A Novel Crosslinking Method for Improving the Anti-Calcification Ability and Extracellular Matrix Stability in Transcatheter Heart Valves

Figures

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  FIGURE 1. Rib crosslinking method of the bovine pericardium (BP). (A) Schematic illustrations of the mechanism by which Rib stabilizes collagen in the ECM. (B) Effect of Rib crosslinking time on the macroscopic appearance of biomaterials. Scale bars: 3 cm.

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  FIGURE 2. Decellularization process of the BP. (A) H&E staining of the native BP. (B) H&E staining of the decellularized BP. (C) Quantitative determination of the DNA content (n = 4) in the native BP and decellularized BP (scale bars: 200 µm; *p < 0.05, **p < 0.01, and ***p < 0.001; ns represents no significant difference).

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  FIGURE 3. Changes in the reactive groups of the BP. (A) Ninhydrin assay to estimate the conversion of amine groups (n = 3) in the BP. (B) Representative 1H-NMR spectrum of the hydrolyzed product of the BP. Fresh BP, brown; Rib-1d, yellow; Rib-4d, green; Rib-8d, blue; Rib-12d, purple (*p < 0.05, **p < 0.01, and ***p < 0.001; ns represents no significant difference).

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  FIGURE 4. SEM images of BP surface structures before implantation. (A,D) Fresh BP. (B,E) Glut-treated BP. (C,F) Rib-treated BP. Scale bars: 100 μm (A–C) and 10 μm (D–F).

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  FIGURE 5. SEM images of BP surface structures after implantation. (A,C) Glut-treated BP was implanted into rats for 90 days. (B,D) Rib-treated BP was implanted into rats for 90 days. Scale bars: 100 μm (A,B) and 10 μm (C,D).

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  FIGURE 6. Resistance to the collagenase enzymatic challenge of the BP with Masson’s trichrome (MT) staining. (A,D) Fresh BP. (B,E) Glut-treated BP. (C,F) Rib-treated BP. Collagen are stained blue. Scale bars: 500 μm (A–F).

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  FIGURE 7. Resistance to the elastase enzymatic challenge of the BP with elastic van-Gieson (EVG) staining. (A,D) Fresh BP. (B,E) Glut-treated BP. (C,F) Rib-treated BP. Elastin are stained black strips (green arrow). Scale bars: 100 μm (A–F).

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  FIGURE 8. ECM stability of the BP in vitro study. (A,B) Relative weight loss of the BP (n = 6) after 24 h collagenase and 48 h elastase digestion. (C) Young’s modulus of the BP (n = 12) in uniaxial tensile testing. (D) Water content of the BP (n = 4). (E) Thickness of the BP (n = 12). (F) Thermal shrinkage temperature of the BP (n = 6) (*p < 0.05, **p < 0.01, and ***p < 0.001; ns represents no significant difference).

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  FIGURE 9. Cytocompatibility of the biomaterial. (A) Cell viability was tested by MTT assay at 1, 3, and 5 days (n = 3). (B–D) HUVEC growth on fresh BP, Glut-treated BP, and Rib-treated BP after 7 days of culture. Scale bars: 50 μm.

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  FIGURE 10. ECM integrity and histological staining after implanted into rats for 90 days. (A,B) H&E staining of the BP. Nuclei were stained purple, and the cytoplasm was stained pink. The thickness of the fibrous capsule (black double-arrow). (C,D) MT staining of the BP and the collagen are stained blue, and new capillary-like tubes are stained red (black arrow). (E,F) Victoria Blue staining of the BP. Elastin fibers were stained dark blue stripes (black arrow). Scale bars: 500 μm (A–D) and 100 μm (E,F).

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  FIGURE 11. Immunological characterization of the inflammatory response of BP implanted into rats for 90 days. (A,B) IHC images. Macrophages were labeled with the CD68 marker and stained red (white arrow). (C,D) IHC images. T cells were labeled with the CD3 marker and stained red (white arrow). Scale bars: 100 μm (A–D).

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  FIGURE 12. Calcification of the BP post-implantation into male juvenile rats for 90 days. (A,B) Macro image of the calcium nodules (black arrow). (C,D) von Kossa staining. Calcification deposits were stained black (black arrow). (E,F) ICP-7400 quantification of calcium and phosphorus contents in Glut- or Rib-treated BP (n = 12) (*p < 0.05, **p < 0.01, and ***p < 0.001) Scale bars: 500 µm.