Biomaterials 27 (2006) 3645–3651 Structural requirements for stabilization of vascular elastin by polyphenolic tannins Jason C. Isenburg, Nishant V. Karamchandani, Dan T. Simionescu, Narendra R. Vyavahare à Cardiovascular Implant Research Laboratory, Department of Bioengineering, Clemson University, 501 Rhodes Engineering Research Center, Clemson, SC 29634, USA Received 5 October 2005; accepted 9 February 2006 Available online 9 March 2006 Abstract Elastin-associated degeneration and calcification are potential causes of long-term failure of glutaraldehyde (Glut) fixed tissue bioprostheses used in cardiovascular surgery. This vulnerability may be attributed to the inability of Glut to cross-link and adequately protect vascular elastin from enzymatic attack. Tannic acid (TA), a poly galloyl glucose (Glc), is compatible with Glut fixation, binds to vascular elastin, improves resistance to degradation and reduces in vivo calcification. While these results provided evidence of a beneficial interaction between elastin and TA, the nature and mechanisms of these interactions are unclear; moreover, TA–elastin binding exhibits a partial instability after long-term interaction with vascular elastin which could contribute to issues of implant toxicity. In present studies, we used resistance to elastase, mechanical properties, and cell viability assays to evaluate the elastin-stabilizing potential and cytotoxicity of TA derivatives and individual TA components such as acetylated TA (AcTA), pentagalloylglucose (PGG), free gallic acid (Gall) and Glc. Our comparative study demonstrates that polyphenolic hydroxyl groups are the main structural groups essential to the interaction between TA and elastin. Furthermore, we show that PGG, the core structure of TA, possesses the same unique elastin- stabilizing qualities of TA, yet it is much less cytotoxic than TA and thus could be potentially useful as an elastin-stabilizing agent for cardiovascular bioprostheses. r 2006 Elsevier Ltd. All rights reserved. Keywords: Glutaraldehyde; Pentagalloylglucose; Elastase; Tannic acid; Aorta; Cross linking 1. Introduction Progressive degeneration and calcification of glutaralde- hyde (Glut) fixed vascular tissues used in cardiovascular surgery restrict their long-term clinical performance [1–3]. This limited biological stability may be attributed to the inability of Glut to cross-link and adequately protect vascular elastin from enzymatic attack. This lack of reactivity towards elastin could be detrimental as elastic fibers are pivotal components in naturally derived replace- ment devices such as tissue-derived vascular grafts and the aortic segment of stentless bioprosthetic heart valves (BHVs). Furthermore, it has been shown that pure elastin, with or without Glut fixation, may be vulnerable to enzymatic degradation [4] and calcification [5,6]. Since these are common problems associated with failure of tissue-derived cardiovascular devices, a tissue pretreatment targeting elastin stabilization may significantly extend the clinical durability of such devices. In previous research we have shown that tannic acid (TA, patent pending), a naturally occurring plant polyphenol binds to pure elastin and in doing so, protects it from in vitro digestion by elastase [4]. In addition, treatment of Glut-fixed porcine aorta with TA resulted in improved resistance to degradation and in vivo calcification in a rat subdermal model [7]. We have also provided evidence for the partial instability of TA after long-term interaction with vascular elastin [7]. This undesirable property may limit long-term elastin stabilization and may also contribute to implant toxicity, as it has been shown that the by-products of TA hydrolysis are cytotoxic [8,9]. TA is composed of a central glucose (Glc) molecule derivatized at its hydroxyl groups with one or more galloyl ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.02.016 à Corresponding author. Tel.: +1 864 656 5558; fax: +1 864 656 4466. E-mail address: narenv@clemson.edu (N.R. Vyavahare).