Improved Cytocompatibility and Reduced Calcification of Glutaraldehyde-Crosslinked Bovine Pericardium by Modification With Glutathione

Figures

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  FIGURE 1. Validation of the decellularisation process of the borcine pericardiums (BPs) (A) HE staining of native BP (Fig.1A,top) and decellularized BP (A). The black bar indicates 100 um (B) Quantitative determination of DNA in native BP and decellularized BP (n = 4; mean values ±s.d.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns represents no significant difference).

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  FIGURE 2. Qualitative and quantitative detection of aldehyde groups: (A): results of different treatments of BPs stained with Schiff reagent, The color intensity is positively correlated with the content of aldehyde groups after the color reaction, The conditions of each group are as follows: H0:GLUT treated group; H1:GLUT+1 mmol/L GSH treated group; H2:GLUT+2 mmol/L GSH treated group; H4:GLUT+4 mmol/L GSH treated group; H8:GLUT+8 mmol/L GSH treated group; (B) is the qualitative content of aldehyde groups from different BP groups respectively (n = 4; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns represents no significant difference).

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  FIGURE 3. Biomechanical properties of BP from GLUT-BP, GLUT + GSH, and GLUT + GLY. (A) is mechanical properties of BP. Use the Young’s modulus (YM), ultimate tensile stress (UTS) and tensile stress (TS) to evaluate the mechanical properties of BPs. (B) is thermal shrinkage temperature, (C) is the BPs’ relative weight loss after collagenase treatment, (D,E) is thickness and water content of BPs from each group (n = 10; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns represents no significant difference).

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  FIGURE 4. Histological staining (HE staining、Masson staining)and SEM for ECM evaluation of BPs from GLUT-BP (A,D), GLUT + GLY (B,E), GLUT + GSH (C, F), The black bar indicates 500 um. The (G,H,I) is the SEM of GLUT (A), GLUT + GLY (B), GLUT + GSH (C) respectively, Each group shows a large picture (10.0kv, Scale bars = 10um) and a small picture on the bottom left corner (1.0kv, Scale bars = 100um). (J) is the porosity of BPs from each group (n = 4; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns represents no significant difference).

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  FIGURE 5. Cytocompatibility of BPs in vitro, A-E is the cell fluorescence staining of Fresh-BP (A), GLUT-BP (B), GLUT + GLY (C), GLUT + GSH (D), and DMEM+10%FBS (E) by 6-CDCFDA after 5 days of culture on the BPs surface respectively. (F) is the count of endothelial cells of BPs. The cell proliferation rate (n = 4, mean values ±s.d.) of EAHY926 cells were determined using the MTT assay kit (G). (H) is the hemolysis percentage of BPs (n = 4; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns represents no significant difference).

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  FIGURE 6. In vivo biocompatibility of BPs from GLUT-BP (A–D), GLUT + GLY (E–H), GLUT + GSH (I–L). The (M–O) is the Fibrous capsule thickness and count of T cell and macrophage cell (n = 4; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns represents no significant difference).

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  FIGURE 7. After 8 weeks of subcutaneous implantation in the rat, the BPs were taken out for Von kossa calcium staining and calcium content determination. The (A–C) is the calcium staining in the GLUT group and the GLUT + GLY and GLUT + GSH treatment groups. The black scale bars are 500 um. The Fig.7 (D) is the calcium content determination in the GLUT group and the GLUT + GLY and GLUT + GSH (n = 10; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001,∗∗∗∗p < 0.0001).