Prevention of porcine aortic wall calcification by acellularization: necessity for a non-glutaraldehyde-based fixation treatment.
OBJECTIVE
Acellularization prevents cell-mediated calcification of the aortic wall, but the inflammatory response towards the unfixed tissue is problematic. Two additional fixation methods, applied after tissue acellularization, were studied.


METHODS
Porcine aortic wall samples were randomized into four groups: (1) Standard fixation with glutaraldehyde (GA); (2) acellularization by a combined method of enzymes (DNase, RNase) and a detergent (Triton X-100); (3) acellularization followed by standard GA fixation; (4) acellularization followed by photo-oxidation. Samples were implanted into the wall of both jugular veins of six juvenile sheep. Tissue was explanted after three months and evaluated by X-radiography, light and electron microscopy, and calcium content (cc) measurement (atomic absorption spectrometry). Auto-fluorescence of elastic fibers was used to identify the relationship between calcific deposits and elastin.


RESULTS
GA-fixed aortic wall samples showed clear mineralization (cc 41.6 +/- 17.8 microg/mg), occurring predominantly at the level of cell remnants, as confirmed by electron- and fluorescence microscopy, locating calcific deposits in between elastic fibers. Acellularized aortic wall fragments were calcified significantly less, but an important (non-infectious) inflammatory response caused elastolysis and subsequent calcification of the elastic fibers (cc 5.6 +/- 2.8 microg/mg). Acellularized and GA-fixed fragments revealed important, inhomogeneously spread calcific deposits (cc 24.7 +/- 10.0 pg/mg). Photo-oxidized samples remained free from calcification (cc 0.82 +/- 1.6 microg/mg).


CONCLUSIONS
Acellularization is a promising tool in the prevention of porcine aortic wall calcification, but additional tissue fixation is necessary to prevent structural degeneration. GA fixation after acellularization causes important inhomogeneous tissue mineralization. Photo-oxidation combines optimal tissue fixation with superior anticalcification characteristics.
Meuris
Bart
B
Department of Cardiac Surgery, Katholieke Universiteit Leuven, Leuven, Belgium.
Verbeken
Erik
E
Flameng
Willem
W
D000818:Animals; D001021:Aortic Valve / Q000473:pathology / Q000528:radiation effects; D001705:Bioprosthesis; D002118:Calcium / Q000032:analysis; D003851:Deoxyribonucleases; D003902:Detergents; D004547:Elastic Tissue / Q000473:pathology; D004549:Elastin / Q000032:analysis; D005404:Fixatives; D005615:Freezing; D005976:Glutaral; D006350:Heart Valve Prosthesis; D008027:Light; D008854:Microscopy, Electron; D017830:Octoxynol; D016877:Oxidants; D011474:Prosthesis Design; D011897:Random Allocation; D012260:Ribonucleases; D012756:Sheep; D013552:Swine; D014021:Tissue Preservation / Q000379:methods
D003902:Detergents; D005404:Fixatives; D016877:Oxidants; D017830:Octoxynol; D004549:Elastin; D003851:Deoxyribonucleases; D012260:Ribonucleases; D002118:Calcium; D005976:Glutaral