Fibrin deposition on bovine pericardium tissue used for bioprosthetic heart valve drives its calcification

Abstract

Background: Bioprosthetic heart valves (BHVs) are less thrombogenic than mechanical prostheses; however, BHV thrombosis has been proposed as a risk factor for premature BHV degeneration.

Objectives: We aimed to explore whether fibrin deposition on bovine pericardium tissue could lead to calcification.

Method: Fibrin clot was obtained by blending three reagents, namely, CRYOcheck™ Pooled Normal Plasma (4/6), tissue factor + phospholipids (Thrombinoscope BV), and 100 mM calcium (1/6), and deposited on pericardium discs. Non-treated and fibrin-treated bovine pericardium discs were inserted into the subcutaneous tissue of 12-day-old Wistar rats and sequentially explanted on days 5, 10, and 15. Calcium content was measured with acetylene flame atomic absorption spectrophotometry. Histological analysis was performed using hematoxylin-eosin staining, Von Kossa staining, and immunohistochemistry.

Results: Calcification levels were significantly higher in fibrin-treated bovine pericardium discs compared to those in non-treated bovine pericardium discs (27.45 ± 23.05 µg/mg vs. 6.34 ± 6.03 µg/mg on day 5, 64.34 ± 27.12 µg/mg vs. 34.21 ± 19.11 µg/mg on day 10, and 64.34 ± 27.12 µg/mg vs. 35.65 ± 17.84 µg/mg on day 15; p < 0.001). Von Kossa staining confirmed this finding. In hematoxylin-eosin staining, the bovine pericardium discs were more extensively and deeply colonized by inflammatory-like cells, particularly T lymphocytes (CD3⁺ cells), when pretreated with fibrin.

Conclusion: Fibrin deposition on bovine pericardium tissue treated with glutaraldehyde, used for BHV, led to increased calcification in a rat model. BHV thrombosis could be one of the triggers for calcification and BHV deterioration.

Keywords: bioprosthetic heart valve; calcification; fibrin; pericardium tissue; thrombosis.

Figure 1

Fibrin deposition on bovine pericardium: preparation, implantation, and scanning electron microscopy analysis. (A) Non-treated pericardium tissue surface (magnification = ×600; scale bars = 20 μm). (B) Fibrin-treated pericardium tissue surface with 10 µl of fibrin solution (magnification = ×3,000; scale bars = 5 μm). (C) Fibrin-treated pericardium tissue surface with 20 µl of fibrin solution (magnification = ×600; scale bars = 20 μm). (D) Fibrin-treated pericardium tissue surface with 20 µl of fibrin solution (magnification = ×3,000; scale bars = 5 μm). (E) fibrin-treated pericardium tissue surface with 50 µl of fibrin solution (magnification = ×3,000; scale bars = 5 μm). (F) Protocol for pericardium tissue preparation and implantation, after washing.

Figure 2

Dosage of tissue calcium levels between fibrin-treated and non-treated bovine pericardium tissues after subcutaneous implantation in rats for 15 days.

Figure 3

Panel of histological analyses. Hematoxylin–eosin staining for (A) non-treated pericardium tissue at day 5, (B) non-treated pericardium tissue at day 15, (C) fibrin-treated pericardium tissue at day 5, and (D) fibrin-treated pericardium tissue at day 15. Von Kossa staining for (E) non-treated pericardium tissue at day 5, (F) non-treated pericardium tissue at day 15, (G) fibrin-treated pericardium tissue at day 5, and (H) fibrin-treated pericardium tissue at day 15.

Figure 4

IHC analysis with anti-CD3. (A) Non-treated pericardium tissue at day 15 (magnification = ×20; scale bars = 50 μm). (B) Fibrin-treated pericardium tissue at day 15 (magnification = ×20; scale bars = 50 μm). (C) Quantification of CD3⁺ cells in implanted pericardium treated or not treated with fibrin.