Reduction of xeno‐antigens in porcine pulmonary heart valves by decellularization and glycolytic enzymatic treatment

Abstract

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.

Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches

“…The risk to include a molecule with high potential to acute response, as the recombinant protein, should carefully be evaluated, avoiding clinical disasters. Hilfiker and collaborators demonstrated the antigenicity reduction by decellularization also suggesting a humanized immunoassay do determine a quantitative antigenicity …”

Heart Valve Matrices: State of the Art and Perspectives

“…Термоли-зин в качестве единственного агента может удалять клетки только с поверхности ткани, поэтому его применение ограничено [51]. Высокоспецифичный фермент α-галактозидаза применяется как компонент методики для децеллюляризации ксе-ногенной ткани и отвечает за удаление эпитопа α-Gal [23,52].…”

“…Поэтому, к сожалению, недостаточность децеллюляризации (технология предполагала гипотонический лизис клеток и об-работку ДНазой) проявилась только на клиническом этапе -больные погибали из-за быстрой дегенерации и разрыва донорской части стенки аорты вследствие острого воспаления [64]. В настоящий момент проблема ксенотрансплантации по-прежнему не решена [67,52].…”

Generation of glycans depleted decellularized porcine pericardium, using digestive enzymatic supplements and enzymatic mixtures for food industry

“…Indeed, carbohydrases such as α-galactosidase or peptide:N-glycosidase F (PNGase F) have been applied for the removal of the α-Gal epitope and N-glycans, respectively, from xenogeneic tissues. [33][34][35] Another carbohydrase with broader specificity, α-amylase from porcine pancreas, has been used for the production of porous foams derived from decellularized porcine left ventricle. 36 Besides carbohydrates removal, enzymes have been largely applied for tissue decellularization.…”

“…Elseways, carbohydrates removal using enzymatic treatments has been limited to enzymes with specificity against the known immunogenic carbohydrates or specific types of glycosylations. Indeed, carbohydrases such as α‐galactosidase or peptide:N‐glycosidase F (PNGase F) have been applied for the removal of the α‐Gal epitope and N‐glycans, respectively, from xenogeneic tissues 33‐35 . Another carbohydrase with broader specificity, α‐amylase from porcine pancreas, has been used for the production of porous foams derived from decellularized porcine left ventricle 36 …”