Biomaterials 28 (2007) 496–503 Glycosaminoglycan-targeted fixation for improved bioprosthetic heart valve stabilization $ Jeremy J. Mercuri, Joshua J. Lovekamp, Dan T. Simionescu, Narendra R. Vyavahare à Department of Bioengineering, Cardiovascular Implant Research Laboratory, Clemson University, 401 Rhodes Engineering Research Center, Clemson, SC 29634, USA Received 26 June 2006; accepted 5 September 2006 Available online 9 October 2006 Abstract Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO 4 ) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG- degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue. r 2006 Published by Elsevier Ltd. Keywords: Glutaraldehyde; Periodate; Carbodiimide; Hexosamine; Subdermal implant 1. Introduction In 2001, nearly 300,000 patients worldwide underwent valve-replacement surgery to manage valvular heart disease [1]. Dysfunctional valves are replaced by either mechanical valves fabricated from pyrolytic carbon or chemically crosslinked biological tissue such as porcine aortic valves or bovine pericardium often called bioprosthetic heart valves (BHVs). Glutaraldehyde (GLUT), an aliphatic dialdehyde, has been routinely used for fixation of BHVs. It has the ability to react with the free amine groups in tissue components, specifically collagen, to create tissue-stabilizing crosslinks. Although GLUT crosslinking provides tissue stability against biological breakdown, minimal immunogenicity, and sterility, it is generally recognized that this fixative contributes to loss of cell viability, leaflet calcification and structural dysfunction due to increased tissue stiffness. Approximately 20–30% of GLUT-fixed BHVs become dysfunctional within 10 years and more than 50% fail due to degeneration within 12 years post-operatively [2]. Glycosaminoglycans (GAGs), an integral component of native leaflets, are critical to valvular biomechanics. GAGs lack the amine functionality necessary for GLUT cross- linking to occur. Unlike collagen, these extracellular matrix components are not stabilized within the GLUT-fixed porcine valve leaflet. As a result GAGs are unremittingly lost from BHVs during in vitro fatigue experiments, storage, as well as when implanted in vivo [3–5]. Alternative fixation techniques using carbodiimides [1], epoxides [6], acyl azides [7], dye-mediated fixation [8], ultraviolet irradiation [9] and sodium periodate [10] have ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2006.09.005 $ This work was supported by a grant from NIH (HL070969). à Corresponding author. Tel.: +1 864 656 5558; fax: +1 864 656 4466. E-mail address: narenv@clemson.edu (N.R. Vyavahare).