The hybrid crosslinking method improved the stability and anti-calcification properties of the bioprosthetic heart valves

Figures

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  FIGURE 1. General procedure and reaction principle of the preparation of the hybrid crosslinked bovine pericardium. The method was followed by Neomycin to stabilize GAGs, PE to stabilize collagen fibers, and Tannic acid to stabilize elastin. GAGs: Glycosaminoglycans. PE: Polyethylene glycol diglycidyl ether.

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  FIGURE 2. Histological staining images of the bovine pericardium. The structure of the leaflets remained intact and compact after the GA and NPTD crosslinked compared to the natural pericardium. The images show the HE staining (A,E,I), Masson’s trichrome staining (B,F,J), elastic Van-Gieson staining (C,G,K) and Alcian blue staining (D,H,L).

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  FIGURE 3. SEM micrograph of the surface and transection of the bovine pericardium. The tissue feature of the NA was the loose porous and rough (A,B,C). The feature of flat and compact structure in the NPTD group (G,H,I) was similar to that of the GA group (D,E,F).

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  FIGURE 4. The water contact angle, thermal denaturation temperature (Td) and biomechanical properties of the bovine pericardium. The water contact angle of the leaflets decreased significantly after NPTD crosslinked compared with those of the NA and GA groups (A). Td of the NPTD samples was distinctly improved compared to those of the NA and GA samples (B). The thinness of the leaflets decreased after NPTD crosslinked and were close to those of the GA group (C). The Young’s modulus was significantly improved in the NPTD group compared with the GA group (D). The Maximun Tensile Stress and Maximum Load among the three groups was no significant difference (E,F). The representative stress/strain curves of the groups (G). Statistical analysis was performed by Kruskal–Wallis test with Tukey’s post hoc test.

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  FIGURE 5. The cytocompatibility and Hemocompatibility of the bovine pericardium. The human umbilical vein endothelial cells (HUVECs) proliferation and morphology was assessed by 6-CDCFDA live cell fluorescent staining (A,B,C), the HUVECs almost completely covered the surface of the NA and NPTD groups on day 5 while only a few cells in the GA group (A,B,C). The cytotoxicity of the leaflets was determined by MTT assay, the relative growth ratios of GA group was significantly lower than that of the NA and NPTD groups (G). The platelet adhesion on the surface of the samples was evaluated by SEM imaging (D,E,F) and LDH assay (I). Red arrows indicate adherent platelets. The quantification of LDH in the NPTD group was significantly lower than that of the NA and GA groups. The hemolysis ratio of LDH in the NPTD and NA group was significantly lower than that of the GA group (H). Statistical analysis was performed by one-way analysis of variance (ANOVA).

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  FIGURE 6. Histological features and quantitative analysis after enzymatic hydrolysis of cross-linked pericardium. The images of Masson’s trichrome staining after collagenase hydrolysis (A,D). The images of elastic Van-Gieson staining after elastinase hydrolysis (B,E), Red arrows show preserved elastic fibers in the NPTD group. The images of Alcian blue staining after hyaluronidase hydrolysis (C,F). The quantitative analysis of mass loss and residual GAGs content in each group after enzymatic hydrolysis (G–I). Statistical analysis was performed by one-way analysis of variance (ANOVA).

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  FIGURE 7. The stability of the pericardium after 90 days of storage in vitro. The images show the Masson’s trichrome staining (A,D,G), elastic Van-Gieson staining (B,E,H) and Alcian blue staining (C,F,I). Red arrows show preserved elastic fibers in the NPTD group. The residual content of collagen in the GA and NPTD groups was significantly higher than that in the NA group (J). The residual content of elastin and GAGs in the NPTD groups was significantly higher than that in the NA and GA group (K,L). Statistical analysis was performed by one-way analysis of variance (ANOVA).

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  FIGURE 8. Characterization of the host response of various samples after subcutaneous implantation for 1 week and 3 weeks. The images show the immunohistochemical staining of CD68 and CD3 (A,B,C,D), on the right side of the corresponding image is the semi-quantitative result (E–H). In the first week, CD3+ cells were not significantly different between the GA and NPTD groups, while in the third week, there were fewer CD3 positive cells in the NPTD group than in the GA group. The results showed the reduced CD68+ inflammatory cell infiltration in the NPTD group compared to the GA and NA groups at 1 week and 3 weeks. Statistical analysis was performed by one-way analysis of variance (ANOVA).

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  FIGURE 9. The gross specimens, Histological characteristics and quantitative analysis after subcutaneous implantation for 90 days. The pericardium in the NA group was severely degraded in vivo, while the pericardium in the GA and NPTD groups was well preserved (F). The images show the HE staining (A), Masson’s trichrome staining (B), elastic Van-Gieson staining (C) and Alcian blue staining (D), Red arrows depict the preserved elastic fibers. The Alizarin red staining show that the GA group had more calcium deposition than the NPTD group (E), Red arrows depict the location of calcification. The calcium quantitative analysis showed that the calcium content of the NPTD group was significantly lower than that of the GA group (G). The elastin and GAGs quantitative analysis showed that the preserved content of the NPTD group was significantly higher than that of the GA group (H,I). Statistical analysis was performed by one-way analysis of variance (ANOVA).