PolyurethaneâCardiolipin Nanoparticle-Modified Decellularized

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Polyurethane-Cardiolipin Nanoparticle-Modified Decellularized Scaffold-Based Vascular Patches for Tissue Engineering Applications Haomiao Zhu, Lei Fu, Lei He, Jun Zhang, Luxia Zhang, Lutao Yang, Yajuan Li, Yue Zhao, Yutong Wang, Hong Mo, and jian Shen ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

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ACS Applied Bio Materials

Polyurethane-Cardiolipin Nanoparticle-Modified Decellularized Scaffold-Based Vascular Patches for Tissue Engineering Applications Haomiao Zhu,†,§ Lei Fu,†,§ Lei He,† Jun Zhang,† Luxia Zhang,† Lutao Yang,† Yajuan Li,† Yue Zhao,† Yutong Wang,‡ Hong Mo,†,* Jian Shen†,*

†Jiangsu

Collaborative Innovation Center of Biomedical Functional Materials, National

and Local Joint Engineering Research Center of Biomedical Functional Materials, Jiangsu Engineering Research Center for Biomedical Function Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China ‡College

of Materials Science and Engineering, Nanjing Forestry University, Nanjing

210037, China

§

These authors contributed equally to this work.

*Authors to whom all correspondence should be addressed E-mail Address: [email protected] or [email protected] Tel: +86-25-83598031, Fax: +86-25-83598031

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Vascular patches based on a decellularized scaffold (DCS) have received considerable attention for treatment of vascular defects caused by cardiovascular diseases. In present work, we fabricated a polyurethane-cardiolipin/polyurethane composite film (PU-CL/PU) by cosedimentating PU-CL nanoparticles in a PUsaturated ethanol solution onto a PU film, and we evaluated the biocompatibility of the composite film. We also fabricated a PU-CL/PU/DCS vascular patch (CLVP) and investigated its in vivo performance in a mouse model. The PU-CL/PU film showed improved biocompatibility features, such as a prolonged in vitro coagulation time, improved nonhemolytic properties, enhanced resistance to platelet adhesion, reduced cytotoxicity, and enhanced affinity for endothelial progenitor cells. The B ultrasound and the Doppler spectrum results indicated that the CLVP maintained blood vessel patency 30 days after implantation. In addition, endothelialization at the surgical site was achieved. Therefore, the CLVP may have great potential for the treatment of diseased or damaged blood vessels. Keywords: vascular patch, decellularized scaffold, cardiolipin, polyurethane, biocompatibility

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 INTRODUCTION Cardiovascular diseases are the principal cause of death globally, which affect more than ten million patients.1–3 Many of these diseases require surgical treatment with vascular patches to repair blood vessel defects.4–7 Autologous vascular patches are considered the best choice for treating vascular defects because they are nonimmunogenic and have the same mechanical properties as the recipient’s vascular tissue. However, suitable donor sites may not be available due to diseases or previous harvest, which largely limits their clinical use.8,9 Vascular patches based on synthetic polymers,

including

expanded

poly(tetrafluoroethylene)

and

poly(ethylene

terephthalate), have a high postoperative failure rate due to their susceptibility to calcification, inflammation and thrombosis.10 Vascular patches derived from allogeneic and xenogeneic tissues have recently attracted considerable attention due to their good source and high availability.11–14 Although immune response is a concern for allogeneic and xenogeneic tissues, it can be eliminated by removing cellular antigens through decellularization.13 Additionally, the decellularized products (namely, decellularized scaffolds) have a high mechanical similarity to native tissues due to the retention of structural components, which can provide geometry support for regenerating tissues.15,16 Moreover, the retained extracellular matrices (ECMs) can constitute a physiological microenvironment, transmit biochemical and cellular signals, and guide cell adhesion and tissue integration. However, the decellularized scaffold (DCS) cannot be used as a vascular patch directly due to its porous structure. Therefore, it generally needs coating with a polymeric film

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such as polyurethane (PU) film.2,5 Polyurethanes (PUs) are extensively used in biomedical applications, including catheters and aortic devices, due to their excellent mechanical properties and good biocompatibility.17,18 However, the long-term cell and blood compatibilities of PU surfaces are a major hurdle that hinders their widespread use for biomedical purposes.19–21 To overcome these shortcomings, researchers have modified surfaces with molecules or moieties such as peptides and phospholipids.5,22 Cardiolipin (CL) is an unusual phospholipid with two units of negative charge in the head and four long acyl chains in the tail. It exists in the inner (20%) and outer (5–10%) membrane of mitochondria of eukaryotic cells and plays a vital role in some membrane-dependent processes.23–25 Therefore, introducing CL onto the surface of a vascular patch may be an effective approach to improve biocompatibility. In addition, nanoparticle coating onto the surface can mimic the texture of the blood vessel luminal surface, which benefits cell adhesion and promotes endothelialization.10 CL-modified PU (PU-CL) nanoparticle coating may be a better choice to improve the biocompatibility and facilitate endothelialization. To our knowledge, this has not been reported in the literature. Herein, in present work, we prepared PU-CL nanoparticles and introduced them onto a PU film surface to obtain a composite PU-CL/PU film, and evaluated its in vitro biocompatibility. We also fibricated a PU-CL/PU/DCS vascular patch (CLVP) by cosedimentating PU-CL nanoparticles in a PU-ethanol solution onto a PU-coated DCS, and investigated its in vivo performance in a mouse model. The PU-CL/PU composite

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film demonstrated improved biocompatibility, including prolonged in vitro coagulation time and plasma recalcification time, improved nonhemolytic properties, enhanced anti-platelet adhesion ability, reduced cytotoxicity, and enhanced affinity for endothelial progenitor cells (EPCs). The B ultrasound and the Doppler spectra showed that the CLVP maintained the patency of the surgical artery at 30 days postimplantation.

In

addition,

the

HE-staining

cell

slice

demonstrated

that

endothelialization at the surgical site was achieved. Therefore, the CLVP may have potential applications in vascular repair.

 MATERIALS AND METHODS Materials.

Poly(tetramethylene

ether)

glycol

(PTMG),

1,1'-methylenebis-(4-

isocyanatobenzene) (methylene diphenyl diisocyanate, MDI) and dimethylolpropionic acid (DMPA) were obtained from Sigma-Aldrich China Corporation. PU (Mn = 65000, PDI = 2.16), ethyl acetate, pyrrolidinone, sodium dodecyl sulphate (SDS), N,Ndimethylformamide (DMF), dibutyltin dilaurate (DBTL) and CL were obtained from Aladdin Reagent Shanghai Limited Company. Pyrrolidinone and ethyl acetate were purified by distillation and then dried over 4 Å molecular sieves. Fresh whole blood, EPCs, DCS, fresh platelet-rich plasma, mice, red blood cells (RBCs) and platelet-poor plasma were obtained from Nanjing Gulou Hospital. The water used in this work was deionized. The phosphate-buffered saline (PBS, pH = 7.4) used in this work was calcium- and magnesium-free. All biomaterials were sterilized with 75% ethanol at 25 °C before use. The PU-CL nanoparticles were prepared according to the previously

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reported method.5 Preparation of the pure PU film. A piece of square glass (2 cm × 2 cm) was soaked in a PU-saturated solution in DMF for 1 min and then dried in vacuum at 40 °C. A pure PU film was obtained by dip-coating three times and was approximately 0.2 mm thick. Preparation of the PU-CL/PU composite film. The PU-CL/PU composite film was prepared according to the previously reported method, by using PU-CL nanoparticles as coating particles.10 The PU-CL/PU composite film was approximately 0.2 mm thick. Scanning electron microscopy (SEM). Surface morphologies of the films were obtained with JSM-7600F SEM (JEOL Co., Japan). X-ray photoelectron spectroscopy (XPS). Spectroscopies were performed on D/max 2500VL/PC X-ray photoelectron spectroscope (Japan) at 1486.6 eV (focused monochromatized Al-Kα radiation), with a radiation area diameter of 500 μm. The residual chamber pressure was 10-8 Pa. The curves were recorded at a constant pass energy of 20 eV. Static water contact angle measurements. The water contact angles were obtained with DSA100 contact angle apparatus (Krüss Co., Germany) at room temperature. Each test was performed in triplicate. Platelet adhesion tests. The platelet adhesion experiments of the films were performed according to the previously reported method.10 Each test was performed in triplicate. Plasma recalcification time measurements. The plasma recalcification times of the films were tested according to the previously reported method.10 Each test was performed in triplicate.

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In vitro coagulation time measurements. The coagulation times of the films were tested according to the previously reported method.10 Each test was performed in triplicate. Hemolysis assays. The hemolysis assays of the films were performed according to the previously reported method.10 Each test was performed in triplicate. Red blood cell morphology measurements. The effects of the films on the morphologies of RBCs were performed according to the previously reported method.10 Each test was performed in triplicate. In vitro cytotoxicity tests. The cytotoxicities of the films were evaluated according to the previously reported method.10 Each test was performed in triplicate. Cell attachment and proliferation measurements. The attachment and proliferation experiments of EPCs on the films were performed according to the previously reported method.10 Each test was performed in triplicate. Preparation of the vascular patches CLVP and PUVP. The vascular patches CLVP and PUVP were prepared according to the previously reported method.5 When preparing the CLVP, PU-CL nanoparticles were used as coating particles. In vivo study. The in vivo performance of CLVPs and PUVPs was investigated according to the previously reported method.5 Histological analysis. Thirty days after implantation, the mouse with CLVP was sacrificed. The CLVP was cut into a section and soaked in 4% formalin for 24 h, and then the section was cut into blocks (5 mm × 5 mm). The blocks were embedded in paraffin. The cells were stained with hematoxylin and eosin (H&E) to analyze the

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formation of endothelial tissue. Statistical analysis. Data were presented as the mean ± standard deviation (SD). The significant difference between groups was analyzed with one way ANOVA and the Bonferroni post hoc test. Statistical significance was accepted for p < 0.05.

 RESULTS Surface characterization. SEM images of the pure PU film and PU-CL/PU are presented in Figure 1. A large number of spherical particles were observed on PUCL/PU whereas none were observed on the pure PU film, indicating that PU-CL nanoparticles were successfully incorporated into the surface of the PU film. Their mean diameter was approximately 300 nm, consistent with those shown in Figure S1 (Supporting Information).

Figure 1. SEM surface morphology images: (A) pure PU film, (B) PU-CL/PU

The XPS spectra of the pure PU film and PU-CL/PU are shown in Figure 2. The PUCL/PU exhibited a characteristic peak of a phosphorous oxygen bond (P2p) at 135 eV, which is consistent with the previously reported data.2,5 In contrast, the pure PU film did not display this P2p characteristic peak because CL contains phosphorus and PU 8


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does not contain it. This indicates that PU-CL nanoparticles were successfully introduced onto the surface of the PU film. P2P

PU-CL/PU

Pure PU

130

132

134

136

138

140

Binding Energy (eV) Figure 2. XPS spectra of the pure PU film and PU-CL/PU

Water contact angle analysis. The water contact angle images of the pure PU film and PU-CL/PU were 97º and 48º, respectively, as shown in Figure 3. The considerable decrease in the angle suggested that the hydrophilicity of PU-CL/PU was substantially improved by PU-CL nanoparticle modification.

Figure 3. Water contact angles: (A) pure PU film, (B) PU-CL/PU

Blood compatibility. SEM images of platelet adhesion on the films are presented in Figure 4. A large number of platelets adhered to the pure PU film (Figure 4A), and 9



some of them were deformed (Figure 4B). In comparison, no platelets adhered to the PU-CL/PU (Figure 4C and 4D). This indicates that the PU-CL/PU film had stronger anti-platelet adhesion ability due to the surface modification with PU-CL nanoparticles.

Figure 4. SEM images of platelet adhesion on the films exposed to PRP for 60 min: (A) pure PU film (×500), (B) pure PU film (×1500); (C) PU-CL/PU (×500), (D) PU-CL/PU (×1500)

The plasma recalcification times (PRT) of the pure PU film and PU-CL/PU are illustrated in Figure 5A. The PRT for the pure PU film and PU-CL/PU were 10.9 and 20.0 min, respectively. The PRT of the PU-CL/PU was significantly prolonged when compared with that of the pure PU film, indicating that the blood compatibility of the film was noticeably improved due to the introduction of PU-CL nanoparticles. The in vitro coagulation times for the films are presented in Figure 5B–D. The APTT of the pure PU film and PU-CL/PU were 88 and 116 s, respectively. The APTT of PU-CL/PU was prolonged by 28 s due to PU-CL nanoparticle modification. Both the PT and TT 10


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showed a similar tendency to the APTT. The PT for the pure PU film and PU-CL/PU were 16 and 23 s, respectively. The TT for the pure PU film and PU-CL/PU were 18 and 25 s, respectively. These data indicate that the introduction of PU-CL/PU nanoparticle effectively improved the antithrombogenicity of the film. 25

140

A

PRT

120

20

B

APTT

15 Time (s)

10

80 60 40

5

20

0

20

20

0 ol

re

tr

PU

C

-C

on

L/

PU re Pu

PU L/ -C

L/ PU

0 PU

5

ol

5

tr

PU

10

-C

10

15

PU

15

PU

Time (s)

25

on

D

TT

25

C

re

30

C

PT

Pu

on C

PU

30

PU

ol tr

PU L/ -C

re Pu

C

on

tr

PU

ol

0

Pu

Time (min)

100

Time (s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (A) plasma recalcification time (PRT); in vitro coagulation times for the pure PU film and PU-CL/PU: (B) APTT, (C) PT, (D) TT.

The hemolysis rates of the pure PU film and PU-CL/PU were 1.62 and 0.86, respectively, as listed in Table S1. They were both nonhemolytic according to ASTM F756-17 since their hemolysis rates were less than 2%. The morphologies of RBCs exposed to 0.9% saline, the pure PU film and PU-CL/PU are presented in Figure S5. Compared with normal RBCs (Figure S5A), some RBCs exposed to the pure PU film were deformed or even ruptured (Figure S5B) while those exposed to PU-CL/PU 11



displayed normal morphology (Figure S5C). This indicates that the hemocompatibility of the film was improved by introducing PU-CL/PU nanoparticles. In vitro cytotoxicity. The viabilities of EPCs in extracts of the films for different incubation time are illustrated in Figure 6. For the pure PU film, the cell viabilities were 94.7% at 6 h, 88.6% at 12 h, and 73.6% at 24 h, respectively. For PU-CL/PU, the cell viabilities were 98.7% at 6 h, 97.8% at 12 h, and 95.6% at 24 h, respectively. At every corresponding time point, the cell viability of PU-CL/PU was higher than that of the pure PU film. In addition, the cell viabilities of the two films decreased with the increasing incubation time, and the cell viability of the pure PU film declined faster. This suggests that PU-CL/PU was more compatible with the cells than the pure PU film. 6h 12h 24h

125

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 75 50 25 0

C

o tr on

l p

e ur

U

PU PU

-C

P L/

Figure 6. The cell viabilities in extracts of the pure PU film and PU-CL/PU.

Cell attachment and proliferation. The images of the attachment and proliferation of EPCs for the films are shown in Figure S6. More EPCs adhered to the PU-CL/PU than to the pure PU film at 4, 12, 24 and 48 h of incubation, demonstrating that the PU-

12


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CL/PU had better affinity for EPCs than the pure PU film. Additionally, as the incubation time increased, more EPCs adhered to PU-CL/PU. In vivo study. The B ultrasound images for the PUVP and CLVP at 2 days postimplantation are illustrated in Figure 7A and 7C, respectively. In addition, the Doppler spectra for the PUVP and CLVP at 2 days post-implantation are presented in Figure 7B and 7D, respectively. In Figure 7A, the B ultrasound for the PUVP appeared black, revealing that there was no blood flowing through the surgical artery and the artery was blocked. In addition, as shown in Figure 7B, the Doppler spectrum for the PUVP did not exhibit any signal, which implies that blood did not circulate in the surgical artery. In contrast, in Figure 7C, the B ultrasound for the CLVP appeared red and orange, suggesting that blood flowed through the surgical artery and the artery was not blocked. Additionally, the Doppler spectrum for the CLVP showed strong signals, indicating that blood circulated in the surgical artery (Figure 7D). These findings demonstrated that the CLVP possessed better biocompatibility than the PUVP.

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Figure 7. At 2 days post-implantation, (A) B ultrasound of the PUVP, (B) Doppler spectrum of the PUVP, (C) B ultrasound of the CLVP, (D) Doppler spectrum of the CLVP.

At 30 days post-implantation, the CLVP surgical site appeared red and orange, showing that blood was flowing through the surgical artery (Figure 8A). In addition, the strong signals on the Doppler spectrum indicate that the surgical artery was not blocked (Figure 8B). In Figure 8C, a confluent layer of endothelial cells was observed at the surgical site, indicating that endothelialization was achieved.

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Figure 8. At 30 days post-implantation, (A) B ultrasound of the CLVP, (B) Doppler spectrum of the CLVP, (C) HE staining cell slice from the surgical artery with CLVP.

 DISCUSSION The high incidence of cardiovascular diseases necessitates efficient and effective development of applicable vascular grafts for surgical treatment of diseased blood vessels. DCS-based vascular grafts have received much attention due to their good sources, nonimmunogenicity and comparable mechanical properties to the recipient’s blood vessels.2,4,5,9,12 For instance, Bai et al. used the decellularized carotid artery of a mouse as an allograft in a murine model. The surgical blood vessel demonstrated high patency and good endothelialization effect.9 In this work, the DCS was derived from canine carotid artery. The vascular patch based on the DCS, as a xenograft, maintained 30-day patency of the surgical artery. In addition, endothelialization at the surgical site was achieved. We also investigated the physical properties of the PU-CL/PU film before it was used to coat the DCS. Hydrophobic surfaces easily induce nonspecific protein adsorption, followed by platelet adhesion and aggregation, leading to coagulation cascades.26,27 In this work, the PU surface was hydrophobic, with a water contact angle of 97°. Thus, it was necessary to change its hydrophobicity. In PU-CL nanoparticles, the hydrophilic headgroup of the CL is outward and the hydrophobic alkyl chains are inward. As a result, after the film was modified with PU-CL nanoparticles (PU-CL/PU), its surface was hydrophilic, with a water contact angle of 48°. Therefore, PU-CL/PU demonstrated enhanced resistance to platelet adhesion (Figure 4). In addition, the nanoparticle modification did not adversely influence the thermal stability of the film since the pure 15



PU film and PU-CL/PU had the almost same initial decomposition temperature of approximately 322 °C (Figure S4). Similarly, the nanoparticle modification did not impair the mechanical properties of the film (Figure S7). CL exists in the inner and outer membrane of mitochondria of eukaryotic cells and thus is considered biocompatible. Compared with the pure PU film, the PU-CL/PU demonstrated improved biocompatibility, including prolonged in vitro coagulation time and enhanced affinity for EPCs. For example, the APTT for the film increased from 88 to 116 s due to PU-CL nanoparticle modification. In our previous work, we used phosphatidylcholine-PU nanoparticles to modify pure PU film to obtain a phosphatidylcholine-PU/PU film. This film also shows improved biocompatibility because phosphatidylcholine, a main component of phospholipid bilayer of cell membranes, is biocompatible.5 For instance, the APTT for the pure PU film is 91.7 s. After surface modification with phosphatidylcholine-PU nanoparticles, the APTT increased to 117.2 s. This significantly improves the antithrombogenic ability of the film. CL and phosphatidylcholine are phospholipids present in cell membranes. Thus, surface modification with them provides biomembrane mimicry for the materials and can improve their biocompatibility.5,28 Improving antithrombogenic ability is crucial to inhibiting the coagulation cascade and preventing thrombus formation for a diseased blood vessel. In a normal blood vessel, the endothelium, located in the innermost layer, can mediate the balance between thrombogenicity and nonthrombogenicity, thereby maintaining blood vessel patency. In a diseased blood vessel, the endothelium cannot work properly, which may

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lead to the coagulation cascade and thrombus formation.29,30 In this work, the antithrombogenic ability of the material was obviously improved, and thus the vascular patch inhibited the coagulation cascade, maintaining blood vessel patency (Figure 8A and 8B). In addition, enhancing the affinity for EPCs can contribute to endothelialization. EPCs are a subset of stem cells with the phenotypes and functions of endothelial cells and can eventually differentiate into mature endothelial cells. Although EPCs can home to the diseased or damaged sites of a blood vessel, EPCs circulate in bloodstream at relatively low concentration. Hence, enhancing the ability to attract EPCs can efficiently recruit them and accelerate endothelialization.31,32 In this work, the cell affinity of the surface was enhanced by surface modification with PU-CL nanoparticles. The patch could effectively capture EPCs from bloodstream and facilitate endothelialization, which was achieved at 30 days post-implantation (Figure 8C).

 CONCLUSIONS PU-CL nanoparticle modification improved the biocompatibility of the material. The improvement in surface hydrophilicity enabled the PU-CL/PU film to effectively inhibit platelet adhesion. The enhancement in antithrombogenic ability enabled the CLVP to prevent the coagulation cascade and maintain the patency of the surgical artery. The enhancement in the cell affinity enabled the CLVP to accelerate endothelialization. Therefore, the CLVP may have great potential in repairing diseased or damaged blood vessels.

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 ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Preparation of PU-CL nanoparticles; Characterization of PU-CL nanoparticles; TEM, SEM and the size distribution of PU-CL nanoparticles; 31P NMR spectra of PU-CL nanoparticles; EDS spectrum of PU-CL nanoparticles; TGA curves of the pure PU film and PU-CL/PU; The hemolysis assays for the pure PU film and PUCL/PU; Morphologies of red blood cells; Cell attachment and proliferation; Mechanical properties of the pure PU film and PU-CL/PU.

 AUTHOR INFORMATION Corresponding Authors *Dr. Hong Mo: [email protected] *Prof. Jian Shen: [email protected] ORCID Haomiao Zhu: 0000-0003-0045-9033 Lei Fu: 0000-0003-3822-2613 Hong Mo: 0000-0002-6374-3632 Author Contributions §

H.Z. and L.F.: These authors contributed equally to this work.

Notes 18


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The authors declare no competing financial interest.

 ACKNOWLEDGEMENTS The authors are grateful for the assistance Dr. Cheng Liu and Prof. Tong Qiao have provided.

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Nanoparticles

onto

a

Decellularized

Scaffold

Facilitates

Endothelialization. J. Biomater. Tiss. Eng. 2018, 8 (7), 979−988. (3) Radke, D.; Jia, W.; Sharma, D.; Fena, K. Tissue Engineering at the BloodContacting Surface: A Review of Challenges and Strategies in Vascular Graft Development. Adv. Healthc. Mater. 2018, 7 (15), 1701461. (4) Tu, Q. F.; Zhang, Y.; Ge, D.X.; Wu, J.; Chen, H. Q. Novel Tissue-Engineered Vascular Patches Based on Decellularized Canine Aortas and Their Recellularization in Vitro, Appl. Surf. Sci. 2008, 255 (2), 282−285. (5) Zhang, J.; Liu, C.; Feng, F. L.; Wang, D. W.; Lu, S. S.; Wei, G.; Mo, H.; Qiao, T. A PC-PU Nanoparticle/PU/Decellularized Scaffold Composite Vascular Patch:

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Synergistically Optimized Overall Performance Promotes Endothelialization. Colloid. Surface. B 2017, 160, 192−200. (6) Chantawong, P.; Tanaka, T.; Uemura, A.; Shimada, K.; Higuchi, A.; Tajiri, H.; Sakura, K.; Murakami, T.; Nakazawa, Y.; Tanaka, R. Silk Fibroin-Pellethane® Cardiovascular Patches: Effect of Silk Fibroin Concentration on Vascular Remodeling in Rat Model. J. Mater. Sci-Mater. Med. 2017, 28 (12), 191. (7) Lehr, E. J.; Rayat, G. R.; Chiu, B.; Churchill, T.; McGann, L. E.; Coe, J. Y.; Ross, D. B. Decellularization Reduces Immunogenicity of Sheep Pulmonary Artery Vascular Patches. J. Thorac. Cardiov. Sur. 2011, 141 (4), 1056−1062. (8) Lee, H.; Yang, J. H.; Jun, T. G.; Cho, Y. H.; Kang, I. S.; Huh, J.; Song, J. Y. Augmentation of the Lesser Curvature with an Autologous Vascular Patch in Complex Aortic Coarctation and Interruption. Ann. Thorac. Surg. 2016, 101 (6) 2309−2314. (9) Bai, H.; Dardik, A.; Xing, Y. Decellularized Carotid Artery Functions as an Arteriovenous Graft. J. Surg. Res. 2019, 234, 33−39. (10)Zhang, J.; Wang, Y. T.; Liu, C.; Feng, F. L.; Wang, D. W.; Mo, H.; Si, L.; Wei, G.; Shen,

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