Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens.
OBJECTIVE
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-alpha1,3-galactose (alpha-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), alpha-galactosidase (5 U/ml) or phospholipase (PL) A2 (150 U/ml). Tissues underwent a 96-h washout under gentle agitation at 27 degrees C, and then evaluated by light microscopy for % cellularity, and by immunohistochemistry and Western blot for alpha-gal, bovine MHC I and smooth muscle alpha-actin.


RESULTS
Standard treatment of BP resulted in only partial removal histological cellularity and persistence of alpha-gal, MHC I and alpha-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 alpha-galactosidase selectively removed alpha-gal from BP.


CONCLUSIONS
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 alpha-galactosidase.
Gonçalves
Ana C
AC
Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Griffiths
Leigh G
LG
Anthony
Russell V
RV
Orton
E Christopher
EC
D000199:Actins / Q000302:isolation & purification*; D000818:Animals; D015478:Antigens, Heterophile / Q000302:isolation & purification*; D002417:Cattle; D003584:Cytological Techniques / Q000379:methods; D004187:Disaccharides / Q000302:isolation & purification*; D000939:Epitopes; D015395:Histocompatibility Antigens Class I / Q000302:isolation & purification*; D010496:Pericardium / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*
D000199:Actins; D015478:Antigens, Heterophile; D004187:Disaccharides; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; C055075:galactosyl-(1-3)galactose