BACKGROUND AND AIM OF THE STUDY: 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). CONCLUSION: 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.
BACKGROUND AND AIM OF THE STUDY: 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). CONCLUSION: 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.