Optimizing α-Gal Epitope Removal in Porcine Dermal Matrix: Enzyme Selection and Tissue Form Matter

Decellularization does not completely remove the matrix-bound α-Gal epitopes in porcine acellular dermal matrix (pADM), and the presence of residual α-Gal epitopes could elicit adverse immunological reactions and cause potential early failure of xenografts. The present study had evaluated the effectiveness of decellularization and α-galactosidase treatment to eliminate the matrix-bound α-Gal epitopes in pADM, as well as the effect of tissue form (intact pADM vs. microparticle). Decellularization eliminated ∼80% of α-Gal epitopes in porcine dermis, and pADM retained ∼20% of the matrix-bound α-Gal epitopes. While Aspergillus α-galactosidase and Coffea α-galactosidase both hydrolyzed the terminal alpha-galactosyl moiety from oligosaccharides, only Coffea α-galactosidase was effective in eliminating the matrix-bound α-Gal epitopes in intact pADM. Aspergillus α-galactosidase did not work for intact pADM, even at an enzyme activity more than an order of magnitude higher than that of Coffea α-galactosidase used. The different efficacy between Aspergillus α-galactosidase and Coffea α-galactosidase was associated to the accessibility to the matrix-bound α-Gal epitopes in intact pADM. When intact pADM was micronized into fine microparticles, Aspergillus α-galactosidase and Coffea α-galactosidase eliminated the matrix-bound α-Gal epitopes equally well. Thus, the tissue form had significant influence on the efficacy of enzymic cleavage. The findings of the study offer valuable insight for enzyme selection and process development for efficient α-Gal antigen reduction in xenogeneic grafts or tissue scaffolds.