Background Limited availability of decellularized allogeneic heart valve substitutes restricts the clinical application thereof. Decellularized xenogeneic valves might constitute an attractive alternative; however, increased immunological hurdles have to be overcome. This study aims for the in vivo effect in sheep of decellularized porcine pulmonary heart valves (dpPHV) enzymatically treated for N‐glycan and DNA removal. Methods dpPHV generated by nine different decelluarization methods were characterized in respect of DNA, hydroxyproline, GAGs, and SDS content. Orthotopic implantation in sheep for six months of five groups of dpPHV (n = 3 each; 3 different decellularization protocols w/o PNGase F and DNase I treatment) allowed the analysis of function and immunological reaction in the ovine host. Allogenic doPHV implantations (n = 3) from a previous study served as control. Results Among the decellularization procedures, Triton X‐100 & SDS as well as trypsin & Triton X‐100 resulted in highly efficient removal of cellular components, while the extracellular matrix remained intact. In vivo, the functional performance of dpPHV was comparable to that of allogeneic controls. Removal of N‐linked glycans and DNA by enzymatic PNGase F and DNase I treatment had positive effects on the clinical performance of Triton X‐100 & SDS dpPHV, whereas this treatment of trypsin & Triton X‐100 dpPHV induced the lowest degree of inflammation of all tested xenogeneic implants. Conclusion Functional xenogeneic heart valve substitutes with a low immunologic load can be produced by decellularization combined with enzymatic removal of DNA and partial deglycosylation of dpPHV.

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Background Heart valve replacement therapy with allogenic decellularized heart valve matrices allows an in vivo autologisation by repopulation of the foreign matrix with autologous cells. This method, which is superior based on experimental evidence to traditional heart valve replacements either of mechanical or glutaraldehyde fixated heart valve grafts, is current object of a multicentric international clinical trial (ESPOIR) granted by the EU. To circumvent the limited availability of allogenic heart valve matrices, we aim to the reduction of immunogenic epitops common to xenogeneic materials by high efficient decellularization paralleled by enzymatic treatment of the glycocalyx. Methods Porcine pulmonary heart valves of landrace pigs harvested at the local slaughterhouse were subjected to detergent and proteinase based decellularization protocols widely described in the literature. In a second step decellularized matrices were exposed to α1‐3 Galactosidase or PNGase F digestion. Alterations in αGal epitope levels and glycocalyx structures were investigated by inhibitory ELISAs with anti‐αGal antibodies (M86) and IgGs of unconditioned human sera on crushed matrix, and histochemical stains on cryosections exploiting lectins as isolectin B4 (staining αGal), wheat germ agglutinin (staining GalNAc), and Datura stramonium lectin (staining GlcNAc). Equally treated GalT‐KO pig and human pulmonary artery tissues served as controls. Results Inhibitory ELISA results show that all decellularization protocols reduced αGal epitope levels significantly, some down to <30% of native porcine tissue. Further reduction down to control levels was achieved by α1‐3 Galactosidase digestion. The absence of preformed non‐ αGal xenoantibodies in human IgG of unconditioned donors is evident by equal binding of human IgG to decellularized tissues of GalT‐KO and human origin. Histochemistry reveals that decellularization results in reduced Isolectin B4 (αGal) and WGA (GalNAc) staining, but not of DSL (GlcNAc) staining. Additional enzymatic treatment of decellularized matrix with PNGase F reduces binding of all lectins, whereas digestion with α1‐3 Galactosidase only affects Isolectin B4 staining (αGal). Conclusion The reduction of αGal epitope levels is strongly dependent on the decellularization method. A further decrease can be achieved by enzymatic digestion of carbohydrates present on the decellularized matrix. Sugar structures of the glycocalyx are believed to be strongly immunogenic, therefore, their general removal by PNGase F digestion may produce non‐immunogenic heart valve matrices. However, the lack of preformed human non‐ αGal xenoantibodies in non conditioned sera requires the testing for immunogenicity in an in vivo system. Thus, in future decellularized and enzymatically treated GalT‐KO matrices will be tested in a humanized mouse model.

Background Xenotransplantation leads to hyperacute or acute graft reaction due to interaction of preformed human antibodies, mainly αGal‐antibodies, with carbohydrate structures present on non‐human tissue. By removing these carbohydrate structures from porcine decellularized pulmonary heart valves (PHV), grafts might be generated that allow heart valve replacement therapy as with allogeneic PHV matrices. Methods Thus, after detergents based decellularization (0.5%SDS/0.5%Triton‐X100) cell free PHV matrices were enzymatically treated with α1‐3,6‐galactosidase or PNGase F. The potential impact on the glycocalyx was investigated by histochemical stains utilizing isolectin B4 (IL‐B4), wheat germ agglutinin (WGA), Datura stramonium lectin (DSL), and Ricinus communis agglutinin (RCA I) on decellularized only, and decellularized and enzymatically treated specimens. Native PHV tissue served as controls. Results All used lectins stained native heart valve tissue. Decellularization resulted in reduced IL‐B4 and WGA staining, whereas cell removal had no effect on DSL and RCA I staining. Enzymatic PNGase F treatment resulted in a further decrease of IL‐B4 and WGA staining whereas initially not affected DSL stain was reduced as well. Enzymatic treatment with α1‐3,6‐galactosidase led to a reduction of IL‐B4 stain only. Conclusion Decellularization per se is able to reduce αGal epitopes as demonstrated by IL‐B4 staining. This reduction of αGal epitopes can be enhanced by α1‐3,6‐galactosidase digestion as expected. Enzymatic treatment with PNGase F that recognizes GlcNacβ(1‐N)Asn sites results in removal of carbohydrate structures as αGal and N‐acetyl‐glucosamines to a high degree as demonstrated by IL‐B4, WGA, and DSL stains. In summary, detergent based decellularization followed by PNGase F treatment resembles an efficient way to remove immunogenic epitopes from porcine pulmonary heart valve matrices, thus potentially enabling the generation of xenogeneic PHV matrices for clinical application.