Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens

Background and aim of study: Assessment of decellularization of xenogeneic biological scaffolds for tissue engineering has relied primarily on histological cellularity, though this may not ensure the removal of known xenogeneic antigens such as galactose-α1,3-galactose (α-gal) and MHC I. Methods: Bovine pericardium (BP) underwent standard (Std) decellularization consisting of hypotonic lysis and treatment with DNAse/RNAse. In addition to Std decellularization, tissues were treated for 24 h with either 0.5% Triton X-100, 0.5% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), α-galactosidase (5 U/ml) or phospholipase (PL) A₂ (150 U/ml). Tissues underwent a 96-h washout under gentle agitation at 27°C, and then evaluated by light microscopy for % cellularity, and by immunohistochemistry and Western blot for α-gal, bovine MHC I and smooth muscle α-actin. Results: Standard treatment of BP resulted in only partial removal histological cellularity and persistence of α-gal, MHC I and α-actin. Adding SD treatment resulted in apparent acellularity, but persistence of xenogeneic antigens. Only the addition of SDS resulted in complete histological acellularity and removal of xenogeneic antigens. Treatment with α-galactosidase selectively removed α-gal from BP. Conclusion: Histological cellularity is not an adequate end-point for assuring removal of antigenicity from xenogeneic biological scaffolds. However, known xenogeneic antigens can be targeted for removal by novel decellularization treatments such as α-galactosidase.