Background and aim of the study: Previous studies have shown that ethanol pretreatment of glutaraldehyde (GA)-fixed porcine aortic valve cusps (GPAV) significantly reduces bioprosthetic leaflet calcification. The anticalcification mechanism is due to extraction of cholesterol and phospholipids, and a permanent alteration in collagen structure. It was noted in experimental implants that ethanol-pretreated GPAV occasionally show low levels of calcification. The study aim was to investigate whether this was due to unreacted aldehyde residues and other reducible compounds resulting from GA cross-linking.
Methods: GPAV were cross-linked in GA (0.6%) and stored at pH 7.4 in 0.2% GA. Cusps were pretreated with ethanol (80%, pH 7.4) for 24 h. Experimental groups included ethanol-pretreated cusps and GA-fixed controls that were pretreated with either sodium borohydride or sodium cyanoborohydride. Differential scanning calorimetry was used to measure shrink temperature as a measure of cross-linking. Subdermal implants of valve cusp tissue were carried out in 21-day-old Sprague-Dawley male rats. Implants were retrieved at 21 days

and samples assessed for the extent of calcification using chemical analyses for Ca, and microscopy studies.
Results: Ethanol pretreatment significantly inhibited calcification compared with controls (13.3 ± 5.6 versus 119.2 ± 6.6 mg Ca/mg tissue; p <0.001). However, sodium borohydride reduction under optimized conditions combined with ethanol pretreatment optimally reduced calcification (1.16 ± 0.1 mg Ca/mg; p <0.05), whereas levels after sodium cyanoborohydride treatment (23.6 ± 10.4 mg Ca/mg) were not significantly different to those after ethanol alone. Neither reducing agent was effective in inhibiting calcification without ethanol pretreatment. Furthermore, the reducing agents had no significant effect on shrink temperature.
Conclusion: Inhibition of GPAV calcification with ethanol pretreatment can be enhanced through the optimized use of reducing agents. This indicates that reducible aldehyde-related moieties are likely responsible for breakthrough calcification, even after ethanol pretreatment.
The Journal of Heart Valve Disease 2004;13:487-493