Carbodiimide Treatment Dramatically Potentiates the Anticalcific Effect of Alpha-Amino Oleic Acid on Glutaraldehyde-Fixed Aortic Wall Tissue

Background Bifunctional amines were previously found to act as bridging molecules between the terminal ends of incomplete glutaraldehyde (GA) cross-links. The additional cross-links thus formed between ε-NH2 groups of tissue were seen to significantly inhibit bioprosthetic calcification. In the current study, the potential ability of α-amino oleic acid (AOA) to act as a bridging molecule between ε-NH2- and COOH-dependent cross-links was hypothesized to similarly augment the anticalcification effect of the AOA molecule. Methods Porcine aortic wall tissue from Medtronic Freestyle valve bioprostheses incorporating the AOA anticalcification process additionally underwent carboxyl-group cross-linking with Jeffamine (poly[propylene glyco]-bis-[aminopropyl ether]) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Tissue was subdermally implanted into 5-week-old Long-Evans rats for 60 days. Standard 0.2% GA-fixed tissue served as a control. To further assess the impact of storage solution on AOA tissue, samples were either stored in GA (0.2%GA) or EDC (25 mmol/L carbodiimide) before implantation. Tissue calcification was assessed by atomic absorption spectroscopy and histochemical staining. Results Aldehyde end-capping with AOA achieved only a modest reduction of calcification in GA-treated aortic wall tissue (−20.0%; p< 0.05). Replacing GA with EDC as a storage solution led to a further 32.4% (p< 0.01) mitigation of calcification in Freestyle tissue. Incorporating an intermediate EDC/Jeffamine cross-linking step achieved a distinct additional reduction of calcification by 40.4% (p< 0.05). Overall, aortic wall calcification was 59.7% (p< 0.0001) lower if commercial Freestyle tissue underwent an additional EDC/Jeffamine cross-linking step and subsequent storage in EDC. Relative to control GA-fixed tissue, this represented a 67.8% (p< 0.0001) reduction. Incorporation of AOA was essential for the beneficial effect of the additional EDC/Jeffamine cross-linking step. Conclusions Potentially utilizing both the amino- and the carboxyl moieties of AOA for tissue binding dramatically reduces aortic wall calcification of GA-fixed tissue.