at least three times.
GA: “GA” indicated the DHVs crosslinked by glutaraldehyde. Briefly,
DHVs were immersed into 0.625% GA solution (diluted with PBS) for 48
h at 37 ◦ C, following by washing with PBS for at least three times.
All the above heart valves were preserved in sterilized PBS (con­
taining 100 U mL− 1 penicillin and 100 μg mL− 1 streptomycin) at 4 ◦ C for
further experiments.
2.5. Scanning electron microscopy (SEM)
The heart valves were dehydrated by gradient ethanol, following by
CO2 critical point drying. Briefly, the heart valves were placed into
increasing gradient ethanol solution (30%, 50%, 70%, 90%, 100%) for
15 min at each concentration. After then, the heart valves were
completely dried by CO2 critical point drying procedure. The morphol­
ogies of dried heart valves were observed by SEM (S-4800, Hitachi,
Japan).
2.6. Differential scanning calorimetry (DSC)
Thermal denaturation temperature (Td) of collagen was evaluated by
DSC (Q20, TA, USA) measurement. Briefly, small pieces of heart valves
(~10 mg) were cut from the same region, blotted dry and placed in the
sealed pans. The heart valves were equilibrated at 20 ◦ C and heated at
10 ◦ C min− 1 up to 110 ◦ C under N2 atmosphere. Td was recorded as the
maximum value of the endotherm peak.
2.7. Collagenase degradation
The heart valves were lyophilized, and the obtained weight were
recorded as W0. Valves were cut into small pieces, followed by being
immersed into collagenase I solution (75 U mL− 1, dissolved in PBS) at
37 ◦ C for 24 h and 48 h. After then, valves were washed by PBS and
lyophilized. The dry weight after collagenase degradation was recorded
as W1. The weight persistence after degradation (%) was calculated by
( )
the following formula: W1 × 100%.
W0
2.8. Uniaxial tensile test
Uniaxial tensile test was conducted via an electronic universal testing
machine (3343, Instron, USA) according to our previous work [30,31].
Briefly, the samples were cut into 15 mm × 5 mm strips along the
collagen fiber direction (circumferential direction) for tensile test. The
extension rate was 10 mm min− 1. The elastic modulus was obtained
from the stress-strain curve within the strain of 0–10%. Five samples for
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Materials Science & Engineering C 128 (2021) 112329
each group were tested.
2.9. Adhesion assay of platelets
Blood collected from rabbit was citrated with sodium citrate (1:9).
Platelet-rich plasma (PRP) was prepared by centrifugation of the citra­
ted blood at 1000 rpm for 10 min. The heart valves pieces (8 mm × 8
mm) were placed into 48-well plates and incubated with PBS for 2 h at
37 ◦ C, followed by replacing with 400 μL fresh PRP and incubating for
another 2 h at 37 ◦ C. After then, PRP was discarded, and the heart valves
were fixed with 2.5% GA at 4 ◦ C overnight. For SEM observation, the
heart valves were dried by CO2 critical point drying as mentioned
before. For LDH assay, the heart valves were lysed with 0.5% Triton X-
100 (v/v) for 30 min at room temperature. The supernatant was
collected and detected by LDH kit (C0016, Beyotime, China) according
to the manuals.
2.10. Hemolysis rate assay
Citrated rabbit blood was diluted with normal saline (1:6). The heart
valves pieces (1 cm × 1 cm) were placed in 24-well plates and incubated
with normal saline for 1 h at 37 ◦ C. After then, the normal saline was
discarded, and 500 μL fresh diluted citrated blood was added. The plate
was incubated at 37 ◦ C for 1 h with continuous shaking. After incuba­
tion, the supernatant was collected by centrifugation at 1000 rpm for 5
min. The absorbance at 540 nm was detected by microplate reader
(Infinite M200 PRO, TECAN, Switzerland). Deionized water and normal
saline were utilized as positive and negative control, respectively. The
hemolysis rate was calculated as the following equation:
( )
Hemolysis rate =
OD valves–OD negative
× 100%
OD positive–OD negative
2.11. In vitro biocompatibilities
2.11.1. In vitro cytotoxicity
To prepare sterilized SMHVs and SMHVs-REDV, MADHVs were
sterilized in 75% ethanol for 24 h followed by replacing with sterilized
PBS for at least 5 times. Hydrogel precursor and REDV peptide solution
were filtrated by 0.22 μm filter. DHVs and GA were sterilized in 75%
ethanol as mentioned above. After then, the heart valves were immersed
into DMEM medium with high glucose at the concentration of 0.1 g
mL− 1 for 24 h at 37 ◦ C in the cell incubator to obtain the extract.
HUVECs were seed in a 96-well plate at a density of 2000 cells/well and
cultured for 8 h followed by replacing with 200 μL obtained extract
medium. Cells were cultured for another 1, 3 and 5 days. The culture
medium was changed every two days. Cell viability was detected by
CCK-8 kit according to the manuals. Cells cultured by fresh medium
were set as the control (Blank).
2.11.2. HUVECs proliferation
Valves were prepared and sterilized as mentioned above. Before
HUVECs seeding, the valves were cut into 1 cm × 1 cm pieces and placed
into 48-well plates, followed by placing a sterilized customized glass
ring on the top of each valve to prevent them from floating. HUVECs
were seeded on the valves by adding 1 mL cell suspension (20,000 cells/
mL) and cultured for another 1, 3 and 5 days. Cell viability at each time
point was tested by CCK-8 kit. After CCK-8 testing, valves were rinsed
thoroughly and fixed by 4% paraformaldehyde. The fixed valves were
dried by CO2 critical point drying and observed by SEM (S-4800, Hita­
chi, Japan). Cells on the samples were stained with Hoechst (1:1000)
and TRITC-phalloidin (1:500), respectively. Images were obtained
under a fluorescence microscope (Olympus IX81, Japan).
Y. Luo et al.
2.12. Subcutaneous implantation
“SMHVs-REDV-EC” indicated SMHVs-REDV valves on which endo­
thelia cells were seeded at the density of 50,000 cells on each side and
cultured for 7 days. Animal experiments were performed according to
the Guidelines for Animal Care and Use Committee of Zhejiang Uni­
versity. Sterilized SMHVs-REDV and GA valves were prepared as
mentioned above. Before implantation, valves were cut into 1 cm × 1 cm
pieces. 3 groups of valves (n = 6 for each group) were implanted sub­
cutaneously and randomly for each Sprague− Dawley (SD) rats. Briefly,
200 g male SD rats were anesthetized by intraperitoneal injection 3%
pentobarbital sodium (at the dose of 1 mL kg− 1), after which the dorsal
hair of SD rats was shaved. Three surgical incisions were created for each
rat on the back using surgical scissors. After implantation, the incisions
were sewed with surgical suture. After 30 days of implantation, valves
wrapped by a newly formed capsule were excised and fixed with 4%
paraformaldehyde for further characterization.
2.13. Calcium deposition assay
Briefly, the wrapped tissues were removed by tweezers carefully
from the freshly harvested valves. After which the obtained valves were
lyophilized, weighted and hydrolyzed with 6 M HCl for 24 h at 96 ◦ C.
The supernatants were filtered and diluted as 1:10 in deionized water
before ICP-OES (730-ES, Varian, USA) analysis. The calcium contents
were normalized to the dry weights of the implanted valves.
2.14. Histological and immunohistological assay
Freshly fixed valves were dehydrated and embedded into the paraffin
before being sectioned into 3 μm sections for future staining. H&E
staining was utilized to visualize the cells. Alizarin red staining was
utilized to verify the calcium deposition in the implanted valves.
For immunohistochemical (IHC) staining, the sections were depar­
affinized and rehydrated followed by antigen retrieval. After then, the
sections were incubated with primary antibodies at 4 ◦ C overnight.
Rabbit antirat F4/80 antibody (Affinity Biosciences; dilution 1:100) and
rabbit antirat CD3 antibody (Servicebio; dilution 1:100) were utilized to
label macrophages and T cells, respectively. The conjugated primary
antibodies were visualized by HRP-labeled goat antirabbit IgG (Serv­
icebio; dilution 1:200) and DAB kit (Servicebio). Cell nuclei were stained
with hematoxylin. Image J software was used to analyze the positive
cells quantitatively.
2.15. Statistical analysis
All experimental results were presented as mean ± standard devia­
tion (SD). A one-way analysis of variance was performed to assess the
significant difference among different groups. p value <0.05 was
considered statistically significant.
3. Results
3.1. Optimization of hybrid hydrogel composition and REDV peptide
density
The hybrid hydrogels with different ratios of MAHA and SBMA were
prepared to optimize the anti-fouling property. As shown in Fig. S2a,
fewer adhered cells were observed on pure MAHA (its successful syn­
thesis was confirmed in Fig. S1) substrate compared with TCP control,
which indicated the excellent anti-fouling property of MAHA. Moreover,
with the adding of SBMA, the hybrid hydrogel substrates showed better
anti-fouling property even compared with pure MAHA substrate.
Quantitative result of adhered cells was shown in Fig. S2b. 3% MAHA-
10% SBMA was chosen as the optimized composition of hybrid hydrogel
for the further experiments.
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REDV peptide was utilized to facilitate the adhesion of endothelial
cells. As shown in Fig. S2c, obvious increasing adhesion of cells can be
observed after the grafting of REDV peptide. The substrate modified by
2 mg mL− 1 REDV was proved to be effective on promoting cell adhesion
on the surface of anti-fouling hydrogel film. Quantitative result of
adhered cells was shown in Fig. S2d. The number of adhered cells
increased by nearly 15-fold after the grafting of REDV peptide. The
grafting densities of REDV peptide on the surface of hydrogel were
measured by QCM-d. As shown in Fig. S3b, the grafting densities of
REDV after incubation in 2 mg mL− 1 and 4 mg mL− 1 peptide solution
were 460.2 ± 118.2 ng cm− 2 and 501.9 ± 127.9 ng cm− 2, respectively.
No significant difference can be observed between these two groups,
which indicated that REDV incubation concentration at 2 mg mL− 1 was
enough for peptide grafting and HUVECs adhesion. Representative
QCM-d curves were shown in Fig. S3a.
To evaluate the selective adhesion of endothelial cells of REDV
peptide, SMCs and HUVECs were coculture on the surface of hydrogel
substrates at the ratio of 1/1. As shown in Fig. S4a, increasing adhesion
of SMCs (red) can be observed with the grafting of REDV peptide. For
hydrogel substrate without REDV modification, neither HUVECs nor
SMCs exhibited excellent adhesion performance. As shown in Fig. S4b,
the number of adhered HUVECs was higher (3-fold) than that of SMCs
for the substrate grafted with REDV peptide.
3.2. Characterization of MADHVs
MA was introduced into DHVs by the reaction between amino group
and anhydride, therefore, 1H NMR spectra were utilized to verify the
successful modification of MA into DHVs directly. As shown in Fig. S5a,
two obvious peaks (5.3 ppm and 5.6 ppm) revealed the successful
grafting of vinyl groups in MADHVs compared with DHVs.
The content of remnant amino groups can be considered as the
modification degree of MA. As shown in Fig. S5b, the higher amino
group content indicated the lower modification degree of MA. Gener­
ally, the amino content decreased with the increase of MA feed ratio.
However, the amino content kept constant despite the feed ratio
increased from 0.5 to 5.0, which indicated the complete reaction be­
tween amino group and MA. MADHVs-0.5 was chosen for the further
experiments.
3.3. Morphologies of heart valves
As shown in Fig. 1, obvious fiber can be observed on the surface of
DHVs, however, the surface of SMHVs became smooth after being
coating by hybrid hydrogel films with no fibers exposed in the air. The
cross-section images showed clear boundary lines between heart valves
and hybrid hydrogel films. Modification of REDV peptide had tiny in­
fluence on the morphologies of SMHVs.
The mass of hydrogel which was integrated onto SMHVs-REDV was
16.0 ± 2.6 mg per piece of heart valve. As shown in Fig. S6, SMHVs-
REDV was stained into light blue. Due to the difference of glycosami­
noglycan contents between hydrogel and heart valve, the interface be­
tween hydrogel and heart valve can be observed.
3.4. Collagen stability
To evaluate the stability of collagen, collagenase digestion treatment
was conducted to simulate the degradation behavior in vivo. As shown in
Fig. 2a, after the collagenase treatment for 24 h and 48 h, the weight
persistence ratios of SMHVs, SMHVs-REDV and GA group were much
higher than DHVs group, which indicated the collagen stability in
SMHVs was enhanced by the hybrid hydrogel coating treatment. Over­
all, similar collagen stability can be achieved in SMHVs and SMHVs-
REDV compared with GA, with nearly 90% weight persistence ratios.
The result implied that hybrid hydrogel coating treatment might be
benefit for improving the stability of collagen after implantation.
Y. Luo et al. Materials Science & Engineering C 128 (2021) 112329

Fig. 1. Morphologic characterization of heart valves. (a) surface and (b) cross section images of heart valves observed by SEM in group DHVs, SMHVs and SMHVs-
REDV, respectively.

Fig. 2. Collagen stability and mechanical property of heart valves. (a) Weight persistence ratio after collagenase treatment for 24 h and 48 h and (b) the thermal
shrinkage temperature of collagen in group DHVs, SMHVs and SMHVs-REDV and GA, respectively; (c) Elastic modulus and (d) fracture strain of heart valves in group
Native, DHVs, SMHVs and SMHVs-REDV and GA, respectively. *, ** and *** indicate p < 0.05, 0.01 and 0.001, respectively.

DSC analysis was conducted to determine the thermal shrinkage
temperature of collagen, a key parameter related to collagen stability. As
shown in Fig. 2b, the thermal shrinkage temperature of SMHVs (76.6 ±
0.9 ◦ C) and SMHVs-REDV (78.7 ± 5.1 ◦ C) were significantly higher than
that of DHVs (66.6 ± 0.2 ◦ C), which indicated an excellent stability after
hybrid hydrogel coating treatment. Though the thermal shrinkage
temperature of GA (87.8 ± 1.7 ◦ C) was much higher. Representative DSC
curve was shown in Fig. S7.

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3.5. Mechanical properties
As shown in Fig. 2c, as expected, the elastic moduli of SMHVs (18.7
± 0.6 MPa), SMHVs-REDV (19.3 ± 2.2 MPa) and GA (20.5 ± 8.2 MPa)
were significantly higher than that of DHVs (13.2 ± 1.0 MPa). Similar
tendency can also be observed in terms of fracture strain (Fig. 2d).
However, no significant difference of fracture tensile strength (Fig. S8a)
can be observed among the five groups. Representative stress-strain
curves were presented in Fig. S8b.

Fig. 3. Hemocompatibility of heart valves. (a) SEM images, (b) LDH quantification of adhered platelets and (c) hemolysis rate of heart valves in group DHVs, SMHVs
and SMHVs-REDV and GA, respectively. *** indicates p < 0.001.

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3.6. Hemocompatibility performance
To evaluate the hemocompatibility performance of the heart valves,
platelets adhesion assay and hemolysis rate assay were conducted. As
shown in Fig. 3a, obvious platelets adhesion in DHVs and GA can be
observed by SEM. After hybrid hydrogel coating, the surface was
endowed with superior anti-fouling property, on which no obvious
platelet adhesion can be observed. Quantitative result of adhered
platelets was shown in Fig. 3b. The LDH activity of DHVs and GA was
much higher than that of the SMHVs and SMHVs-REDV, which was
consistent with the SEM images.
The hemolysis rates of DHVs, SMHVs, SMHVs-REDV and GA were
1.4 ± 0.9%, 1.5 ± 0.3%, 2.1 ± 1.4% and 2.5 ± 0.7%, respectively. No
significant difference can be observed among these four groups. All the

Fig. 4. In vitro biocompatibility of heart valves. Cell viabilities of HUVECs after culturing with the extraction (a) or seeding (b) on the surface of heart valves in group
DHVs, SMHVs, SMHVs-REDV and GA, respectively; (c) Representative SEM images of HUVECs after growth on the surface of the above heart valves for 5 days. ***
indicates p < 0.001.

7
Y. Luo et al.
hemolysis rates that were low enough to be acceptable for further ex­
periments. Representative images were shown in Fig. S9.
3.7. In vitro biocompatibility
3.7.1. In vitro cytotoxicity
As shown in Fig. 4a, the cell viabilities of DHVs, SMHVs and SMHVs
increased with culture time, and no significant difference was observed
among them, which demonstrated that hybrid hydrogel coating treat­
ment showed no cytotoxicity. On the contrast, GA treatment showed
obvious cytotoxicity to endothelia cells with only 70% cells survival
after being cultured for 5 days.
Fig. 5. Morphologic characterization of heart valves after subcutaneous implantation for 30 days. (a) General view and (b) H&E staining images of heart valves in
group SMHVs-REDV, SMHVs-REDV-EC and GA, respectively.
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3.7.2. HUVECs proliferation on heart valves
HUVECs were seeded on the surface of heart valves to evaluate the
endothelialization ability potential of the heart valves, and the cell vi­
abilities were measured by CCK-8 kit. As shown in Fig. 4b, the cell vi­
abilities of DHVs and SMHVs-REDV were much higher than those of the
other two groups after 5 days of culture, which demonstrated that REDV
modification of SMHVs was necessary for facilitating the adhesion and
proliferation of HUVECs. As expected, obvious cells can be observed on
the surface of group DHVs and SMHVs-REDV under SEM (Fig. 4c), which
was consistent with CCK-8 result. Fluorescence images of HUVCEs were
shown in Fig. S10. Large number of HUVECs were stained on the surface
of group DHVs and SMHVs-REDV and nearly no cells was stained on the
other two groups.
Y. Luo et al. Materials Science & Engineering C 128 (2021) 112329

3.8. In vivo biocompatibility and immune response 3.9. In vivo calcification

After 30 days of subcutaneous implantation, the valves were har­
vested and implemented with histological staining and immunohisto­
chemical staining. For all the three groups, no obvious loss or shrinkage
of heart valves was observed from the image in Fig. 5a, which was
consistent with the results of collagen stability. For GA group, H&E
staining images revealed that the inflammatory cells might have been
recruited on the interface between heart valves and surrounding tissues.
However, fewer cells can be observed on the interface between heart
valves of the other two groups and their surrounding tissues (Fig. 5b).
After then, immunohistochemical staining was conducted to eval­
uate the immune response to all groups after 30 days of subcutaneous
implantation. Macrophages and T cells were stained by F4/80 antibody
and CD3 antibody, respectively. Increasing numbers of F4/80 and CD3
positive cells were only observed in GA group compared with the other
two groups (Fig. 6a, c). As expected, significant difference in number of
both macrophages and T cells were observed between hybrid heart
valves and GA treated heart valves (Fig. 6b, d). The results demonstrated
that hybrid hydrogel coated heart valves elicited much mitigated im­
mune response than GA group. The number of F4/80 positive macro­
phages (Fig. 6a, b) was much higher than that of CD3 positive T cells
(Fig. 6c, d).
The heart valves were implanted subcutaneously for 30 days to
evaluate in vivo calcification. Alizarin red staining was conducted to
evaluate the calcium deposition on the harvested valves. As shown in
Fig. 7a, obvious calcium deposition (deep red) can only be observed in
group GA, in which inevitable calcification occurred. ICP-OES analysis
was performed to determine the calcification quantitatively. As shown
in Fig. 7b, the content of calcium of group SMHVs-REDV, SMHVs-REDV-
EC and GA were 3.3 ± 1.2, 4.8 ± 1.5 and 17.3 ± 4.5 mg per gram of dry
samples, respectively. The results suggested that hybrid hydrogel
coating treated heart valves had superior anti-calcification potential
compared with GA treated ones.
4. Discussion
Due to the urgent need for valve replacement surgery, BHVs has
become one major type of valve substitutes for the last half-century.
Despite the superior properties of BHVs, BHVs may still suffer from
deterioration and dysfunction due to the inevitable calcification [32].
Currently, most of commercial BHVs products are crosslinked with GA,
which is benefit for stabilizing the heart valves. However, several
drawbacks, such as cytotoxicity [7,9], immune response [6,7,33] and
calcification [33,34], were frequently met in GA crosslinked BHVs.
Several non-GA crosslinkers, such as tannic acid [13], quercetin [14]

Fig. 6. Immune response elicited by heart valves after subcutaneous implantation for 30 days. (a) Representative IHC images stained with F4/80 and (b) quantitative
analysis of macrophages surround the implants; (c) Representative IHC images stained with CD3 and (d) quantitative analysis of T cells surround the implants. Cell
number of T cells and macrophages were calculated by Image J software. 0.01 mm2 square areas near the interface of the implants were chosen randomly and the
cells inside were counted and normalized to the cells per mm2. *** indicates p < 0.001.

9
Y. Luo et al.
Fig. 7. In vivo calcification after subcutaneous implantation for 30 days. (a) Alizarin red staining images and (b) quantitative analysis of calcium content by ICP-OES.
** indicates p < 0.01.
and carbodiimide [15], have been utilized to replace or coordinate with
GA to reduce the calcification and improve the biocompatibility of
BHVs. Besides, several studies have revealed that GA-crosslinked BHVs
facilitated the adhesion of platelets and protein, which indicated a risk
of coagulation [35]. In summary, construction of BHVs capable of anti-
calcification and deactivating coagulation is benefit for prolong the
lifespan.
Previous studies have demonstrated that anti-fouling hydrogel
hybrid porcine pericardium exhibited improved anti-calcification
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Materials Science & Engineering C 128 (2021) 112329
performance and biocompatibility [16]. However, the ability to repel
non-specific adhesion of protein and platelet still need to be improved.
Zwitterionic hydrogels have been widely used in biomedical field due to
the superior anti-fouling property [18–20,36]. Zwitterions monomers or
polymers can be introduced onto the target surface through covalent
bonding, such as ATRP [23] and free radical polymerization [16]. In
present study, MADHVs was prepared according the previous research
[6,8]. SBMA monomer was polymerized into hydrogel on the surface of
MADHVs with newly formed chemical bond between hydrogel and heart
Y. Luo et al.
valves. Furthermore, to improve the endothelialization potential of
hydrogel films coated heart valves, REDV peptide was chosen as the
endothelial cell-affinity molecule. MAHA was added as the supplier of
remnant double bonds to facilitate the grafting of REDV peptide.
SMHVs-REDV exhibited serval advantages. Firstly, the preparation
procedure is simple and convenient, which is benefit for the further
product development. Secondly, hybrid hydrogel is coated onto the
surface of heart valves through stable covalent bonding. Finally, anti-
calcification, anti-fouling and endothelialization properties can be
realized simultaneously.
SBMA/MAHA hybrid hydrogel precursor was cured by UV light on
the surface of MADHVs. It's necessary to ensure that amino groups of
DHVs can be methacrylated successfully. The content of free amino
groups was detected as a parameter of vinyl groups grating density. 1H
NMR spectra also demonstrated that vinyl groups were grafted into
DHVs successfully.
REDV peptide has been utilized as endothelial cell-affinity peptide to
improve the endothelialization potential of implanted biomaterials,
especially for cardiovascular devices [37,38]. Thiol-end REDV peptide
was grafted onto the surface of SMHVs through “click” reaction, which
was confirmed by the increasing adhesion of HUVECs. HUVECs
exhibited excellent adhesion performance than SMCs on the REDV-
modified cell culture platform, which demonstrated endothelial cells
affinity of REDV.
Generally, native heart valves are covered by a layer of endothelial
cells, named as valve endothelial cells, which play a key role in modu­
lating homeostasis of heart valves [39]. After decellularization, ECM
fibers were exposed in the air, which were covered by newly cured
hybrid hydrogel film in SMHVs.
Collagen is one of the main components of heart valves, which plays
a key role in load-bearing capability. Therefore, the stability of collagen
determines the structural and mechanical function of heart valves post-
implantation. Commercial BHVs products are mainly crosslinked by GA
because of the abundance of amino groups, by which collagen can be
stabilized via Schiff base reaction. Previous researches have revealed
that methacrylated DHVs can be crosslinked through radical polymeri­
zation, which endowed the heart valve with similar collagen stability
compared with GA crosslinked ones [6,8]. In present study, SMHVs
showed similar collagenase digestion resistance compared with GA
group, which indicated an obvious improvement of collagen stability
after hybrid hydrogel coating. It's hypothesized that potential cross­
linking may be introduced because of the existence of vinyl groups after
UV exposure. The hydrogel film acted as a barrier preventing SMHVs
from collagen digestion. As expected, the thermal shrinkage tempera­
tures of SMHVs and GA were significantly higher than that of DHVs.
Results of in vivo subcutaneous implantation for 30 days confirmed the
excellent collagen stability of SMHVs with intact structure reserved.
Biomechanical properties are crucial for maintaining the normal
functions of BHVs post-implantation due to millions of opening and
closing procedures. According to the results, hybrid hydrogel coating
treatment significantly enhanced the biomechanical properties of heart
valves compared with DHVs. After UV exposure, hybrid hydrogel film
was fabricated via free radical polymerization. Meantime, potential
crosslinking may be introduced among the collagen fibers, which was
beneficial to collagen stability and biomechanical properties.
It is believed that blood compatibility is essential for implanted
cardiovascular devices, including heart valve substitutes. Undesirable
protein absorption, platelet adhesion and blood coagulation result in the
dysfunction of medical device, which should be amended for clinical
usage. Despite the good hemocompatibility of BHVs, patients post-
surgery may still suffer from thrombosis [40]. Studies showed that
protein absorption and platelet adhesion cannot be avoided on the
surface of GA crosslinked heart valves [16,35]. In present study, SBMA/
MAHA hybrid hydrogel film was introduced onto the surface of
MADHVs, by which the exposed fibers were covered. It has been proved
that zwitterionic hydrogels possess superior anti-fouling property
11
Materials Science & Engineering C 128 (2021) 112329
because of the solvation and hydrogen bonding of the positive and
negative charge groups, which induces the formation of hydration layer,
thus resists non-specific protein absorption [17]. According to the re­
sults, SMHVs exhibited superior resistance to platelet adhesion
compared with GA group, which should be attributed to the anti-fouling
property of zwitterions. Hemolysis rate experiments indicated that all
the heart valves were acceptable. In conclusion, hybrid hydrogel film
coating endowed SMHVs with significant improvement of blood
compatibility.
Re-endothelialization is the common approach to improve the
hemocompatibility of cardiovascular implants [10]. Valve endothelial
cells play an important role in valve development and disease. Intact
layer of endothelial cells can act as a physical barrier which prevents the
undesired protein absorption and platelet adhesion [41]. In addition, re-
endothelialization can prevent the adhesion and proliferation of SMCs
followed by the aggregation of platelet and leucocytes, thus inducing
potential thrombus and inflammation [42]. Currently, residual aldehyde
groups existed in GA crosslinked heart valves have been proved to be
responsible for the inability for re-endothelialization due to the severe
cytotoxicity [6,8]. In present study, the extracts of DHVs, SMHVs and
SMHVs-REDV showed no obvious cytotoxicity. HUVECs tended to
adhere and proliferate on the surface of DHVs and SMHVs-REDV.
Because of the superior anti-fouling property of hybrid hydrogel film,
HUVECs couldn't adhere and proliferate on the surface of SMHVs. All the
above results demonstrated that grafting of REDV peptide facilitate the
growth of HUVECs. In vivo implantation results confirmed the endo­
thelialization potential of SMHVs-REDV (Fig. S11), no matter pre-
seeding HUVECs was conducted or not. However, no obvious loss of
endothelialization potential of group GA has been observed, which may
result from the lack of long-term subcutaneous implantation.
Commercial BHVs products are mainly made from porcine heart
valve or bovine pericardium, which elicit inevitable immune response as
soon as the completion of valve replacement surgery. Studies have
proved that there is closed relationship between immune response and
calcification [6–8,11,34]. T cells and macrophages are usually chosen as
two typical indicators to evaluate the immune response of implants.
Infiltration and aggregation of inflammatory cells, following by secre­
tion of inflammatory cytokines, induce fibrosis and calcification of BHVs
[43]. In present study, all the harvested heart valves were wrapped with
newly formed capsule. CD3 and F4/80 antibodies were utilized as the
surface makers of T cells and macrophages, respectively. Both of SMHVs-
REDV and SMHVs-REDV-EC exhibited fewer CD3 and F4/80 positive
cells infiltration, indicating that hybrid hydrogel film coating was
benefit for easing immune response.
Calcification is the main obstacle which limits the lifespan of com­
mercial BHVs, which stiffen the heart valves, subsequently impeding the
normal opening and closing procedures, and finally causing the deteri­
oration and dysfunction of BHVs. The mechanism of calcification still
remains poorly understood. Cell debris, remnant aldehyde group and
enriched negative carboxyl groups are considered as potential factors for
calcification [12,32,44]. In present study, the calcification was evalu­
ated by subcutaneous implantation model of SD rats. After 30 days of
implantation, SMHVs-REDV and SMHVs-REDV-EC exhibited significant
fewer calcium deposition than GA group, which indicated that SMHVs-
REDV and SMHVs-REDV-EC have superior anti-calcification capability.
5. Conclusion
In this study, one kind of hybrid hydrogel coated heart valve was
fabricated based on free radical polymerization. SMHVs showed excel­
lent collagen stability, biomechanical properties compared with DHVs.
The coating of anti-fouling hybrid hydrogel endowed SMHVs with su­
perior hemocompatibility. Moreover, SMHVs-REDV exhibited better
biocompatibility compared with GA group. Subcutaneous implantation
results confirmed that SMHVs-REDV suffered from reduced calcification
and mitigated immune response. In conclusion, SMHVs-REDV is
Y. Luo et al. Materials Science & Engineering C 128 (2021) 112329

expected to realize anti-calcification and endothelialization simulta­
neously, which is benefit for improving the main drawbacks of com­
mercial BHVs products.
CRediT authorship contribution statement
Yu Luo: Conceptualization, Methodology, Investigation, Formal
analysis, Writing - Original Draft.
Shenyu Huang: Investigation
Lie Ma: Conceptualization, Methodology, Supervision, Funding
acquisition, Writing - Review & Editing
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We acknowledge the financial support by the National Key R&D
Program of China (2016YFC1101000).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.msec.2021.112329.
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