Surface functionalization and grafting of heparin and/or rgd by an aqueous-based process to a poly(carbonate-urea)urethane cardiovascular graft for cellular engineering applications Henryk J. Salacinski, 1 George Hamilton, 1 Alexander M. Seifalian 1 Tissue Engineering Center, University Department of Surgery, Royal Free and University College Medical School, University College London and the Royal Free Hospital, London NW3 2QG, United Kingdom Received 23 January 2002; revised 30 July 2002; accepted 14 November 2002 Abstract: An aqueous-based process is reported for surface functionalization and grafting of anticoagulant and cell attach- ment moieties, such as heparin and/or arginine– glycine– aspartate (RGD) onto the lumenal surface of a prefabricated cardiovascular graft (5 mm i.d.) made of poly(carbonate- urea)urethane (MyoLink™). It is a three-stage process, all aqueous: (1) hydroxylation using an azobis compound, partic- ularly 2,2'-azobis(2-methylpropionamidine)dihydrochloride, which abstracts hydrogen via an electron transfer process from the polyurethane surface (strong oxygen purging); (2) grafting using the as-generated hydroxide groups to allow attachment of an acrylamide monomer using a conventional ceric ion technique (strong nitrogen purging); and (3) moiety attach- ment, preactivated with [1-ethyl-3-(3-dimethylaminopropyl- )carbodiimide] in acidic solution. The technique was validated by attaching heparin and RGD/heparin to the MyoLink™ polymer. Following bonding, the graft segments were exposed to prolonged physiologic shear force in a flow circuit (10 h). The grafts first were analyzed by X-ray photoelectron spectros- copy (XPS) to determine the degree of attachment of the moi- eties and then by materials methods to assess whether any degradation of the graft material itself had occurred since polyurethanes with carbonate amorphous segments are readily susceptible to hydrolytic degradation following functionaliza- tion processes. XPS showed the moieties were present on the surface at a concentration of 10%. The S2p 3/2 states of sulfur indicated that there were high degrees of ionic covalent bond- ing, indicating high degrees of moiety bioactivity. Heparin was found to be present from the sulfur signal, namely NSO 3 . RGD was found to be present from the nitrogen signal present at the binding energy of 399 eV. Macroscopic analysis and ESEM showed no signs of polyurethane degradation or small protu- berances indicative of microgel formation. Quality control (QC) showed that the internal diameters and wall thicknesses of all the respective grafts postbonding remained within normal batch release limits (5  0.1mm, i.d.; 0.9  0.05 mm, wall thickness). Gel permeation chromatography (GPC) showed there were no statistical differences between the control, which was nonbonded (MN 45,300, MW 98,500, D 2.17) and all of the bonded samples, respectively (MN 41,800, MW 104,000, D 2.45). Radial tensile strength (RTS) analysis also showed that all of the respective samples postbonding (1.48N/mm) remained within batch release specifications (1N/mm). A simple aque- ous polymer surface functionalization and grafting technique has been developed for covalent bonding of anticoagulant and cell-attachment moieties onto poly(carbonate-urea)urethane(s) and has been validated by surface and materials analyses. The moieties were attached uniformly and were bioactive at a high surface density. No degradation in terms of a loss in mechan- ical properties was evident following bonding of the polyure- thane. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 688 – 697, 2003 Key words: surface functionalization; grafting; covalent bond- ing; polymer; cardiovascular graft; cellular engineering; hepa- rin; arginine– glycine–aspartate (RGD); X-ray photoelectron spectroscopy (XPS); gel permeation chromatography (GPC); radial tensile testing (RTS); 2,2'-azobis(2-methylpropiona- midine)dihydrochloride; ceric ion; acrylamide monomer; N-(3- aminopropyl)methacrylamide hydrochloride INTRODUCTION The processes of polymer functionalization and grafting have become of great importance in surgical applications. 1 Vascular bypass surgery with polymeric grafts, especially in below-knee sites, have had high failure rates 2 due to the inherent thrombogenicity of the polymer surface 3 and the inherent lack of compli- ance. 4 One strategy is to attach anticoagulant moi- eties; 5 another is the development of a stable, conflu- ent layer of human endothelial cells (EC); 6 and a third is to develop a compliant polymer. 7 Numerous techniques to covalently or ionically bond such moieties to the surface of polymers ex- ist, 5,8,9 but the majority rely on attaching the molecule in question into the backbone of the polymer 9 or via spacer arms during the synthesis. 10 Correspondence to: A.M. Seifalian; e-mail: A.Seifalian@RFC. UCL.AC.UK Contract grant sponsor: CardioTech Ltd., Wrexham, Wales, UK (HJS) Contract grant sponsor: Public Health Grants; contract grant numbers: M142, M172 Contract grant sponsor: Royal Free Hospital Special Trust- ees; contract grant number: 493 (for laboratory equipment) © 2003 Wiley Periodicals, Inc.