POSS‐PEG‐NHS and GSH Cocrosslink DPAV to Prepare Anticalcification Bioprosthetic Scaffold With Enhanced Reendothelization, Anti‐inflammation, and Anti‐ROS

Figures

• 

  FIGURE 1. Synthesis and characterization of POSS‐PEG‐NHS. (a) Schematic illustration of the three‐step synthesis route for POSS‐PEG‐NHS. (b) 1H NMR spectrum confirming the chemical structure of POSS‐PEG‐NHS. (c) FTIR spectrum of POSS‐PEG‐NHS and its synthetic intermediates.

• 

  FIGURE 2. Construction and characterization of scaffolds. (a) HE and Masson staining of PAV and DPAV scaffolds. (b) Schematic diagram reveals reaction principle that POSS‐PEG‐NHS and GSH modified DPAV. (c) Macroscopic appearance of prepared scaffolds. (d) FTIR of the scaffolds. (e) XPS of DPAV and PPN‐GSH‐AV scaffolds.

• 

  FIGURE 3. Mechanical properties and morphology characterization of the scaffolds. (a) Elongation at break, (b) Young's modulus, and (c) tensile strength of the scaffolds. (d) Surface morphology visualized by SEM. (e) WCA measurements. (f) Representative WCA images. (g) Surface roughness quantified by AFM. (h) 2D and 3D AFM topographical images. (Data were presented as mean ± SD, *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001).

• 

  FIGURE 4. The hemocompatibility of scaffolds, including PLTs adhesion, hemolysis, and thrombosis. (a) SEM images of platelet adhesion on the scaffold surfaces. (b) Representative hemolysis assay images. (c) Macroscopic views of thrombus formation on the scaffolds (from top to bottom are DPAV, PPN‐AV, PPN‐GSH‐AV and GAV). (d) Quantification of adherent platelets by LDH assay. (e) The relative hemolysis rate of scaffolds was quantified by the OD540 nm value of supernatant. (f) Thrombus formation quantified by absorbance of the lysate at OD540 nm. (Data were presented as mean ± SD, *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001).

• 

  FIGURE 5. Reendothelialization capacity and cytotoxicity of scaffolds. (a) Live and dead Raw264.7 cells were captured by confocal microscopy on day 7. (b) Proportions of live and dead HUVECs of scaffolds. (c) Proportions of live and dead Raw264.7 of scaffolds. (d) Live and dead Raw264.7 cells were captured by confocal microscopy on day 7. (e) Cytotoxicity toward HUVECs at 48 h. (f) Cytotoxicity toward Raw264.7 cells at 48 h. (g) Adhesion and proliferation of HUVECs on the surface of scaffolds on days 1, 3, 5, 7, 9, 11. (h) Adhesion and proliferation of Raw264.7 cells on the surface of scaffolds on days 1, 3, 5, 7, 9, 11. (Data were presented as mean ± SD, *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001.).

• 

  FIGURE 6. Anti‐ROS and anti‐inflammation properties of scaffolds in Raw264.7 cells. (a) Representative fluorescence intensity of ROS on scaffolds was captured by confocal microscopy. (b) Relative expression quantification of ROS on scaffolds. c Relative content of H2O2 of scaffolds was quantified by absorbance. (d) Relative expression quantification of Arg 1. (e) Relative expression quantification of iNOS. (f) The fluorescence intensity of Arg 1 and iNOS of scaffolds was captured by confocal microscopy on day 5. (Data were presented as mean ± SD, *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001.).

• 

  FIGURE 7. The overall performances of scaffolds in vivo. (a) HE, Masson, and Vonkossa staining of scaffolds. (b) IL‐1ß, IL‐6, and IL‐10 immunohistochemical staining of scaffolds. (c) The mean number of scaffolds is used to determine recellularization capacity. (d) IL‐1ß relative expression of each cell. (e) IL‐6 relative expression of each cell. (f) IL‐10 relative expression of each cell. (Data were presented as mean ± SD, *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001.).

• 

  FIGURE 8. The anti‐inflammatory and anti‐ROS properties of scaffolds in vivo. (a) The immunofluorescence staining for CD3 (T‐cell marker). (b) CD68 (macrophage marker). (c) CD206 (highly expressed in M2 macrophages). (d) Arg‐1 (highly expressed in M2 macrophages) and iNOS (highly expressed in M1 macrophages). (e) ROS (indicates the degree of oxidative stress).

• 

  FIGURE 9. Overall performances of scaffolds in vitro pulsatile flow testing. (a) Total view, closed and open state pictures in pulsatile flow system of interventional valves. (b) EOA of interventional valves. (c) Regurgitant volume ratio of interventional valves. (d) Mean pressure difference of interventional valves. (e) EOA of surgical valves. (f) Regurgitant volume ratio of interventional valves. (g) Mean pressure difference of surgical valves. (h, j) Total view, closed and open state pictures in pulsatile flow system of surgical valve prepared from DPAV and PPN‐GSH‐AV. (i, k) Pressure and flow rate variation curves in valve pulsatile flow testing of DPAV and PPN‐GSH‐AV.