Biofunctionalized Decellularized Tissue-Engineered Heart Valve with Mesoporous Silica Nanoparticles for Controlled Release of VEGF and RunX2-siRNA against Calcification

Figures

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  Figure 1. Preparation scheme. (A) MSN@VEGF-PEI-siRNA synthesis scheme. (B) Cell uptake of nanoparticles. (C) Preparation of modified hybrid valve.

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  Figure 2. Characteristics of MSN@VEGF−PEI-siRNA delivery system. (A) I: MSN, II: MSN@VEGF−PEI, III: MSN@VEGF−PEI−siRNA, IV: Double steaming water. The Dundar effect was used to measure the dispersion of the synthesized material. Cytotoxicity was detected by concentration gradient in each group (MSN, MSN@VEGF−PEI, MSN@VEGF−PEI−siRNA). Concentrations of 0.75 mg/mL and 1 mg/mL showed higher cell activity. (B) Representative image of SEM (C) and TEM (D). Zeta potential (E) and elemental mapping image of MSN (F). Energy dispersive spectroscopy of MSN, reveals the basic elemental composition of nanoparticles (G). Uptake of valve interstitial cells (H) and particle size analysis. The nanoparticle particle size was 50 nm (I). Cumulative release curve of siRNA (J) and VEGF (K). Significant differences (* p < 0.05, ** p < 0.01), n = 3.

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  Figure 3. Anti-calcification of the MSN@VEGF-PEI-siRNA delivery system; MSN@VEGF-PEI-siRNA silencing efficiency is higher than that of conventional transfection reagents. mRNA expression of calcification genes associated with valve interstitial cells was detected after transfection with MSN@VEGF-PEI-siRNA and Lipo 3000+siRNA (A). Representative image of Alizarin red staining (B) and ALP staining (C). Quantitative analysis of alizarin red staining (D) and ALP staining (E). Determination of calcium content (F). Significant differences (* p < 0.05, ** p < 0.01), n = 3. scale bar = 20 µm.

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  Figure 4. Physiological behavior of endothelial cells after treatment with MSN@VEGF-PEI-RunX2 delivery system; MSN@VEGF-PEI-RunX2 promotes endothelial cell proliferation, adhesion, migration, and tube formation. Scratch assay (A) and tube formation assay (B). The migration rate of endothelial cells (C). Analysis of tube formation assay—total tube length (D) and number of new tubes (E). Proliferation assay (F) and adhesion assay (G) of endothelial cells. Significant differences (* p < 0.05, ** p < 0.01), n = 3. scale bar = 50 µm.

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  Figure 5. Characterization of the hybrid valve and function test after cell implantation. Representative image of the hybrid valve under a SEM (A). Cumulative release curve of siRNA and VEGF from hybrid valve (B). Proliferation and adhesion test of the hybrid valve (C). mRNA expression of the calcified gene was detected after implantation of valvular interstitial cells (D). Hemolysis rate of the hybrid valve (E). Calcium content was detected after the implantation of valve interstitial cells (F). Significant differences (* p < 0.05, ** p < 0.01), n = 3.

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  Figure 6. Mechanical properties of the hybrid valve. Representative image of modified hybrid valves (A). Diagram of circumferential direction and radial direction (B). Elastic modulus (C), fracture strain (D), fracture strength (E), and ultimate tensile strength (F) of the hybrid valve. Significant differences (* p < 0.05, ** p < 0.01), n = 3.

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  Figure 7. CD31 staining of PAV, DPAV, DPAV-MSN-PEI-siRNA, and DPAV-MSN@VEGF-PEI-siRNA (A) and analysis of CD31 staining (B). Significant differences (* p < 0.05, ** p < 0.01), n = 6.