In vivo efficacy for novel combined anticalcification treatment of glutaraldehyde-fixed cardiac xenograft using humanized mice

The animal immune response against Galα1,3-Galβ1-4GlcNAc-R(α-Gal) epitopes gives an important cause for the failure of glutaraldehyde(GA)-fixed cardiac xenografts. This study aimed to assess the in vivo effect of our novel combined anticalcification treatment, which includes immunologic modification, using α1,3-galactosyltransferase knock-out mice to mimic human immunologic environment. Bovine pericardia were cross-linked with GA and treated with decellularization, immunologic modification with α-galactosidase, space-filler with polyethylene glycol, organic solvent, and detoxification. The bovine pericardia were subcutaneously implanted into humanized and wild type mice, and titers of anti α-Gal IgM and IgG were evaluated at various time intervals. In vivo calcification and immunohistochemistry staining was assessed for the explanted xenografts several months after implantation. In humanized mice, titers for anti α-Gal IgM and IgG increased as the period of implantation increased, and reduced with our anticalcification treatments. The humanized mice had more in vivo calcification in GA-fixed xenografts treated with our anticalcification protocol compared with wild type mice. In humanized mice, in vivo calcification reduced with our combined anticalcification treatment, and the immunohistochemistry of the harvested xenografts proved the compatible findings with the results of in vivo immunogenicity and calcification. Humanized mice are effective model for the assessment of in vivo calcification, and our combined anticalcification treatments reduced in vivo calcification as well as in vivo immunogenicity in humanized mice group, suggesting that the animal immune reaction is the cause for calcification. Our novel combined anticalcification strategies of decellularization, immunologic modification, space-filler, organic solvent, and detoxification have possible promise to prolong the lifespan of cardiac xenograft.