Butterfly-Inspired Multiple Cross-Linked Dopamine-Metal-Phenol Bioprosthetic Valves with Enhanced Endothelialization and Anticalcification

Abstract

Valve replacement is the most effective means of treating heart valve diseases, and transcatheter heart valve replacement (THVR) is the hottest field at present. However, the durability of the commercial bioprosthetic valves has always been the limiting factor restricting the development of interventional valve technology. The chronic inflammatory reaction, calcification, and difficulty in endothelialization after the implantation of a glutaraldehyde cross-linked porcine aortic valve or bovine pericardium often led to valve degeneration. Improving the biocompatibility of valve materials and inducing endothelialization to promote in situ regeneration can extend the service life of valve materials. Herein, inspired by the hardening process of butterfly wings, this study proposed a dopamine-metal-phenol strategy to modify decellularized porcine pericardium (DPP). This is a strategy to make dopamine (DA) coordinate trivalent metal chromium ions (Cr(III)) with antiplatelets (PLTs) and anti-inflammatory properties, and then cross-link it with tea polyphenols (TP) to generate a valve scaffold that is mechanically comparable to glutaraldehyde-cross-linked scaffolds but avoids the cytotoxicity of aldehyde and presents better biocompatibility, hemocompatibility, anticalcification, and anti-inflammatory response properties.

Keywords: cross-linking; dopamine; endothelialization; tea polyphenols; transcatheter heart valve replacment.

Figure 1

Screening of suitable reaction concentrations of dopamine. (A) general view of the scaffolds modified with DA at different concentrations. (B) Strain–stress curve of scaffolds. (C) Young’s modulus of scaffolds. (D) Tensile strength of scaffolds. (E) Cytotoxicity of scaffolds measured by the OD450 nm value. (F) PLTs adhered on the scaffolds observed by SEM. (G) Images of thrombus of scaffolds. (H) Relative content of PLTs quantified by LDH assay kits on the surface of scaffolds. (I) Relative content of thrombus on the surface of scaffolds. (J) Representative images of hemolysis of scaffolds. (K) Relative hemolysis rate of scaffolds. Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2

Construction of the scaffolds. (A) Total view of scaffolds modified with different Cr(III). (B) Representative strain–stress curves of scaffolds. (C) Young’s modulus of scaffolds. (D) Tensile strength of scaffolds. (E) Cytotoxicity of scaffolds. (F) Total view of scaffolds cross-linked with TP at different concentrations. (G) Cytotoxicity of leachate from scaffolds at 1 week. (H) Representative strain–stress curves of scaffolds. (I) Young’s modulus of scaffolds. (J) Tensile strength of scaffolds. (K) Cytotoxicity of scaffolds. (L) FTIR spectrum showing the specific peaks of scaffolds. (M) XPS revealed the specific absorption peak of Cr(III) of DA/Cr and DA/Cr/TP scaffolds. Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3

Characteristics of the scaffolds. (A) Representative strain–stress curves of scaffolds. (B) Young’s modulus of scaffolds. (C) Tensile strength of scaffolds. (D) Proliferation of HUVECs of scaffolds on different time points (2, 4, 6, 8, 10 day). (E) Roundness measured by AFM of scaffolds. (F) Hydrophilicity reflected by WCA of scaffolds. (G) Surface morphology captured by SEM. (H) AFM showsthe 2D and 3D presentative images of scaffolds. (I) Images of WCA of scaffolds. Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4

Hemocompatibility of the scaffolds. (A) Images of PLTs adhered on the surface of scaffolds were captured by SEM. (B) Images of thrombus of scaffolds. (C) Representative images of hemolysis of scaffolds. (D) Relative contents of PLTs adhered on the scaffolds were quantified by the LDH assay kit. (E) Relative contents of thrombus of scaffolds were quantified by OD540 nm value. (F) Relative hemolysis rates of scaffolds. Data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 5

Endothelialization capacity and anti-inflammatory properties of the scaffolds. (A) Adhesion and proliferation of HUVECs on the surface of scaffolds on days 4 and 10. (B) Fluorescence intensities of iNOS and Arg-1 of scaffolds were captured by confocal microscope on day 5.

Figure 6

Recelluarization, anticalcification, and anti-inflammation of scaffolds under hemodynamic environment. (A) General view of abdominal aortic replacement and 4 weeks after replacement. (B) Staining for HE, Masson, and von Kossa. (C) Immunological staining of TNF-a, IL-6, and IL-1ß of scaffolds.

Figure 7

Inflammatory cell infiltration of scaffolds in a model of abdominal aortic replacement. (A) Immunological staining for CD3 (T-cell marker), CD31 (endothelial cell marker), CD68 (macrophage marker), iNOS (highly expressed in M1 macrophages), and Arg-1 (highly expressed in M2 macrophages) of scaffolds under a hemodynamic environment for 4 weeks.