https://doi.org/10.1007/s00392-025-02625-4
1Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden Department of Cardiac Surgery Dresden, Deutschland; 2LMU Munich Chair of Molecular Animal Breeding and Biotechnology, Gene Center, and Center for Innovative Medical Models Munich, Deutschland; 3Medizinische Fakultät Carl Gustav Carus der TU Dresden Forschungsabteilung der Klinik für Herzchirurgie Dresden, Deutschland; 4Institute Biofunctional Polymermaterials Leibniz-Institut für Polymerforschung Dresden e.V. Dresden, Deutschland
Introduction: Xenogeneic pericardium is the main source for biological aortic valve (AV) prostheses. Immune responses to residual αGal epitopes and epitopes exposed by glutaraldehyde (GA)-fixation are responsible for deterioration and the need for reoperation. Genetically modified (GM) porcine pericardium shows up as an attractive alternative that avoids immunologic interactions, which may result in the absence of degeneration. The clinical application of GM materials depends on the activation of human defense pathways. In our pilot study, properties such as calcification potential and hemocompatibility are presented as baseline parameters for estimating the biocompatibility of GM porcine pericardium.
Materials and methods: Pericardia of different GM pigs (A: GGTA1-knockout (KO) with additional CCL2-KO and transgenes for human PD-L1 and CD47; B: GGTA1-KO with transgene for human CD46) were implemented (native or GA-fixed). Pericardia from slaughterhouse wild-type (WT) pigs were used as controls. αGal epitopes were quantified based on IHC and IF stainings using isolectin B4. Collagen fiber density was verified using Picrosiriusred staining. The calcification potential was determined in vitro. Via uniaxial tensile testing, elastic moduli were determined. For analysis of hemocompatibility, tissues were incubated with fresh human blood (1.5 U/ml heparin, 2h, 37°C). Inflammation (complement C5a, granulocyte activation [CD11b]) and hemostasis (prothrombin fragment F1+2, platelet activation [CD62P]) were analyzed via flow cytometry or ELISA. Blood cell deposits and matrix infiltration were determined in IHC stainings and scanning electron microscopy (SEM).
Results: Biomechanic properties and collagen fiber density of GM porcine pericardia did not differ from control tissues. Also the calcification potential in vitro with 1.1 ± 0.6 vs. 1.6 ± 1.6 µg/mg dry weight did not differ between GM vand WT samples. αGal expression was neither detectable via IHC nor isolectin staining in GM pericardia. Compared to WT control, complement and granulocyte activation were significantly lower for the native GM pericardia, but increased after GA-fixation, independent of the genetic background. Granulocyte loss tended to be different between the two GM pericardia, with a trend of lowergranulocyte loss in GM tissue A compared to B. Coagulation activation of native tissues was excessively high, but low after GA-fixation. Pericardium surface deposits in SEM supported the hemocompatibility findings. IHC staining revealed slightly more infiltration of immune cells in native WT than in nativ and GA-fixed GM and GA-fixed WT samples.
Conclusions: Biomechanics and fiber density did not differ between GM and WT porcine pericardium. The GA-fixed GM and WT pericardia had equal potential for calcification in buffer. In contrast, the lower inflammatory potential of the GM pericardium indicates the superior biocompatibility of the GM pericardium. The initial data suggest that the inflammatory reaction is affected by additional genetic modifications beside αGal depletion. Modes of preparation and fixation protocol steps can directly impact bioprostheses durability based on GM pericardium. For definite consideration of GM pericardium for in vivo testing hemocompatibility evidences are currently further testet.
MFM and SMT contributed equally