BACKGROUND AND AIMS OF THE STUDY: Due to its superb crosslinking activity, glutaraldehyde (GA) is still the most widely used fixative for bioprosthetic heart valves. At the same time, however, GA is also believed to be partly responsible for tissue calcification and the lack of surface re-endothelialization, both of which may contribute to valve degeneration. Although excess GA has previously been extracted from thin leaflet tissue, this treatment proved insufficient for the detoxification of thick aortic wall tissue of stentless valves or root prostheses. METHODS: In order to establish a detoxification procedure which thoroughly extracts biologically active GA from aortic wall tissue, we used a highly sensitive bioassay where endothelial cells were seeded onto glutaraldehyde-fixed aortic wall discs following various detoxification procedures. Absolute cell numbers and morphologic shape were correlated with shrinkage temperature and shrinkage extent of the tissue to determine the potential of the treatments to reverse crosslinks. To optimize treatment conditions, pH (3.2 versus 4.5), temperature (22 degrees C versus 37 degrees C) and incubation time (48 h versus one week) were varied. In order to identify an optimal detoxification agent, 12 different amino-reagents from four chemical groups were compared: low pKa aromatic amines, amino acids, low pKa N-heterocyclic compounds and amino sugars. RESULTS: Amino-reagent treatment required warm temperature (37 degrees C), prolonged reaction time (one week) and a pH of 4.5 to achieve long-term cell growth on glutaraldehyde-fixed aortic wall. All 12 amino-reagents were able to detoxify aortic tissue satisfactorily; and all mildly reversed crosslinks, although there were differences between candidates. When summarized data were ranked correlating cell growth and quality with shrinkage temperature and shrinkage extent, seven reagents had a rank sum above the overall mean value, and five below with statistically significant differences between candidates. The additional stabilization of the detoxification reaction through borohydride-reduction had no further effect on tissue biocompatibility and crosslinks. CONCLUSIONS: Efficient detoxification of thick aortic wall tissue is possible if a one-week incubation in an acetic acid buffer-based amino-reagent is carried out at 37 degrees C.
BACKGROUND AND AIMS OF THE STUDY: Due to its superb crosslinking activity, glutaraldehyde (GA) is still the most widely used fixative for bioprosthetic heart valves. At the same time, however, GA is also believed to be partly responsible for tissue calcification and the lack of surface re-endothelialization, both of which may contribute to valve degeneration. Although excess GA has previously been extracted from thin leaflet tissue, this treatment proved insufficient for the detoxification of thick aortic wall tissue of stentless valves or root prostheses. METHODS: In order to establish a detoxification procedure which thoroughly extracts biologically active GA from aortic wall tissue, we used a highly sensitive bioassay where endothelial cells were seeded onto glutaraldehyde-fixed aortic wall discs following various detoxification procedures. Absolute cell numbers and morphologic shape were correlated with shrinkage temperature and shrinkage extent of the tissue to determine the potential of the treatments to reverse crosslinks. To optimize treatment conditions, pH (3.2 versus 4.5), temperature (22 degrees C versus 37 degrees C) and incubation time (48 h versus one week) were varied. In order to identify an optimal detoxification agent, 12 different amino-reagents from four chemical groups were compared: low pKa aromatic amines, amino acids, low pKa N-heterocyclic compounds and amino sugars. RESULTS: Amino-reagent treatment required warm temperature (37 degrees C), prolonged reaction time (one week) and a pH of 4.5 to achieve long-term cell growth on glutaraldehyde-fixed aortic wall. All 12 amino-reagents were able to detoxify aortic tissue satisfactorily; and all mildly reversed crosslinks, although there were differences between candidates. When summarized data were ranked correlating cell growth and quality with shrinkage temperature and shrinkage extent, seven reagents had a rank sum above the overall mean value, and five below with statistically significant differences between candidates. The additional stabilization of the detoxification reaction through borohydride-reduction had no further effect on tissue biocompatibility and crosslinks. CONCLUSIONS: Efficient detoxification of thick aortic wall tissue is possible if a one-week incubation in an acetic acid buffer-based amino-reagent is carried out at 37 degrees C.