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heparin complex surface modified ePTFE vascular graft
Applied Surface Science 241 (2005) 485–492 www.elsevier.com/locate/apsusc
Blood compatibility of chitosan/heparin complex surface modified ePTFE vascular graft A.P. Zhua,b,*, Zhang Mingc, Shen Jianb a
College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225009, PR China b Department of Polymer Science and Engineering, Nanjing University, Nanjing 210093, PR China c Medicine College, Nanjing University, Nanjing 210093 PR China Received in revised form 26 July 2004; accepted 26 July 2004 Available online 9 September 2004
Abstract Vascular grafts made of expanded polytetrafluoroethylene (ePTFE) are widely employed in vascular reconstructive surgery. While they are successful as replacements for large-diameter blood vessels, ePTFE vascular grafts are unsuitable for smalldiameter ones because when the internal diameters of the graft are less than 6 mm, they are found to fail without exception due to blood clot formation. To reduce platelets adhesion onto the ePTFE vascular graft, a novel method of binding of chitosan/heparin (CS/Hp) complex to the surface of vascular graft was developed. The binding of chitosan was achieved by irradiating with ultraviolet light the azide modified chitosan that was coated on the ePTFE surface. By forming complex with this coating of chitosan, heparin was then bonded to the ePTFE surface. In vitro blood compatibility experiments showed that CS/Hp surfacemodified ePTFE vascular grafts exhibited markedly reduced platelets adhesion. The outstanding performance of these grafts was further demonstrated by the in vivo experiments, in which they were found to be still unclogged two weeks post-implantation into dog veins. # 2004 Elsevier B.V. All rights reserved. Keywords: Chitosan/heparin complex; UV photosensitive; ePTFE vascular graft; Blood-compatibility
1. Introduction Vascular grafts made of expanded polytetrafluoroethylene (ePTFE) are widely employed in vascular * Corresponding author. Tel.: +86 5147975568; fax: +86 5147975568. E-mail address: [email protected] (A.P. Zhu).
reconstructive surgery [1]. While they are successful as replacement for large-diameter blood vessels, ePTFE vascular grafts are unsuitable for smalldiameter ones because when the internal diameters of the grafts are less than 6 mm, they are found to fail without exception due to blood clot formation [2,3]. Enhancement of antithrombogenicity at the outer ePTFE surface may lead to a functional arterial graft.
0169-4332/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2004.07.055
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Various approaches for improving graft surface designs have been attempted over the year. These include collagen, gelatin or albumin coating, followed by chemical crosslinking with glutaraldehyde, formaldehyde or diepoxided poly (ethylene glycol), and finally heparin impregnation of these protein-immobilized systems [3–5]. However, these methods have achieved limited success. Chitosan, a commercially available polysaccharide made of 2-aminoglucose obtained by deacetylation of chitin, has been applied to promote the formation of extracellular matrix (ECM) in tissue regenerative therapy [6–10]. Recently, it has been widely investigated in the tissue engineering field for it has been found to support the growth of cells such as epithelial cells [11] Both chitosan films and an absorbed chitosan monolayer are, however, highly thrombogenic, as evidenced by their use in the healing of Dacron-made grafts and their ability to trigger formation of an intense thick blood coagulum [12]. A great deal of research work suggests that the chitosan’s thrombogenic properties could be systematically altered by chemical modification of its surfaces. [13–14]. Heparin-coated materials show excellent biocompatibility as shown by the reduced activation of coagulation, complement and blood cells [15–19]. Heparin remains the gold-standard inhibitor of the process involved in the vascular response to injury [20]. To improve the antithrombogenic property, heparin was selected to form the complex with chitosan. The complex of chitosan/heparin (CS/ Hp) coating was supposed to have good blood compatibility according to the in vivo result that heparin–chitosan scaffolds were observed to stimulate cell proliferation and the formation of a thick, dense and highly vascularized granulation layer [21]. To bond the chitosan on the vascular graft, the photosensitive azide group was firstly introduced into the chitosan molecule; the vascular graft was then coated with the azido–chitosan and then UV irradiated. Heparin could be bonded to the vascular graft by forming a complex with chitosan. This immobilization of the complex of heparin and chitosan onto the vascular graft surface to improve the blood compatibility of the graft has not been reported.
2. Experimental 2.1. Materials Chitosan (food grade) was purchased from Nantong Shuanglin Biological product Co. (China). It was purified before use, firstly, by dissolving it in acetic acid and subsequently separating it using an alkali. The viscosity average molecular weight was 5.2 105 g/mol and the N-deacetylation degree was 90%. ePTFE vascular grafts and heparin (Na salt, 160.9 IU/ mg) were bought from the Sigma Chemical Co. 4Azidobenzoic acid was prepared according to literature [22]. All the other chemicals were of analytical grade and were used without further purification. 4Azidebenzoic bonded chitosan (Az–CS) was synthesized by the method reported in our previous work [23]; 2.5% (mol) azide benzoic groups could be bonded to the chitosan molecules through the reaction between a carboxyl groups of 4-azidobenzoic acid and a free amino group of the chitosan. 2.2. Immobilization of Az–CS onto ePTFE vascular graft surface (ePTFE/CS) ePTFE vascular grafts were cut into 2 cm-length and washed in ultrasonic bath of ethanol. 1% Aqueous acetic acid/methoxyethanol (8:2, v/v) Az–CS solution (0.1%) was cast on the surface of ePTFE vascular graft and dried in a brown color desiccator. ePTFE was irradiated with UV light (8 W mercury l A, 254 nm UV-tube light, China) for 10 min, washed completely with 1% aqueous acetic acid, 0.05% NaOH and water (in that sequence), and finally dried. 2.3. Immobilization of heparin onto the ePTFE/CS vascular graft (ePTFE/CS/Hp) The ePTFE/CS vascular graft was immersed in the heparin solution (0.1 g heparin dissolved in 10 mL acetate buffer solution, pH 4.5) for 24 h, washed with water and then dried. 2.4. Determination heparin amount on ePTFE surface Heparin was determined by the method reported by Park et al. [24]. To obtain the standard curve, various
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concentrations (5–35 mg/mL) of heparin solution (2.0 mL) were prepared in separate 10 mL conical flasks. A volume of 3 mL of toluidine blue solution (25 mg toluidine blue dissolved in 500 mL 0.01 N HCl containing 0.2% NaCl) was added to each flask, which was then shaken to ensure complete reaction. After 30 min, 3 mL n-hexane was added to the solution, mixed, and allowed to phase-separate. The heparin/ toluidine blue complex formed was extracted with the n-hexane and its concentration was determined using a UV–vis-NIR scanning spectrophotometer at 631 nm. The amount of heparin present was then calculated. For measuring the heparin amount on the surface of the ePTFE vascular graft, 2 mL of distilled water and 3 mL of toluidine blue solution were added into an empty 10 mL conical flask. A film of the vascular graft was then immersed into the solution and the system was allowed to react for 30 min. After that, 3 mL of nhexane was added into the flask, which was shaken to hasten the extraction of heparin/toludine complex by the n-hexane. By measuring the absorbance of the aqueous solution at 631 nm, the amount of heparin on the surface of the film of ePTFE/CS/Hp vascular graft could be determined from the standard curve. This experiment was repeated three times and the average results were reported here. 2.5. Characterization Ultraviolet spectroscopy analysis was carried out using UV–vis-NIR scanning spectrophotometer. The electron spectroscopy chemical analysis (ESCA) spectra were obtained using a V.G. ESCALAB MK II spectrometer. The X-ray source was an Mg Ka radiation (1253.6 eV) operated at 12 kV and 20 mA. The take-off angle was fixed at 458 relative to the surface of the sample. The measurements were done in vacuum (<2 10 8 mbar) at room temperature. The surface topography was analyzed by SEM (X-650 Scanning Electron Micro Analyzer).
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blood from clotting) for 3 h at 37 8C under the static conditions. After washing with PBS, the sheets were fixed using 2% gluteraldehyde solution for 30 min; they were then washed with PBS again, immersed into 55, 70, 80, 90, 95 and 100% ethanol solution in sequence and finally dried in a desiccator. The sheets were then gold-sputtered for examination with SEM. Three samples each of the unmodified and CS/Hpmodified grafts were examined, and the typical results obtained were presented here. 2.7. In-vivo blood-compatibility Two segments (about 2 cm long) of each the CS/ Hp-modified and unmodified ePTFE vascular grafts were prepared. Each segment was implanted into the saphenous vein in the abdomen of a dog that has been successfully administered systemic anesthesia. After one or two weeks, the dog was sacrificed by acute exsaunguination under general anesthesia and heparin-administration (2–5 mg/Kg). The implanted vascular graft was then taken out. After washing with PBS, the vascular graft was fixed using 2% gluteraldehyde solution for 30 min; it was washed with PBS again, immersed into 55, 70, 80, 90, 95 and 100% ethanol solution in sequence, and finally dried in a desiccator. The graft was then gold-sputtered for examination with SEM. Angiography experiments were also done to examine the blood-compatibility of ePTFE/CS/Hp vascular graft. The implanted position
2.6. Platelet adhesion in vitro The assay of platelet adhesion could be used to examine the activation of platelets, fibrin clots, etc. The ePTFE vascular graft was cut into sheets (10 mm 10 mm), which were allowed to contact with 4 mL of fresh whole blood (heparin was used to prevent the
Scheme 1. Reaction scheme of immobilization of chitosan on ePTFE surface.
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Fig. 1. ESCA spectrum of ePTFE/CS.
of the grafts for the angiography was the same as that for SEM study. As two segments required one dog, two dogs were sacrificed. The typical results were presented here.
3. Results and discussion 3.1. Immobilization of Az–CS on ePTFE surface The reaction schemes for the synthesis of Az–CS and for the immobilization of chitosan on ePTFE were shown in Scheme 1. Azide groups could be introduced into the chitosan molecule through the reaction between a carboxyl group of 4-azidobenzoic acid and the amino group on the chitosan. Azide
groups (-N3) are known to release N2 under UV irradiation and to convert into highly reactive nitrene groups. Nitrene groups are supposed to undergo insertion reaction with the underlying ePTFE molecules and to crosslink the chitosan chains as well (Scheme 1). Fig. 1 showed the ESCA spectra of ePTFE/CS. The peaks at 693.8 and 292.3 eV are characteristics of fluorine and carbon, respectively, and they were clearly from the ePTFE. The new peaks at 287.0, 285.1 and 400.28 eV were attributed to the C1s peaks of hydrocarbon (C–C–C) and ether (C–O–C), and the N1s peak of nitrogen, respectively; clearly, these peaks were a result of the presence of chitosan. Hence, using ESCA, chitosan has been shown to be bonded to the ePTFE surface.
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3.2. Preparation of ePTFE/CS/Hp Much potential of chitosan as a biomaterial stems from its cationic nature and its high charge density in solution. The charge density allows chitosan to form insoluble ionic complexes with anionic polysaccharides such as GAGs and alginates [25]. Because the chitosan charge density is pH dependent, transfer of these ionic complexes to physiological pH can result in dissociation of a portion of the immobilized polyanion. This property can be used for local delivery of biologically active polyanions such as the GAGs and DNA. For example, heparin released from ionic complexes may enhance the effectiveness of growth factors released by inflammatory cells in the vicinity of an implant [26]. Heparin can effectively form complex with chitosan due to the cationic property of chitosan. UV–vis–NIR scanning spectrophotometer was used to measure the amount of heparin bonded on the ePTFE graft surface through the formation of the chitosan/heparin complex. The obtained value is 10.25 mg/cm2. 3.3. Blood-compatibility 3.3.1. In vitro assay Scanning electron micrographs of the ePTFE vascular graft sheets after being contacted with whole blood were presented in Fig. 2, which clearly show that whereas the surface of the unmodified ePTFE
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sheet was covered with an accumulation of red blood cell, while that of the ePTFE/CS/Hp sheet had almost no sign of any cellular matter on it. This in vitro experiment unequivocally proves that ePTFE/CS/Hp graft could inhibit platelet adhesion and activation. The possible cause for this inhibition could be the heparin that was released from the ePTFE/CS/Hp graft because previous studies on biomaterial surfaces with heparin had shown that heparin could reduce the activation of both the complement and the coagulation systems [17,18]. 3.3.2. In vivo assay Fig. 3 shows the SEM images of ePTFE vascular grafts with internal diameter of 5 mm. From Fig. 3(a), it could be seen that the ePTFE vascular graft does not form blood clot one week post-implantation into a dog. This result is very different from that of the ePTFE/CS/Hp vascular graft two weeks post-implantation into a dog; there was obviously a layer of dense granulation tissue formed in the inner surface of ePTFE/CS/Hp vascular graft as shown in Fig. 3(b). This layer of dense granulation tissue may be caused by endothelia regeneration. In the previous research, the glycosaminoglycan (GAG) had been shown to inhibit the proliferation of smooth muscle cells [27] and chitosan was found to enhance the migratory activity of human umbilical vascular endothelial cell [28]. In addition, in vivo heparin–chitosan scoffolds
Fig. 2. SEM images of vascular grafts after being contacted with blood in vitro (a) ePTFE (700) (b) ePTFE/CS/Hp (1000).
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Fig. 3. SEM images of ePTFE/CS/Hp vascular grafts after being planted into veins of dog (a) planted for a week (1000) (b) planted for two weeks (1000).
were observed to stimulate cell proliferation and formation of a thick layer of dense granulation layer that was highly vascularized [21]. This endothelial regeneration could result in prolonged excellent blood compatibility for the ePTFE vascular graft. The success of ePTFE vascular grafts with internal diameter of less than 6 mm for an idea healing process is regarded as follows: as the luminal surface coating is biodegraded, while it maintains its thrombus-free
character, tissue formation, including endothelial regeneration progress. When this coating is completely biodegraded, generated tissues are expected to replace it. CS/Hp complex immobilized on the ePTFE vascular graft may be able to meet the biodegradable and antithrombotic requirement. Fig. 4 shows the angiograph of ePTFE vascular graft. The arrow in Fig. 4 refers to the position where a blood occlusion had taken place. From Fig. 4, it could
Fig. 4. Angiograph image of vascular graft (a) ePTFE (b) ePTFE/CS/Hp.
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be seen that there had been a complete occlusion on the unmodified ePTFE vascular graft whereas there was none on the ePTFE/CS/Hp vascular graft. This in vivo assay result indicated that ePTFE/CS/Hp vascular graft has excellent blood compatibility. It is very likely that it was the heparin released from the ePTFE/CS/ Hp vascular graft as well as the cooperative effect of chitosan and heparin that made this vascular graft possesses this excellent antithrombogenicity. 4. Conclusions A simple and effective method of introducing a chitosan/heparin complex on ePTFE vascular graft surface was developed. The CS/Hp complex appears to modify the surface of the ePTFE vascular prosthesis, causing it to significantly reduce the deposition and spreading of platelets, and hence greatly improving its biocompatibility. It is likely that it was the heparin released from the ionic complex as well as the cooperative effect of chitosan and heparin that resulted in the excellent blood compatibility of such a modified ePTFE vascular graft. The introduction of chitosan/heparin complex on ePTFE has great potential in applications utilizing small-diameter vascular grafts. References [1] D.K. Wilkerson, M.A. Zatina, in: R.S. Greco (Ed.), Implantation Biology: The Host Response and Biomedical Devices, CRC Press, Boca Raton, FL, 1994, pp. 179–190. [2] D.Y. Tseng, E.R. Edelman, Effects of amide and amine plasma-treated ePTFE vascular grafts on endothelial cell lining in an artificial cieculatory system, J. Biomed. Mater. Res. 42 (1998) 188–198. [3] H. Kito, T. Matsuda, Biocompatible coatings for luminal and outer surfaces of small-caliber artificial grafts, J. Biomed. Mater. Res. 30 (1996) 321–330. [4] Y. Noishiki, M. Chvapil, Healing pattern of collagen-impregnated and preclotted vascular grafts in dogs, Vasc. Surg. 21 (1987) 401–411. [5] J.K. Drury, T.R. Ashton, J.D. Cunningham, R. Maini, J.G. Pollock, Experimental and clininal experience with a geletin impregnated Dacron prosthesis, Am. Vasc. Surg. 5 (1987) 542– 547. [6] M.B. Yaylaoglu, P. Korkusuz, U. Ors, F. Korkusuz, V. Hasirci, Development of Calcium phosphate-gelatin Composite as a bone substitute and its use in drug release, Biomaterials 20 (1999) 711–719.
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[7] J.O. Hollinger, G.C. Battison, Biodegradable bone repair materials, Synthetic Polymers and Ceramics, Clin. Orthop. 207 (1986) 290–305. [8] L.G. Cima, J.P. Vacanti, D. Ingber, D. Mooney, R. Canger, Tissue engineering by cell transplantation using degradable polymer substitutes, J. Biomech. Eng. 113 (1991) 143–151. [9] C.T. Laurencin, S.F. El-Amin, S.E. Ibim, D.A. Wilough, M. Attawia, H.R. Allock, A.A. Ambrocio, A highly porous 3dimensional polyphosphazene polymer matrix for skeletal tissue regeneration, J. Biomed. Mater. Res. 30 (1996) 133–138. [10] R.A.A. Muzzarelli, C. Zucchini, P. Ilari, A. Pugnaloni, M. Mattioli-Belmonte, G. Biagini, Stimulatory effect on bone formation exerted by a modified chitosan, Biomaterials 14 (1993) 925–929. [11] P. Popowicz, J. Kurzyca, B. Dolinska, J. Popowicz, Cultivation of MDCK epithelial cells on chitosan membranes, Biomed. Biochim. Acta 44 (1985) 1329–1333. [12] S. Hirano, Y. Noishiki, The blood compatibility of chitosan and N-acylchitosan, J. Biomed. Mater. Res. 19 (1985) 413–417. [13] S. Hirano, M. Zhang, M. Nakagawa, T. Miyata, Wet spun chitosan-collagen fibers, their chemical N-modification and blood compatibility, Biomaterials 21 (2000) 997–1003. [14] A.P. Zhu, B. Shan, Y.L. Yuan, J. Shen, preparation and blood compatibility of phosphorylcholine bonded O-butyrylchitosan, Polym. Int. (2000) (in press). [15] S. Bannan, A. Danby, D. Cowan, S. Ashraf, P.G. Martin, Low heparinization with heparin bonded bypass circuits: is it a safe strategy? Ann. Thorac. Surg. 63 (1997) 663–668. [16] M. Fukutomi, S. Kobayashi, K. Niwaya, Y. Hamada, S. Kitamura, Changes in platelet, granulocyte, and complement activation during cardiopulmonary bypass using heparincoated equipment, Artif. Organs 20 (1996) 767–776. [17] K. Kagisaki, T. Masai, K. Kadoba, Y. Sawa, F. Nomura, N. Fukushima, H. Ichikawa, T. Ohata, K. Suzuki, S. Taketani, H. Matsuda, Biocompatibility of heparin-coated circuits in pediatric cardiopulmonary bypass, Artif. Organs 21 (1997) 836– 840. [18] S. Svenmarker, E. Sandstrom, T. Karsson, E. Jansson, S. Haggmark, R. Lindholm, M. Appelblad, T. Aberg, Clinical effects of the heparin coated surface in cardiopulmonary bypass, Eur. J. Cardiothorac. Surg. 11 (1997) 957–964. [19] A. Belboul, N. Al-Khaja, Does heparin coating improve biocompatibility? A study on complement, blood cells and postoperative morbidity during cardiac surgery Perfusion 12 (1997) 385–391. [20] T. Chandy, Rao GHR, R.F. Wilson, G.S. Das, Delivery of LMW heparin via surface coated chitosan/peg-alginate microspheres prevents thrombosis, Drug Deliv. 9 (2) (2002) 87–96. [21] J.M. Chupa, A.M. Foster, S.R. Sumner, S.V. Madihally, HW.T. Matthew, Vascular cell responses to polysaccharide materials in vitro and in vivo evaluations, Biomaterials 21 (22) (2000) 2315–2322. [22] H. Akio, G. Yoshitaka, N. Masaharu, Novel photoactivatable nonlinear optical polymers: poly[((4-azidopheny)carboxy) ethyl methacrylate], Macromolecules 25 (1992) 5094–5098. [23] A.P. Zhu, M. Zhang, J. Wu, J. Shen, Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-
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bifunctional crosslinking reagent on PLA surface, Biomaterials 23 (23) (2002) 4657–4665. [24] D. Park, A.Z. Piao, H. Jacobs, T. Okano, S.W. Kim, J. Polym. Sci.: Part A: Polym. Chem. 29 (1991) 1725–1735. [25] A. Dekker, K. Reitsma, T. Beugeling, A. Bantjes, J. Feijien, W.G. Van Aken, Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetra fluoroethylene, Biomaterials 12 (1991) 130–138. [26] H.J. Griesser, R.C. Chatelier, T.R. Gengenbach, G. Johnson, J.G. Steele, Growth of human cells on plasma polymers:
putative role of amine and amide groups, J. Biomater. Sci. Polym. Ed. 5 (1994) 531–554. [27] A. Foster, HW.T. Matthew, Growth of vascular cells on bioactive polysaccharide surfaces, Proc. -NOBCChE 25 (1998) 127–129. [28] Y. Okamoto, M. Watanabe, K. Miyatake, M. Morimoto, Y. Shigemasa, S. Minami, Effect od chitin/chitosan and their oligomers/monomers on migrations of fibroblasts and vascular endothelium, Biomaterials 23 (9) (2000) 1975– 1979.
Blood compatibility of chitosan/heparin complex surface modified ePTFE vascular graft A.P. Zhua,b,*, Zhang Mingc, Shen Jianb a
College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225009, PR China b Department of Polymer Science and Engineering, Nanjing University, Nanjing 210093, PR China c Medicine College, Nanjing University, Nanjing 210093 PR China Received in revised form 26 July 2004; accepted 26 July 2004 Available online 9 September 2004
Abstract Vascular grafts made of expanded polytetrafluoroethylene (ePTFE) are widely employed in vascular reconstructive surgery. While they are successful as replacements for large-diameter blood vessels, ePTFE vascular grafts are unsuitable for smalldiameter ones because when the internal diameters of the graft are less than 6 mm, they are found to fail without exception due to blood clot formation. To reduce platelets adhesion onto the ePTFE vascular graft, a novel method of binding of chitosan/heparin (CS/Hp) complex to the surface of vascular graft was developed. The binding of chitosan was achieved by irradiating with ultraviolet light the azide modified chitosan that was coated on the ePTFE surface. By forming complex with this coating of chitosan, heparin was then bonded to the ePTFE surface. In vitro blood compatibility experiments showed that CS/Hp surfacemodified ePTFE vascular grafts exhibited markedly reduced platelets adhesion. The outstanding performance of these grafts was further demonstrated by the in vivo experiments, in which they were found to be still unclogged two weeks post-implantation into dog veins. # 2004 Elsevier B.V. All rights reserved. Keywords: Chitosan/heparin complex; UV photosensitive; ePTFE vascular graft; Blood-compatibility
1. Introduction Vascular grafts made of expanded polytetrafluoroethylene (ePTFE) are widely employed in vascular * Corresponding author. Tel.: +86 5147975568; fax: +86 5147975568. E-mail address: [email protected] (A.P. Zhu).
reconstructive surgery [1]. While they are successful as replacement for large-diameter blood vessels, ePTFE vascular grafts are unsuitable for smalldiameter ones because when the internal diameters of the grafts are less than 6 mm, they are found to fail without exception due to blood clot formation [2,3]. Enhancement of antithrombogenicity at the outer ePTFE surface may lead to a functional arterial graft.
0169-4332/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2004.07.055
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Various approaches for improving graft surface designs have been attempted over the year. These include collagen, gelatin or albumin coating, followed by chemical crosslinking with glutaraldehyde, formaldehyde or diepoxided poly (ethylene glycol), and finally heparin impregnation of these protein-immobilized systems [3–5]. However, these methods have achieved limited success. Chitosan, a commercially available polysaccharide made of 2-aminoglucose obtained by deacetylation of chitin, has been applied to promote the formation of extracellular matrix (ECM) in tissue regenerative therapy [6–10]. Recently, it has been widely investigated in the tissue engineering field for it has been found to support the growth of cells such as epithelial cells [11] Both chitosan films and an absorbed chitosan monolayer are, however, highly thrombogenic, as evidenced by their use in the healing of Dacron-made grafts and their ability to trigger formation of an intense thick blood coagulum [12]. A great deal of research work suggests that the chitosan’s thrombogenic properties could be systematically altered by chemical modification of its surfaces. [13–14]. Heparin-coated materials show excellent biocompatibility as shown by the reduced activation of coagulation, complement and blood cells [15–19]. Heparin remains the gold-standard inhibitor of the process involved in the vascular response to injury [20]. To improve the antithrombogenic property, heparin was selected to form the complex with chitosan. The complex of chitosan/heparin (CS/ Hp) coating was supposed to have good blood compatibility according to the in vivo result that heparin–chitosan scaffolds were observed to stimulate cell proliferation and the formation of a thick, dense and highly vascularized granulation layer [21]. To bond the chitosan on the vascular graft, the photosensitive azide group was firstly introduced into the chitosan molecule; the vascular graft was then coated with the azido–chitosan and then UV irradiated. Heparin could be bonded to the vascular graft by forming a complex with chitosan. This immobilization of the complex of heparin and chitosan onto the vascular graft surface to improve the blood compatibility of the graft has not been reported.
2. Experimental 2.1. Materials Chitosan (food grade) was purchased from Nantong Shuanglin Biological product Co. (China). It was purified before use, firstly, by dissolving it in acetic acid and subsequently separating it using an alkali. The viscosity average molecular weight was 5.2 105 g/mol and the N-deacetylation degree was 90%. ePTFE vascular grafts and heparin (Na salt, 160.9 IU/ mg) were bought from the Sigma Chemical Co. 4Azidobenzoic acid was prepared according to literature [22]. All the other chemicals were of analytical grade and were used without further purification. 4Azidebenzoic bonded chitosan (Az–CS) was synthesized by the method reported in our previous work [23]; 2.5% (mol) azide benzoic groups could be bonded to the chitosan molecules through the reaction between a carboxyl groups of 4-azidobenzoic acid and a free amino group of the chitosan. 2.2. Immobilization of Az–CS onto ePTFE vascular graft surface (ePTFE/CS) ePTFE vascular grafts were cut into 2 cm-length and washed in ultrasonic bath of ethanol. 1% Aqueous acetic acid/methoxyethanol (8:2, v/v) Az–CS solution (0.1%) was cast on the surface of ePTFE vascular graft and dried in a brown color desiccator. ePTFE was irradiated with UV light (8 W mercury l A, 254 nm UV-tube light, China) for 10 min, washed completely with 1% aqueous acetic acid, 0.05% NaOH and water (in that sequence), and finally dried. 2.3. Immobilization of heparin onto the ePTFE/CS vascular graft (ePTFE/CS/Hp) The ePTFE/CS vascular graft was immersed in the heparin solution (0.1 g heparin dissolved in 10 mL acetate buffer solution, pH 4.5) for 24 h, washed with water and then dried. 2.4. Determination heparin amount on ePTFE surface Heparin was determined by the method reported by Park et al. [24]. To obtain the standard curve, various
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concentrations (5–35 mg/mL) of heparin solution (2.0 mL) were prepared in separate 10 mL conical flasks. A volume of 3 mL of toluidine blue solution (25 mg toluidine blue dissolved in 500 mL 0.01 N HCl containing 0.2% NaCl) was added to each flask, which was then shaken to ensure complete reaction. After 30 min, 3 mL n-hexane was added to the solution, mixed, and allowed to phase-separate. The heparin/ toluidine blue complex formed was extracted with the n-hexane and its concentration was determined using a UV–vis-NIR scanning spectrophotometer at 631 nm. The amount of heparin present was then calculated. For measuring the heparin amount on the surface of the ePTFE vascular graft, 2 mL of distilled water and 3 mL of toluidine blue solution were added into an empty 10 mL conical flask. A film of the vascular graft was then immersed into the solution and the system was allowed to react for 30 min. After that, 3 mL of nhexane was added into the flask, which was shaken to hasten the extraction of heparin/toludine complex by the n-hexane. By measuring the absorbance of the aqueous solution at 631 nm, the amount of heparin on the surface of the film of ePTFE/CS/Hp vascular graft could be determined from the standard curve. This experiment was repeated three times and the average results were reported here. 2.5. Characterization Ultraviolet spectroscopy analysis was carried out using UV–vis-NIR scanning spectrophotometer. The electron spectroscopy chemical analysis (ESCA) spectra were obtained using a V.G. ESCALAB MK II spectrometer. The X-ray source was an Mg Ka radiation (1253.6 eV) operated at 12 kV and 20 mA. The take-off angle was fixed at 458 relative to the surface of the sample. The measurements were done in vacuum (<2 10 8 mbar) at room temperature. The surface topography was analyzed by SEM (X-650 Scanning Electron Micro Analyzer).
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blood from clotting) for 3 h at 37 8C under the static conditions. After washing with PBS, the sheets were fixed using 2% gluteraldehyde solution for 30 min; they were then washed with PBS again, immersed into 55, 70, 80, 90, 95 and 100% ethanol solution in sequence and finally dried in a desiccator. The sheets were then gold-sputtered for examination with SEM. Three samples each of the unmodified and CS/Hpmodified grafts were examined, and the typical results obtained were presented here. 2.7. In-vivo blood-compatibility Two segments (about 2 cm long) of each the CS/ Hp-modified and unmodified ePTFE vascular grafts were prepared. Each segment was implanted into the saphenous vein in the abdomen of a dog that has been successfully administered systemic anesthesia. After one or two weeks, the dog was sacrificed by acute exsaunguination under general anesthesia and heparin-administration (2–5 mg/Kg). The implanted vascular graft was then taken out. After washing with PBS, the vascular graft was fixed using 2% gluteraldehyde solution for 30 min; it was washed with PBS again, immersed into 55, 70, 80, 90, 95 and 100% ethanol solution in sequence, and finally dried in a desiccator. The graft was then gold-sputtered for examination with SEM. Angiography experiments were also done to examine the blood-compatibility of ePTFE/CS/Hp vascular graft. The implanted position
2.6. Platelet adhesion in vitro The assay of platelet adhesion could be used to examine the activation of platelets, fibrin clots, etc. The ePTFE vascular graft was cut into sheets (10 mm 10 mm), which were allowed to contact with 4 mL of fresh whole blood (heparin was used to prevent the
Scheme 1. Reaction scheme of immobilization of chitosan on ePTFE surface.
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Fig. 1. ESCA spectrum of ePTFE/CS.
of the grafts for the angiography was the same as that for SEM study. As two segments required one dog, two dogs were sacrificed. The typical results were presented here.
3. Results and discussion 3.1. Immobilization of Az–CS on ePTFE surface The reaction schemes for the synthesis of Az–CS and for the immobilization of chitosan on ePTFE were shown in Scheme 1. Azide groups could be introduced into the chitosan molecule through the reaction between a carboxyl group of 4-azidobenzoic acid and the amino group on the chitosan. Azide
groups (-N3) are known to release N2 under UV irradiation and to convert into highly reactive nitrene groups. Nitrene groups are supposed to undergo insertion reaction with the underlying ePTFE molecules and to crosslink the chitosan chains as well (Scheme 1). Fig. 1 showed the ESCA spectra of ePTFE/CS. The peaks at 693.8 and 292.3 eV are characteristics of fluorine and carbon, respectively, and they were clearly from the ePTFE. The new peaks at 287.0, 285.1 and 400.28 eV were attributed to the C1s peaks of hydrocarbon (C–C–C) and ether (C–O–C), and the N1s peak of nitrogen, respectively; clearly, these peaks were a result of the presence of chitosan. Hence, using ESCA, chitosan has been shown to be bonded to the ePTFE surface.
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3.2. Preparation of ePTFE/CS/Hp Much potential of chitosan as a biomaterial stems from its cationic nature and its high charge density in solution. The charge density allows chitosan to form insoluble ionic complexes with anionic polysaccharides such as GAGs and alginates [25]. Because the chitosan charge density is pH dependent, transfer of these ionic complexes to physiological pH can result in dissociation of a portion of the immobilized polyanion. This property can be used for local delivery of biologically active polyanions such as the GAGs and DNA. For example, heparin released from ionic complexes may enhance the effectiveness of growth factors released by inflammatory cells in the vicinity of an implant [26]. Heparin can effectively form complex with chitosan due to the cationic property of chitosan. UV–vis–NIR scanning spectrophotometer was used to measure the amount of heparin bonded on the ePTFE graft surface through the formation of the chitosan/heparin complex. The obtained value is 10.25 mg/cm2. 3.3. Blood-compatibility 3.3.1. In vitro assay Scanning electron micrographs of the ePTFE vascular graft sheets after being contacted with whole blood were presented in Fig. 2, which clearly show that whereas the surface of the unmodified ePTFE
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sheet was covered with an accumulation of red blood cell, while that of the ePTFE/CS/Hp sheet had almost no sign of any cellular matter on it. This in vitro experiment unequivocally proves that ePTFE/CS/Hp graft could inhibit platelet adhesion and activation. The possible cause for this inhibition could be the heparin that was released from the ePTFE/CS/Hp graft because previous studies on biomaterial surfaces with heparin had shown that heparin could reduce the activation of both the complement and the coagulation systems [17,18]. 3.3.2. In vivo assay Fig. 3 shows the SEM images of ePTFE vascular grafts with internal diameter of 5 mm. From Fig. 3(a), it could be seen that the ePTFE vascular graft does not form blood clot one week post-implantation into a dog. This result is very different from that of the ePTFE/CS/Hp vascular graft two weeks post-implantation into a dog; there was obviously a layer of dense granulation tissue formed in the inner surface of ePTFE/CS/Hp vascular graft as shown in Fig. 3(b). This layer of dense granulation tissue may be caused by endothelia regeneration. In the previous research, the glycosaminoglycan (GAG) had been shown to inhibit the proliferation of smooth muscle cells [27] and chitosan was found to enhance the migratory activity of human umbilical vascular endothelial cell [28]. In addition, in vivo heparin–chitosan scoffolds
Fig. 2. SEM images of vascular grafts after being contacted with blood in vitro (a) ePTFE (700) (b) ePTFE/CS/Hp (1000).
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Fig. 3. SEM images of ePTFE/CS/Hp vascular grafts after being planted into veins of dog (a) planted for a week (1000) (b) planted for two weeks (1000).
were observed to stimulate cell proliferation and formation of a thick layer of dense granulation layer that was highly vascularized [21]. This endothelial regeneration could result in prolonged excellent blood compatibility for the ePTFE vascular graft. The success of ePTFE vascular grafts with internal diameter of less than 6 mm for an idea healing process is regarded as follows: as the luminal surface coating is biodegraded, while it maintains its thrombus-free
character, tissue formation, including endothelial regeneration progress. When this coating is completely biodegraded, generated tissues are expected to replace it. CS/Hp complex immobilized on the ePTFE vascular graft may be able to meet the biodegradable and antithrombotic requirement. Fig. 4 shows the angiograph of ePTFE vascular graft. The arrow in Fig. 4 refers to the position where a blood occlusion had taken place. From Fig. 4, it could
Fig. 4. Angiograph image of vascular graft (a) ePTFE (b) ePTFE/CS/Hp.
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be seen that there had been a complete occlusion on the unmodified ePTFE vascular graft whereas there was none on the ePTFE/CS/Hp vascular graft. This in vivo assay result indicated that ePTFE/CS/Hp vascular graft has excellent blood compatibility. It is very likely that it was the heparin released from the ePTFE/CS/ Hp vascular graft as well as the cooperative effect of chitosan and heparin that made this vascular graft possesses this excellent antithrombogenicity. 4. Conclusions A simple and effective method of introducing a chitosan/heparin complex on ePTFE vascular graft surface was developed. The CS/Hp complex appears to modify the surface of the ePTFE vascular prosthesis, causing it to significantly reduce the deposition and spreading of platelets, and hence greatly improving its biocompatibility. It is likely that it was the heparin released from the ionic complex as well as the cooperative effect of chitosan and heparin that resulted in the excellent blood compatibility of such a modified ePTFE vascular graft. The introduction of chitosan/heparin complex on ePTFE has great potential in applications utilizing small-diameter vascular grafts. References [1] D.K. Wilkerson, M.A. Zatina, in: R.S. Greco (Ed.), Implantation Biology: The Host Response and Biomedical Devices, CRC Press, Boca Raton, FL, 1994, pp. 179–190. [2] D.Y. Tseng, E.R. Edelman, Effects of amide and amine plasma-treated ePTFE vascular grafts on endothelial cell lining in an artificial cieculatory system, J. Biomed. Mater. Res. 42 (1998) 188–198. [3] H. Kito, T. Matsuda, Biocompatible coatings for luminal and outer surfaces of small-caliber artificial grafts, J. Biomed. Mater. Res. 30 (1996) 321–330. [4] Y. Noishiki, M. Chvapil, Healing pattern of collagen-impregnated and preclotted vascular grafts in dogs, Vasc. Surg. 21 (1987) 401–411. [5] J.K. Drury, T.R. Ashton, J.D. Cunningham, R. Maini, J.G. Pollock, Experimental and clininal experience with a geletin impregnated Dacron prosthesis, Am. Vasc. Surg. 5 (1987) 542– 547. [6] M.B. Yaylaoglu, P. Korkusuz, U. Ors, F. Korkusuz, V. Hasirci, Development of Calcium phosphate-gelatin Composite as a bone substitute and its use in drug release, Biomaterials 20 (1999) 711–719.
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