Notwithstanding their wide exploitation, biological prosthetic heart valves are characterized by limited durability (10–15 years). The treatment of biological tissues with chemical crosslinking agents such as glutaraldehyde accounts for the enhanced risk of structural deterioration associated with the early failure of bioprosthetic valves. To overcome the shortcomings of the currently available solutions, adoption of decellularized biological tissues of animal origin has emerged as a promising approach. The present study aims to assess in vitro cardiovascular scaffolds composed of bovine pericardium decellularized with the novel TRITDOC (TRIton-X100 and TauroDeOxyCholic acid) procedure. The effects of the treatment have been assessed by means of histological, biomolecular, cellular, biochemical and biomechanical analyses. The TRITDOC procedure grants the complete decellularization of bovine pericardial scaffolds while preserving the extracellular matrix architecture and the biomechanical properties. With a dedicated ELISA test, the TRITDOC procedure has been proven to ensure the complete removal of the alphaGal antigen, responsible for hyperacute rejection and for long-term deterioration of xenogenic biomaterials. Static seeding of the acellular pericardial patches with human adipose-derived stem cells resulted in an evenly repopulated scaffold without signs of calcification. The in vitro cyto-/immuno-compatibility response of the TRITDOC-bovine pericardium was compared with glutaraldehyde-treated xenogenic pericardium collected from two bioprosthetic devices currently used in clinical practice: PERIMOUNT MAGNA and TRIFECTA ^TM . TRITDOC-bovine pericardium exhibited lower complement activation, lower cytotoxicity and a lower tendency to secrete pro-inflammatory cytokines compared to the tested commercial bioprostheses. Therefore, TRITDOC-decellularized pericardium could be considered as possible candidate material for the production of prosthetic heart valves.

Proceedings of the XXXIV National Meeting of the Italian Society for the Study of Connective Tissues (SISC)

Introduction. Over the past two decades, the αGal (Galα1–3Galβ1–4GlcNAc–R) epitope, a carbohydrate found in many non-primate mammals, has gained significant relevance in medicine due to its association with an increasing number of allergic reactions to animal-derived foods, drugs, and medical devices. Due to a mutated gene coding for α1,3-galactosyltransferase (α1–3GT), humans lack αGal and, therefore, naturally produce anti-α-Gal antibodies (IgM, IgA, and IgG), especially in the context of a xenotransplantation, which can lead to extreme immunological reactivity, including hyperacute rejection of the transplant. Recently, these uncontrollable immune reactions have driven demand for more accurate procedures to better detect αGal in animal-derived foods or bioprosthetics. The currently most widely used α-Gal-specific monoclonal antibody is an IgM antibody (clone M86), developed in Ggta1 KO mice and isolated from hybridoma tissue culture. As the IgM isotype has limited purification properties, specificity, and sensitivity, we aimed to produce a novel IgG antibody with high affinity and extensive applicability. Methods. An experimental murine IgG1 anti-αGal antibody (IgG-αGalomab) was developed by immunization of Ggta1 knockout (KO) mice, and its affinity was evaluated using ELISA, Western blot, flow cytometry, and immunohistochemistry/immunofluorescence. Results. Compared to IgM-M86, IgG-αGalomab demonstrated ~1200-fold higher binding potency and lower cross-reactivity with competitive molecules, i.e., bovine serum albumin, galactobiose, and lactose. Unlike IgM-M86, IgG-αGalomab showed an increasing affinity over time in the binding tests performed on xenogeneic tissues. Notably, high-affinity for αGal was detected by Western blot at high dilution [1:200,000] of IgG-αGalomab compared to IgM-M86 [1:1000]. By flow cytometry, specificity and dose-dependent response were confirmed using in vitro cultures of porcine and human fibroblasts. Finally, in immunofluorescence and immunohistochemistry analysis, αGal was demonstrated to be detectable by IgG-αGalomab at a dilution of [1:1000], while IgM-M86 was demonstrated to be detectable at [1:100]. Conclusions. Altogether, our newly developed antibody showed high sensitivity and specificity for α-Gal in various applications. Based on its potential binding capacity, IgG-αGalomab could have important applications in precision medicine for predicting, treating, and preventing immune-mediated phenomena of patients in different medical areas.