Background and aim of the study: The extension of glutaraldehyde (GA) crosslinks with diamine bridges was shown previously to reduce bioprosthetic heart valve calcification to a significant degree. The aim of the present study was to investigate whether the additional crosslinking of functional carboxyl groups could augment this anticalcific effect at the low glutaraldehyde concentrations typically used in commercial heart valve production.
Methods: Entire aortic roots of medium-sized pigs were fixed after 48 h of cold storage. Crosslinking of amino-functional groups was achieved either by GA fixation alone (0.2% or 0.7%) or with an interim treatment with the diamine L-lysine (25, 50 or 100 mM; 37°C; 2 days). Carboxyl groups were activated with carbodiimide (N’-{3-dimethylaminopropyl}-N-ethyl carbodiimide hydrochloride (EDC), 240 mM) and crosslinked with an oligomeric diamine (polypropylene glycol-bis-aminopropyl ether (Jeffamine‘), 60 mM, 230D). By permutation of treatments and combinations thereof, a total of 17 groups was compared. Aortic wall discs (12 mm diameter) were implanted subcutaneously into seven-week-old Long-Evans rats for 60 days. Tissue calcification was determined by histology and atomic

absorption spectrophotometry.
Results: There was no significant difference in tissue calcification if either GA or carbodiimide fixation was used alone. Equally, the combined crosslinking with GA and EDC/Jeffamine did not achieve a mitigation of tissue calcification below levels seen in at least one of the two treatments alone. When commercial GA fixation was mildly diamine-enhanced with L-lysine (25 mM), additional EDC/Jeffamine crosslinking of carboxyl groups resulted in a distinct additive effect in both 0.2% (-31%; p <0.0002) and 0.7% (-36%; p = 0.0073) GA-fixed tissue. Relative to conventional GA fixation, this combination mitigated aortic wall calcification by 43% (p <0.0001) and 34% (p = 0.0014) in 0.2% and 0.7% GA-fixed tissue, respectively. An increase in L-lysine concentration to 100 mM further reduced calcification of 0.7% GA-fixed tissue (18.5%; p = 0.016), but had no additional effect on 0.2% GA-fixed tissue (0.6%; p = 0.463).
Conclusion: A distinct reduction in bioprosthetic aortic wall calcification can be achieved by combining diamine-extended conventional GA fixation with a diamine-extended carbodiimide based crosslinking step.
The Journal of Heart Valve Disease 2005;14:538-545