In-vivo subdermal implant in the alpha-Gal knockout mice model for the assessment of bioprosthetic heart valve calcification rate before and after a polyphenols-based treatment

Rationale The presence of preformed antibodies against αGal in the human lead to opsonization of the implanted bioprosthetic heart valve (BHV), leading to deterioration and calcification. Murine subcutaneous implantation of BHVs leaflets has been widely used for testing the efficacy of anti-calcification treatments, however, unlike the situation in humans, leaflets implanted into a murine model will not be able to elicit an αGal immune response because both donor and recipient species constitutively express the epitope. Objective This study evaluates the calcium deposition on commercial BHV using a new humanized murine αGal knockout (KO) animal model. Furtherly, the anti-calcification efficacy of a polyphenol-based treatment was deeply investigated. Methods and Results By using CRISPR/Cas9 approach an αGal KO mouse was created and used for the evaluation of the calcific propensity of original and polyphenols-treated BHV by subcutaneous implantation. The calcium quantification was carried out by plasma analysis; the immune response evaluation was performed by histology and immunological assays. Anti-αGal antibodies level in KO mice increases at least double after 2 months of implantation of original commercial BHV compared to WT mice, conversely, the polyphenols-based treatment seems to effectively mask the antigen to the KO mice’s immune system. Commercial leaflets explanted after 1 month from KO mice showed a four-time increased calcium deposition than what was observed on that explanted from WT. Polyphenol treatment prevents calcium deposition by over 99% in both KO and WT animals. Conclusions The implantation of commercial BHV leaflets significantly stimulates the KO mouse immune system resulting in massive production of anti-Gal antibodies and the exacerbation of the αGal-related calcific effect if compared with the WT mouse. The polyphenol-based treatment applied in this investigation showed an unexpected ability to inhibit the recognition of BHV xenoantigens by circulating antibodies completely preventing calcific depositions compared to the untreated counterpart. ### Competing Interest Statement The authors have declared no competing interest.