fibronectin mixture on titanium surfaces and their blood compatibility

Colloids and Surfaces B: Biointerfaces 81 (2010) 255–262

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Coimmobilization of heparin/fibronectin mixture on titanium surfaces and their blood compatibility Guicai Li, Fengming Zhang, Yuzhen Liao, Ping Yang ∗ , Nan Huang Key Lab. for Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, PR China

a r t i c l e

i n f o

Article history: Received 10 March 2010 Received in revised form 20 May 2010 Accepted 7 July 2010 Available online 13 July 2010 Keywords: Coimmobilization Heparin Fibronectin Biocompatibility Biomaterials

a b s t r a c t Coimmobilization as a versatile biomodification technique has been widely used in the development of biomimetic materials with superior mechanical and biological properties. In this study, a mixture of heparin and fibronectin (Hep/Fn) was tested for its hemocompatibility after either physisorption or covalent coimmobilization to a titanium (Ti) substrate. The process of substrate activation and film deposition was associated with an increase of roughness; successful immobilization in both cases was demonstrated by FTIR. The immobilized heparin amount was probed by toluidine blue O binding, fibronectin by immunochemistry. Both molecules had slightly higher concentrations on the physisorbed film than after covalent coimmobilization. Plasmatic coagulation activation, tested as activated partial thromboplastin time APTT, and platelet adhesion were significantly improved on the covalently coimmobilized samples than on the physisorbed or blank ones. All these results suggest that the covalent coimmobilization of heparin with fibronectin improved the anticoagulant activity of heparin and caused a favorable blood compatibility. We envisage that this method will provide a potential and effective selection for biomaterials surface modification. © 2010 Published by Elsevier B.V.

1. Introduction Biocompatibility is very essential for the biomedical devices in direct contact with blood, such as vascular stents, artificial heart valves and ventricular assist devices. The major problems for these devices are still thrombosis and embolization. Several approaches to improve their blood compatibility, including coating the device materials with biocompatible films (DLC [1], Ti-O [2], polymers [3], etc.) and immobilizing biomolecules (heparin [4–6], chitosan [7], tropoelastin [8], etc.) on them have been explored. Additionally, endothelial cell (EC) seeding and endothelialization [9] are considered as an ideal method to keep the long-term anticoagulation function. However, the previous studies have shown that when the antithrombotic effect is improved with the anticoagulation molecules, the growth of EC will be inhibited [10], whereas when the growth of EC is promoted with extracelluar matrix molecules, the blood compatibility deteriorates [11,12]. Even though these methods have improved the blood compatibility or cytocompatibility of the implants, most studies just focus on one aspect of the biocompatibility, i.e. blood compatibility or endothelialization.

∗ Corresponding author at: Key Lab. for Advanced Technologies of Materials, Ministry of Education, Southwest Jiaotong University, No. 111, North One Erhuan Road, Chengdu 610031, PR China. Tel.: +86 028 8763 4148 802. E-mail address: [email protected] (P. Yang). 0927-7765/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2010.07.016

There are few studies investigating both aspects simultaneously [13], so the development of such biomaterials that can inhibit thrombosis and promote endothelialization simultaneously is very necessary for the long-term implantation of biomedical devices. In this study, heparin and fibronectin were chosen as the constituents for this purpose, because heparin is an important anticoagulant in clinics to minimize thrombus formation on artificial surfaces [14]. It has an incontrovertible effect on inhibiting thrombus formation by catalytically increasing the affinity of antithrombin III (AT III) to thrombin [15]. While fibronectin is an extracellular matrix protein known to promote cell attachment and spreading [16], differentiation [17,18] and phagocytosis [19,20]. The chemical structures of the heparin and fibronectin are shown in Fig. 1 [21,22]. Both of them can form the Hep/Fn mixture by the heparin binding site on fibronectin chains. The coimmobilized films of Hep/Fn mixture were anticipated to offer both anticoagulant and promoting endothelialization function. In the present work, we describe the construction and evaluation of the coimmobilized Hep/Fn films on titanium (Ti) plates for surface modification for blood-contacting implant applications. As a preliminary study of combining these two biomolecules, the focus of analysis here is on blood compatibility, however, the application of fibronectin also promises good growth of endothelial cells, what will be the subject of a further study. Ti plates are chosen as the substrates because Ti and its alloy are the most widely used biomaterials for orthopaedic implants [23,24] and cardiovascular

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Fig. 1. Structure of (a) heparin and (b) fibronectin.

implants [25]. The modification of Ti substrate was characterized, as well as the quantity of the immobilized heparin and fibronectin was investigated. To determine whether the coimmobilization of Hep/Fn mixture improved the blood compatibility, contact activation and adhesion of blood platelets were evaluated in vitro. We anticipate that this Hep/Fn mixture coating will be helpful to improve the biocompatibility of the Ti-based biomaterial devices. 2. Materials and methods

solution of APTE for 10 h at 37 ◦ C with gentle shaking. After reaction, the carriers were washed with the same solvents and kept in a 120 ◦ C oven for 10 h to enhance the binding of APTE with the carrier (sample was labeled as TiOH-A) [26]. The aminolyzed substrates were then dipped in the mixed solution of Hep/Fn (V1:1 ) with or without pre-activation by EDC/NHS for 1 h and subsequently rinsed with PBS (EDC and NHS were diluted in PBS (pH 7.3) and used during half an hour time. V(EDC/NHS) :V(Hep/Fn) = 1:10). These samples were labeled as TiOH-AHFP and TiOH-AHF, respectively. Finally, the samples were dried for 48 h at RT.

2.1. Materials Heparin sodium (Hep, >160 IU/mg, Solarbio Corp., China) was diluted to a concentration of 5 mg/ml with phosphate buffer saline (PBS) solution at pH 7.3. Fibronectin (Sigma–Aldrich) was diluted to a concentration of 30 ␮g/ml with PBS solution at pH 7.3. All the antibodies used in the experiment were purchased from Abcam Ltd (Hong Kong). 1 mM N-hydroxy-2,5dioxopyrolidine-3-sulfonicacid sodium salt (NHS, purity: >99%), 5 mM 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC), 3aminopropyltriethoxysilane (APTE), toluidine blue O (TBO) and acid orange 7 were purchased from Sigma–Aldrich. Hep/Fn mixture was referred to as HF, and Hep/Fn mixture pre-activated by EDC/NHS was referred to as HFP in the following study. All the other reagents were analytical of grade and used as received. 2.2. Methods 2.2.1. Fabrication of Hep/Fn mixture films Commercial high pure titanium (Baoji, China) was used as the substrate. First, Ti plates were cut into small squares and polished, then the Ti discs were sonicated successively in acetone, ethanol, deionized water and finally dried at room temperature (RT). Fig. 2 shows the scheme of the coimmobilization of Hep/Fn mixture. The cleaned Ti plates were immersed in a 1 M NaOH solution at 70 ◦ C for 24 h (sample was labeled as TiOH), then rinsed thoroughly with deionized water and subsequently immersed in a 2% (v/v) ethanol

2.2.2. FTIR of Ti as the function of hydroxyl, APTES and Hep/Fn immobilization The characteristic absorption peaks of the hydroxyl groups, amino groups and the immobilization of Hep/Fn on the modified Ti surface were detected using a Fourier Transform infrared spectrometer (FTIR, NICOLET 5700, USA) at room temperature. A sample was placed onto a diamond accessory sample stage and then scanned. Spectra were recorded with a resolution of 4 cm−1 in the range of 4000–400 cm−1 . 2.2.3. Determination of amino groups of the grafted APTES The surface densities of amino groups were determined by the uptake of an acid dye [27]. Amino groups could form mixture with acid orange 7 at pH 3, and then the mixture dye was desorbed with 1 mM NaOH. The absorbance of the supernatant at 485 nm was then measured. For morphological observation, Ti and the modified Ti surfaces were observed by scanning electron microscopy (SEM, QUANTA 200, FEI, The Netherlands). 2.2.4. Determination of immobilized heparin The sulfonic groups on the surface can form complexes with TBO dye. To determine the surface density of immobilized heparin [28], the method as described by Smith et al. [29] was selected and modified for our purposes. Briefly, the square samples with 8 mm × 8 mm were incubated with 5 ml of a freshly prepared aque-

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Fig. 2. Sketch map of the preparation of Hep/Fn mixture films.

ous solution of toluidine blue (0.01 M HCl, 0.2 wt% NaCl, 0.04 wt% toluidine blue O zinc chloride double salt) and gently shaken for 4 h at room temperature, resulting in complexation of toluidine blue with heparin. Thereafter, samples were washed with demineralized water (twice for 5 min). Subsequently, toluidine blue complexation to heparin was solubilized in 5 ml of a 1:4 (v/v) mixture of 0.1 M NaOH and ethanol, and toluidine blue was released into the fluid phase. After complete decolourization of the coimmobilized films, the absorbance of the fluid phase was measured at 530 nm using an UV spectrophotometer (BIO-TEK instruments, USA). The amount of immobilized heparin was calculated from a calibration curve. 2.2.5. Determination of immobilized fibronectin The amount of Fn was determined by immunochemistry method as following: Ti samples with Hep/Fn mixture coimmobilized were blocked with sheep serum (1/100 dilution in PBS) for 30 min, washed three times with PBS and incubated with mouse anti-human Fn antibody (1/250 dilution in PBS) for 1 h. Subsequently, the samples were rinsed with PBS again and incubated with horseradish peroxidase labeled goat anti-mouse antibody (1/100 dilution in PBS) for another 1 h. Finally, the chromogenic substrate 3,3 5,5-tetramethylbenzidine (TMB) substrate was added and the reaction was stopped with sulfuric acid after 10 min. The absorbance was measured at 450 nm using an UV spectrophotometer. And the amount of fibronectin was calculated from a calibration curve. 2.2.6. In vitro anticoagulation properties – APTT test The activated partial thromboplastin time (APTT) detects the intrinsic coagulation, i.e., an influence on Factor XIIa, XIa, IXa, VIIIa and high molecular weight kininogen (HMWK) [30]. Therefore, the blood plasma APTT test is commonly used to evaluate the in vitro anticoagulation properties of different biomaterials.

In this work, APTT was obtained to evaluate the anticoagulation activities of the coimmobilized films of Hep/Fn mixture. Briefly, citrate anticoagulated human whole blood (30 ml) from a healthy volunteer supplied by Chengdu Blood Center was centrifuged at 3000 rpm for 15 min to separate the blood corpuscles. The platelet-poor plasma (PPP) obtained was used for the APTT test. Samples with the size of 1 cm × 1 cm were placed into a 24well culture plate and added 500 ␮l PPP per well and incubated for 15 min at 37 ◦ C. Then 100 ␮l of actin activated thromboplastin reagent (Huachen, Shanghai, China) was put in a glass tube, and incubated at 37 ◦ C for 1 min. Thereafter, 100 ␮l of PPP solution from the sample well was added in the tube and incubated for 3 min, and then 100 ␮l of the 30 mM CaCl2 solution was added. The clotting time of the plasma solution was recorded with a clotting time analyzer (Clot 1-␣, Germany) at the first sign of fibrin formation. 2.2.7. Platelet adhesion In vitro platelet adhesion testing was performed to investigate the quantity, morphology, aggregation and pseudopodia of the adherent platelets. Anticoagulant citrate dextrose (ACD) fresh blood taken from a healthy adult was centrifuged at 1500 rpm for 15 min and converted to platelet-rich plasma (PRP). The coimmobilized samples and the control Ti were cut into squares of 8 mm × 8 mm and put in the 24-well culture plate, then PRP was added for 500 ␮l/well and incubated at 37 ◦ C for 1 h. After rinsing with PBS, fixed with 2.5% glutaraldehyde solution, dehydrated at increasing alcohol concentrations (50%, 75%, 90%, 100%; Valcohol /Vdemineralized water ) and dealcoholized at increasing isoamyl acetate (50%, 75%, 90%, 100%; Visoamyl acetate /Valcohol ), all the samples were dried with critical point drying (CPD030, BALZERS) and observed with an optical microscope (Leica, Germany) or coated with gold to conduct scanning electron microscopy to evaluate the morphology and quantity of the adherent platelets.

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roughness due to the chain structure of APTE. The surface roughness of Ti was affected by NaOH activation, silanization and immobilization of Hep/Fn. The TiOH surface just had –OH grafted on it, while APTE could be crosslinked with each other and increased the surface roughness. However, the immobilization of Hep/Fn mixture decreased the roughness (Fig. 4(d)) because of the filling of heparin and protein. These results indicate the existence of coimmobilized Hep/Fn films on the modified Ti surface. 3.2. Quantitative characterization of heparin

Fig. 3. The ATR–FTIR spectra of: (a) Ti; (b) TiOH; (c) TiOH-A; (d) TiOH-AHF (P).

2.3. Statistical analysis The data were reported as mean ± SD. The statistical analysis between different groups was performed using Student’s t-test and one-way ANOVAs. The confidence range selected was 95% and the probabilities of P < 0.05 were considered as a significant difference. All experiments were repeated for three times. 3. Results 3.1. Surface characterization of the modified Ti Fig. 3 shows the FTIR spectra of the bare Ti and modified Ti samples. Compared with the original Ti surface (Fig. 3(a)), the TiOH surface (Fig. 3(b)) showed a new peak in 3400 cm−1 , which was approximate to the –OH group. The APTE grafted surface (Fig. 3(c)) displayed new peaks in 2920 cm−1 corresponding to –CH2 and –CH3 , indicating APTE derived on the surface. The peak of –NH2 in 3200 cm−1 was not sensitive to FTIR and could hardly be found in the FTIR spectrum, however, the –NH2 group could be detected by complexation with the anionic dye, acid orange 7 (5 × 10−4 M, pH 3). As shown in Table 1, compared with Ti and TiOH surfaces a significant increase of amino density was seen on the TiOH-A surface (P < 0.05). The enlargement of –OH peaks in 3400 cm−1 and the appearance of an amide I band peak in 1650 cm−1 (Fig. 3(d)) showed the existence of Hep/Fn mixture on Ti. Fig. 4 depicts the SEM images of Ti, activated by NaOH (TiOH), TiOH grafted with APTE (TiOH-A) and TiOH-A coimmobilized with the Hep/Fn mixture. Compared with the Ti surface (Fig. 4(a)) and the TiOH surface (Fig. 4(b)), the TiOH-A surface (Fig. 4(c)) had the largest

The surface densities of heparin on the coimmobilized films are shown in Fig. 5. It can be seen that the amount of heparin on the samples with Hep/Fn mixture immobilization was significantly larger (P < 0.05) than those on control Ti, but there was no significant difference (P > 0.05) between the TiOH-AHF and the TiOH-AHFP samples. The result indicated the successful immobilization of heparin on the TiOH-A surfaces. Byun et al. [31] studied a styrene/p-amino styrene random co-polymer coupled with poly(ethylene oxide) (PEO) to bind heparin, and the amount of immobilized heparin was 4.9 ␮g/cm2 . Our result showed that the amount of heparin on our coimmobilized films was larger than that reported in the literature [31]. Interestingly, heparin immobilization onto the TiOH-AHF exceeded that onto the TiOH-AHFP, despite the pre-activation of carboxylic acid groups on heparin by EDC. 3.3. Quantitative characterization of fibronectin The amount of fibronectin on different samples was determined by the immunochemistry (Fig. 6). It was shown that fibronectin on Hep/Fn mixture coimmobilized samples was significantly more than that on control Ti (P < 0.05). The amount of fibronectin on the sample of TiOH-AHF and TiOH-AHFP was about 4-fold compared with the control Ti. The result clearly indicated that fibronectin was also successfully immobilized. But similar to the result of heparin (Fig. 5), the amount of fibronectin on the samples of TiOH-AHF and TiOH-AHFP was not significantly different (P > 0.05), and preactivation of heparin and fibronectin with EDC and NHS also did not increase the immobilization of fibronectin. 3.4. APTT The APTT was applied in the evaluation of in vitro antithrombogenicity of biomaterials. The APTT values for the coimmobilized films of Hep/Fn mixture and the control Ti and plasma are shown in Fig. 7. It was found that the APTT values of the coimmobilized samples were significantly prolonged compared with Ti and plasma (P < 0.05) because of the influence of heparin on antithrombin III (AT III), indicating that Hep/Fn mixture coimmobilized films kept excellent blood compatibility. Moreover, from Fig. 7 it could be seen that the pre-activation of heparin and fibronectin mixture by

Table 1 Amino density on the surface of Ti, TiOH and TiOH-A. Error bars represent means ± SD. N = 3. ** P < 0.05 (compared with control Ti and TiOH).

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Fig. 4. SEM images of: (a) Ti; (b) TiOH; and (c) TiOH-A; (d) TiOH-AHF (P); 10,000×.

EDC and NHS displayed significant longer APTT values than that not pre-activated (P < 0.05), even though the amount of heparin on the TiOH-AHFP sample was equivalent with that on the TiOHAHF sample (P > 0.05). The result indicates that the pre-activation of heparin and fibronectin mixture by EDC and NHS could promote the anticoagulant properties of the coimmobilized films. Note that the normal ranges of the APTT for a healthy blood plasma were regarded as 34 ± 7 s, however, the APTT values of plasma were about 50 s, because the PPP was frozen for a certain time before used in the experiment.

3.5. Platelet test

Fig. 5. Amount of heparin on reference Ti and TiOH-A samples after contacting with Hep/Fn mixture pre-activated or not pre-activated by EDC and NHS. N = 5, mean ± SD, ** P < 0.05, * P > 0.05.

Fig. 6. Amount of fibronectin on reference Ti and TiOH-A samples after contacting with Hep/Fn mixture pre-activated or not pre-activated by EDC and NHS. N = 5, mean ± SD, ** P < 0.05, * P > 0.05.

The adhesion and activation of platelets on a biomaterials surface could lead to coagulation and results in thrombus formation. Therefore, the in vitro platelet adhesion test can be performed to investigate the blood compatibility of the biomaterials. Typical SEM micrographs of the platelets on all the samples after being contacted with PRP for 1 h are presented in Fig. 8. It could be seen that the bare Ti surface and the TiOH-AHF sample displayed a large number of adherent platelets with many pseudopods

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stretching out and aggregation after incubated in PRP for 1 h, indicating the activation of platelets. In contrast, the number of adhered platelets and the aggregation phenomenon decreased on the surface of the TiOH-AHFP sample. It was considered that the improved antithrombogenicity could be attributed to the pre-activation of Hep/Fn mixture by EDC and NHS. These results proved that the hemocompatibility could be effectively improved by modifying the surfaces with this kind of coimmobilized Hep/Fn films (preactivated by EDC and NHS). The effects of these coimmobilized films of Hep/Fn mixture on the improvement of the antithrombotic property were first revealed.

4. Discussion

Fig. 7. APTT values of control plasma and TiOH-A samples coimmobilized with Hep/Fn mixture (TiOH-AHFP: pre-activated by EDC and NHS; TiOH-AHF: not preactivated by EDC and NHS). N = 5, mean ± SD, ** P < 0.05.

A biocompatible surface is very important for the application of the biomedical devices. These devices, exposing their surface to streaming blood, should not activate coagulation processes or support the adhesion of blood cells; however later on endothelialization of the surface is desired and this process should be

Fig. 8. Representative SEM images of platelets deposited from human plasma onto various substrates during 1 h incubation at 37 ◦ C. Substrates: (a) Ti; (b) TiOH-AHF; (c) TiOH-AHFP. Original magnification: 1000×, 3000×.

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supported by the material. Until now, many researches [9,32] have focused either on the improvement of the blood compatibility or the cytocompatibility of inorganic and organic biomaterials, while less report considering the both aspects [13]. Therefore, construction of a biocompatible surface for the thromboresistance and endothelialization simultaneously is very significant. Here, two biomolecules: heparin and fibronectin are mixed at first and then coimmobilized onto the Ti surfaces. Heparin has excellent anticoagulant properties, while fibronectin could promote the adhesion and spreading of endothelial cells. For the mixture of heparin and fibronectin, two systems were evaluated: either physisorption or covalent immobilization after EDC/NHS activation. We primarily demonstrated that this functionalized surface coimmobilized with EDC/NHS activated heparin/fibronectin mixture performs better blood compatibility than the physisorbed surfaces and pure Ti. APTES covalently links to the hydroxyl groups of the TiOH surface; the thus silanized surface provides reactive amino group for conjugation with carboxyl group on both heparin and fibronectin molecules. Finally, the Hep/Fn mixture can be covalently immobilized on the surface firmly, which will be more stable and perform longer biofunction than the other immobilization methods. It had been shown previously that bonding of fibronectin to an aminosilanized surface does not alter its biological activity [33]. Fibronectin is an adhesion protein which can be secreted by different cells, such as endothelial cells and fibroblasts, etc. Fibronectin can not only promote the attachment and spreading of EC, but also cause the adhesion of platelets by the RGD peptides on itself and the integrin receptor on platelet membrane. Heparin immobilization to fibronectin may prevent platelet adhesion and blood coagulation in the period after implantation when the implants are not yet completely covered by endothelial cells. Additionally, Laemmel et al. reported that immobilization of heparin may prevent intimal hyperplasia, which often leads to graft failure [34]. Heparin mainly performs its anticoagulant properties by binding to AT III and indirectly impacting the intrinsic coagulation pathway (APTT), however, anticoagulant activity of the immobilized heparin depends on different reaction conditions. Both AT III and fibronectin bind to heparin via their anion binding sites [35], thus the bulk concentration of fibronectin could influence the binding of AT III to heparin, AT III binding to heparin could be displaced by fibronectin with an increasing bulk concentration [31]. On the other hand, EDC can activate the carboxylic acid groups, and then heparin and fibronectin were coimmobilized by the covalent binding of the activated carboxylic acid groups and the amino groups of the aminosilanized surface. The concentration of EDC could also influence the anticoagulant properties of heparin. A good thrombin inhibitory activity could be obtained using a suitable molar ratio of EDC:carboxylic acid groups of heparin (0.2–0.4) [36]. However, the effect of heparin immobilization on endothelial cell proliferation, inhibiting [10] or promoting [37], is not consistent and needs further study. In our study, both the quantitative experiment results of heparin (Fig. 5) and fibronectin (Fig. 6) displayed that there was no significant difference (P > 0.05) between the TiOH-AHF and the TiOH-AHFP samples. The slightly lower amount of heparin and fibronectin on TiOH-AHFP than TiOH-AHF may ascribe to the depletion of carboxyl acid groups activated by EDC/NHS, and more heparin and fibronectin molecules combined with each other than that not activated. However, the anticoagulation properties were significantly different (P < 0.05), the TiOH-AHFP had much longer activated partial thromboplastin time, ∼122 s, than the TiOH-AHF, ∼62 s (Fig. 7). Moreover, the platelets on the TiOH-AHFP showed no pseudopods or aggregation compared to the control Ti and the TiOH-AHF, indicating a better blood compatibility of the TiOHAHFP. This is especially remarkable, as activated platelets express binding sites for fibronectin [38–40]. Note that the amount of

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heparin on both surfaces was equivalent, and the bulk concentration of fibronectin was the same, but the anticoagulation activity was significantly different. The reason maybe that after the preactivation of EDC and NHS, the immobilized heparin exposed much more binding sites to the AT III, therefore, the interaction between the heparin and the AT III was enhanced. Additionally, heparin was the anticoagulation molecule, while fibronectin was an adhesion molecules, which had the binding site of the platelets and could cause platelets adhesion and aggregation. From the results we speculate that the presence of EDC and NHS enhanced the anticoagulation activity of heparin while may inhibit the effect of fibronectin. However, the detailed mechanism remains unclear in this study, because the role of EDC and NHS had relationship with the molar ratio and the concentration of themselves [41,42], and also had relationship with the concentration of heparin and the interaction of heparin and fibronectin molecules. Moreover, the binding mechanism of heparin and fibronectin under different conditions was also not clear, which still need our further investigation. We primarily studied the blood compatibility of a newly constructed biofunctional surface here. In order to clarify the surprising effect that equal or lower amount of covalently immobilized Hep/Fn shows better anticoagulant properties than the physisorbed molecules, further investigation will focus on the interaction of AT III and EDC/NHS activated Hep/Fn films, the stability of the coimmobilized films and the growth of endothelial cells on the coimmobilized films. 5. Conclusion The Hep/Fn mixture could be coimmobilized on aminosilanized Ti surfaces, the pre-activation of the mixture by EDC and NHS improved the anticoagulation activity of heparin. The Ti plates coimmobilized with the pre-activated Hep/Fn mixture showed favorable blood compatibility. In our opinion, the present surface biomodification technology may offer a potential application of biomaterial devices that are directly in contact with blood. Acknowledgement The authors gratefully acknowledge assistance of Dr Manfred Maitz, Jie Liu and Leon Kunad and the financial support of Fundamental Research Funds for the Central Universities of China (2010XS32) Key Basic Research Program 2005CB623904, National Natural Science Foundation of China (No. 30870629). References [1] R. Hauert, Diam. Relat. Mater. 12 (2003) 583–589. [2] N. Huang, P. Yang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, G.J. Wang, P.D. Ding, T.F. Xi, Y. Leng, Biomaterials 24 (2003) 2177–2187. [3] S.P. Baldwin, W.M. Saltzma, Polym. Tissue Eng. TRIP 4 (1996) 177. [4] N. Weber, H.P. Wendel, G. Ziemer, Biomaterials 23 (2002) 429–439. [5] F.R. Gong, X.Y. Cheng, S.F. Wang, Y.C. Zhao, Y. Gao, H.B. Cai, Acta Biomater. 6 (2010) 534–546. [6] J.L. Chen, Q.L. Li, J.Y. Chen, C. Chen, N. Huang, Appl. Surf. Sci. 255 (2009) 6894–6900. [7] A.P. Zhu, M. Zhang, J. Wu, J. Shen, Biomaterials 23 (2002) 4657–4665. [8] Y.B. Yin, S.G. Wise, N.J. Nosworthy, A. Waterhouse, D.V. Bax, H. Youssef, M.J. Byrom, M.M. Bilek, D.R. McKenzie, A.S. Weiss, Biomaterials 30 (2009) 1675–1681. [9] L.Y. Chiu, M. Radisic, Biomaterials 31 (2010) 226–241. [10] C. Nojiri, Y. Noishiki, H. Koyanagi, J. Thorac. Cardiovasc. Surg. 93 (1987) 867–877. [11] A. Stemberger, R. Ascherl, G. Blumel, Hamostaseologie 10 (1990) 164–176. [12] P. Parise, G. Agnelli, Blood Coagul. Fibrinolysis 2 (1991) 749–758. [13] S. Meng, Z.J. Liu, L. Shen, Z. Guo, L.L. Chou, W. Zhong, Q.G. Du, J.B. Ge, Biomaterials 30 (2009) 2276–2287. [14] D. Adil, J. Appl. Sci. 74 (1999) 655–662. [15] P.S. Damus, M. Hicks, R.D. Rosenberg, Nature 246 (1973) 355–357. [16] J.R. Potts, I.D. Campbell, Matrix Biol. 15 (1996) 313–320. [17] R. Pankov, K.M. Yamada, J. Cell Sci. 115 (2002) 3861–3863.

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