Tailoring ZE21B Alloy with Nature-Inspired Extracellular Matrix Secreted by Micro-Patterned Smooth Muscle Cells and Endothelial Cells to Promote Surface Biocompatibility

Abstract

Delayed surface endothelialization is a bottleneck that restricts the further application of cardiovascular stents. It has been reported that the nature-inspired extracellular matrix (ECM) secreted by the hyaluronic acid (HA) micro-patterned smooth muscle cells (SMC) and endothelial cells (EC) can significantly promote surface endothelialization. However, this ECM coating obtained by decellularized method (dECM) is difficult to obtain directly on the surface of degradable magnesium (Mg) alloy. In this study, the method of obtaining bionic dECM by micro-patterning SMC/EC was further improved, and the nature-inspired ECM was prepared onto the Mg-Zn-Y-Nd (ZE21B) alloy surface by self-assembly. The results showed that the ECM coating not only improved surface endothelialization of ZE21B alloy, but also presented better blood compatibility, anti-hyperplasia, and anti-inflammation functions. The innovation and significance of the study is to overcome the disadvantage of traditional dECM coating and further expand the application of dECM coating to the surface of degradable materials and materials with different shapes.

Keywords: ZE21B alloy; biocompatibility; cardiovascular materials; dECM coating; hyaluronic acid micro-patterns.

Figure 1

Preparation processes of nature-inspired ECM coating onto ZE21B by self-assembly.

Figure 2

SEM images and EDS spectra of ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples.

Figure 3

FTIR spectra of ZE21B, MgF₂, PDA ECM 6 h, ECM 12 h, and ECM 24 h samples.

Figure 4

Water contact angles’ analysis of ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples (mean ± SD, n = 3, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 5

(A) 2D and 3D AFM images of ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples. (B) The average surface roughness (Rq) of ZE21B, MgF2, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples (mean ± SD, n = 3, * p < 0.05, and ** p < 0.01).

Figure 6

(A) The fluorescence images of FN (green color) and COL IV (red color) distributed on the ECM 6 h, ECM 12 h, and ECM 24 h samples; (B) 3D images of FN and COL IV fluorescence intensity generated by Ipwin32 software.

Figure 7

Tafel curve images of ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples.

Figure 8

(A) Fibrinogen adhesion on ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples (mean ± SD, n = 5, * p < 0.05, ** p < 0.01, and *** p < 0.001); (B) Fibrinogen denaturation on ZE21B, MgF₂, PDA, and different ECM samples (mean ± SD, n = 5, * p < 0.05, and ** p < 0.01).

Figure 9

(A) SEM images of adherent platelets adhesion on ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples; (B) The number of adherent platelets on ZE21B, MgF₂, PDA, and different ECM samples (mean ± SD, n = 8, * p < 0.05, and ** p < 0.01).

Figure 10

SEM images of components of blood adherent on ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples.

Figure 11

Hemolysis rates of ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples (mean ± SD, n = 5, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 12

Fluorescence images of HUVECs in the ZE21B, MgF₂, PDA, ECM 6 h, ECM 12 h, and ECM 24 h samples (The cells were stained with green color, and the nucleus were stained with blue color).

Figure 13

Cell viabilities of HUVECs in the ZE21B, MgF₂, PDA, and different ECM groups at 4 h, 24 h, and 72 h (mean ± SD, n = 10, * p < 0.05, ** p < 0.01, and *** p < 0.001). The control group was RPMI-1640 complete medium (containing 10% serum and 1% penicillin and streptomycin mixture).

Figure 14

NO released by HUVECs in the ZE21B, MgF₂, PDA, and different ECM groups (mean ± SD, n = 3, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 15

Migration experiment of HUVECs: (A) Optical microscope images at 0 h, 12 h, and 24 h; (B) Scratch width statistics result of each sample at 0 h, 12 h, and 24 h (mean ± SD, n = 5); (C) Total healing distance statistics result of each sample at 12 h and 24 h (mean ± SD, n = 5, * p < 0.05, ** p < 0.01, and *** p < 0.001). The control group was RPMI-1640 complete medium (containing 10% serum and 1% penicillin and streptomycin mixture).

Figure 16

(A) Fluorescence images of SMC stained with α-SMA antibody (green) and DAPI (blue) on each sample; (B) Fluorescence intensity 3D images of α-SMA expression of SMC generated by Ipwin32 software.

Figure 17

(A) CMFDA (green) staining of HUVECs and CFDA (red) staining of SMC on the surface of ZE21B, MgF₂, PDA, and ECM samples; (B) After 6 h, the number of HUVECs and SMC on each sample, and the ratio of HUVECs/SMC (mean ± SD, n = 8, * p < 0.05, and ** p < 0.01); (C) After 24 h, the number of HUVECs and SMC on each sample and the ratio of HUVECs/SMC (mean ± SD, n = 8, * p < 0.05, and ** p < 0.01).

Figure 18

(A) Fluorescence images of macrophages stained with CD206 antibody (green), TNF-α antibody (red) and DAPI (blue) on each sample; (B) Fluorescence intensity 3D images of CD206 and TNF-α generated by Ipwin32 software.