Reduction of Calcification of Carbodiimide-Processed Heart Valve Tissue by Prior Blocking of Amine Groups with Monoaldehydes Frank Everaerts 1 , Mirian Gillissen 1 , Mark Torrianni 1 , Peter Zilla 2 , Paul Human 2 , Marc Hendriks 1 , Jan Feijen 3 1 Medtronic Inc., Minneapolis, USA, 2 UCT Cardiovascular Research, University of Cape Town, South Africa, 3 Faculty of Science and Engineering, University of Twente, The Netherlands Calcification of bioprostheses after implantation is a commonly occurring phenomenon that may have sev- eral different causative factors. In order to mitigate the local inflammatory responses and associated enzymat- ic degradation that begins after implantation of the bio- prosthesis, the tissue matrix is usually preserved, primarily by the introduction of cross-links between the collagen molecules in the matrix. Glutaraldehyde (GA)-based cross-linking of valvular bioprostheses is the current standard (1), and these prostheses have a low incidence of thromboembolism and a satisfactory hemodynamic performance. Nevertheless, problems of durability have been reported with this process (2-5). Clinical failure due to cuspal calcification has been reg- istered in many cases (4), with calcium deposition caus- ing valve stiffness, tearing and rupture that result in stenosis and/or insufficiency (6). The exact mechanism of calcification is still not well understood, and numer- ous hypotheses have been described in the literature. Most theories are based on the loss of components from the matrix, such as proteoglycans and glycosaminogly- cans during the reaction with GA (7). These compo- nents act as natural inhibitors of calcification. Other factors mentioned are changes in the morphology and charge distribution in the valve after fixation, and the occurrence of local stress in the valve material (2-4,8). In various studies, post-GA fixation reactions were developed with the aim of reducing calcification, and some of these were successfully introduced into clini- cal practice (9). During the past few years, research has also focused on the use of alternative cross-linking agents (1). Cross-linking based on water-soluble car- bodiimide (EDC) in the presence of N-hydroxysuccin- imide (NHS) has been reported to be a particularly promising alternative to GA (10-12). Calcification after explantation has been reported to be less than that Presented in part at the Second Biennial Meeting of the Society for Heart Valve Disease, 28th June-1st July 2003, Palais des Congrès, Paris, France Address for correspondence: Frank Everaerts, Medtronic Bakken Research Center, Endepolsdomein 5, 6229 GW Maastricht, The Netherlands e-mail: Frank.Everaerts@medtronic.com © Copyright by ICR Publishers 2006 Background and aim of the study: Failure of implant- ed bioprostheses due to calcification is a commonly occurring phenomenon. In order to prevent calcifica- tion, many alternative cross-linking methods to glu- taraldehyde (GA) have been developed and evaluated. Methods: In a novel approach an improved carbodi- imide (EDC) cross-linking method that comprises a two-step process was developed. First, the available amine groups in (tissue) collagen were blocked with a monoaldehyde, followed by an EDC-activated cross-linking reaction of the carboxyl groups in the tissue with a poly(propylene glycol) bis 2-(amino- propyl) ether (Jeffamine™). Results: Samples processed via this method have shown a significantly reduced calcification in a sub- dermal juvenile rat model compared to samples with standard GA treatment. In the present study, heart valve tissue was blocked with various monoalde- hydes, followed by reaction with Jeffamine using carbodiimide cross-linking chemistry. Leaflet calcifi- cation was almost eliminated using different alde- hydes, whereas wall calcification was maximally 95% reduced when propionaldehyde was used as block- ing agent, as compared to a carbodiimide cross- linked control without Jeffamine and blocked amine groups. Conclusion: Amine blocking and cross-linking tech- nology appears promising in the design of the next generation of tissue valves. Calcification was signifi- cantly reduced compared to GA cross-linking. The mechanistic insight of decreased wall calcification is still unknown, and research investigations are ongo- ing. The Journal of Heart Valve Disease 2006;15:269-277