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

Abstract

Bioprosthetic heart valves (BHVs) used in clinics are fabricated via glutaraldehyde (GLUT) crosslinking, which results in cytotoxicity and causes eventual valve calcification after implantation into the human body; therefore, the average lifetime and application of BHVs are limited. To address these issues, the most commonly used method is modification with amino acids, such as glycine (GLY), which is proven to effectively reduce toxicity and calcification. In this study, we used the l-glutathione (GSH) in a new modification treatment based on GLUT-crosslinked bovine pericardium (BP) as the GLUT + GSH group, BPs crosslinked with GLUT as GLUT-BP (control group), and GLY modification based on GLUT-BP as the GLUT + GLY group. We evaluated the characteristics of BPs in different treatment groups in terms of biomechanical properties, cell compatibility, aldehyde group content detection, and the calcification content. Aldehyde group detection tests showed that the GSH can completely neutralize the residual aldehyde group of GLUT-BP. Compared with that of GLUT-BP, the endothelial cell proliferation rate of the GLUT + GSH group increased, while its hemolysis rate and the inflammatory response after implantation into the SD rat were reduced. The results show that GSH can effectively improve the cytocompatibility of the GLUT-BP tissue. In addition, the results of the uniaxial tensile test, thermal shrinkage temperature, histological and SEM evaluation, and enzyme digestion experiments proved that GSH did not affect the ECM stability and biomechanics of the GLUT-BP. The calcification level of GLUT-BP modified using GSH technology decreased by 80%, indicating that GSH can improve the anti-calcification performance of GLUT-BP. Compared with GLUT-GLY, GLUT + GSH yielded a higher cell proliferation rate and lower inflammatory response and calcification level. GSH can be used as a new type of anti-calcification agent in GLUT crosslinking biomaterials and is expected to expand the application domain for BHVs in the future.

Keywords: anti-calcification; biomaterial modification; cytocompatibility; glutaraldehyde crosslinking; glutathione detoxification.

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).

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).

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).

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).

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).

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).

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).