Abstract
BACKGROUND:The expanded polytetrafluoroethylene (ePTFE) vascular grafts hold promise for enhanced healing, extended suture retention, kink reduction and compression resistance. But thrombus formation still limits its use for revascularization of small-caliber vessels. It is the surface of ePTFE vascular graft that contacts with the blood. The current study focused on surface modification of ePTFE materials to improve its blood compatibility.
OBJECTIVE: To characterize the heparin/alginate (H/A) gel modified ePTFE vascular graft and investigate the hemocompatibility and histocompatibility of the graft.
DESIGN: Observation experiment.
SETTING: Laboratory for Nanomedicine and Biosensor, Biomedicine Engineering Center, Harbin Institute of Technology.
MATERIALS: The GORE-TEX ePTFE vascular grafts were 4 mm in internal diameter. Sodium alginate and 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were purchased from Sigma. Heparin sodium salt was obtained from Calbiochem. Nafion and chitosan were purchased from Aldrich company. Human α-thrombin and AT III were purchased from Haematologic Technologies, Inc. S-2238 was purchased from Chromogenix.
METHODS: This study was performed at the Laboratory for Nanomedicine and Biosensor, Biomedicine Engineering Center, Harbin Institute of Technology between May 2006 and June 2007. The graft was first modified with Nafion and then Chitosan/Nafion/Chitosan multilayer. Following the impregnation of heparin and alginate, covalent crosslinking was performed using ethylenediamine and EDC. Some characterization methods were employed: stastic water contact angle for the hydrophilicity; SEM for the surface morphology; ATR-FTIR for the surface chemical characteristics; APTT and PT, percent hemolysis and Chromogenic assay for the hemocompatibility of the ePTFE vascular graft after modification.
MAIN OUTCOME MEASURES: ①Static water contact angles. ②Characterization of the surface morphology and platelet adhesion by SEM. ③ATR-FTIR ④APTT and PT. ⑤Percent hemolysis ⑥Chromogenic assay for heparin activity.
RESULTS: ①ATR-FTIR revealed the presence of -CO-NH- at 1626 cm-1. ②The water contact angle was greatly decreased from (125±1)° to (84±2)°. ③The prolonged APTT and PT, low percent hemolysis(0.065%) and low amount of platelet adhesion assay showed the H/A gel impregnated graft had good blood compatibility. ④Chromogenic assay showed the modified graft was less thrombogenic than the bare one, and the H/A coating had good stability in. PBS buffer.
CONCLUSION: The H/A modified ePTFE vascular graft has great potential in applications utilizing small-diameter vascular grafts.






INTRODUCTION

Polymer materials in contact with blood are widely used in cardiovascular health care, but an "ideal" prosthetic vascular graft unfortunately does not exist. The expanded polytetrafluoroethylene (ePTFE) vascular grafts hold promise for enhanced healing, extended suture retention, kink reduction and compression resistance. But thrombus formation and neointimal hyperplasia still exist and limit its use [1-4]. Nowadays, there are various ways to improve the hemocompatibility of ePTFE vascular grafts, including endothelialization, modification of albumin or fibronection, etc. ePTFE is very inert and hydrophobic, so Nafion impregnation into the ePTFE interstices was facilitated. Biocompatibility studies on Nafion have shown no acute or chronic foreign body response[5]. Heparin has a significant anticoagulant efficiency. Ionic binding of heparin [6] and covalent binding has been studied since the 1980s with limited success [7]. Yet, the ionically bonded heparin is gradually replaced in blood by proteins, while the covalent binding of heparin requires a preactivation of the material surface which can damage the device and the anticoagulant activity of heparin is often decreased. Therefore, the immobilization technique based on consecutive adsorption makes it possible to coat solid surfaces with ionically attached assemblies consisting of an eligible number of molecular layers of heparin and cationically charged polyelectrolytes arranged according to a specifically designed architecture [8-9]. But release of heparin from the surface may cause massive platelet adhesion and aggregation. Another strategy to prevent thrombus formation is to create a microenvironment of heparin at the surface of blood contacting devices [10]. Alginate is an acidic polysaccharide and alginate gels are known to be biocompatible, degradable, and nontoxic [11].
Therefore, we proposed a new technique to modify the surface of a small diameter ePTFE vascular prosthesis with covalently crosslinked heparin/alginate (H/A) gel to increase its hemocompatibility.

MATERIALS AND METHODS

Materials
The GORE-TEX ePTFE vascular grafts (W.L Gore & Associates, Inc., Flagstaff, AZ) were 4 mm in internal diameter and 22-30 μm in internodal distance. Sodium alginate (medium viscosity) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were purchased from Sigma(USA).

Heparin sodium salt (porcine intestinal mucosa, Mr 12 000-14 000) was obtained from Calbiochem. Nafion (Naf)(5% w/v mixture in water and lower aliphatic alcohols) and chitosan (Ch) (medium molecular weight, 75%-85% deacetylation) were purchased from Aldrich. Human α-thrombin and AT III were purchased from Haematologic Technologies, Inc. S-2238 was purchased from Chromogenix.

Methods
Impregnation of covalently crosslinked gel of heparin and alginate onto an ePTFE vascular graft
The ePTFE graft was first cleaned by ethanol for half an hour and then dried in an oven at 60 ℃ for 1 hour. The cleaned graft was initially impregnated with 0.1wt% Nafion/ethanol solution. After 1-hour incubation with Nafion, the graft was dried overnight. Then, it was alternatively impregnated with an aqueous chitosan solution (2 mg/mL, 0.3% (v/v) acetic acid and 0.5 mol/L NaCl) and 0.1 wt% Nafion/ethanol solution, and allowed for 15-minute incubation followed by three washings in deionized water after each impregnation.
Thereafter, 1% wt heparin and 1% wt sodium alginate were dissolved in water and then ethylenediamine (3.1×10-2 mol/L) and EDC (0.33 mol/L) were added. The ePTFE graft coated with (Naf/Ch)4 multilayer was then impregnated with this solution. Then the graft was oriented vertically at 25 ℃ for 3 days. Then, the impregnated graft was washed with CaCl2 (2.5 mmol/L) and NaCl (143 mmol/L) until the absorbance of the supernatant measured at a wavelength of 210 nm was reduced less than 0.01. The graft was then washed with purified water and dried at room temperature. The coating weight was calculated. This process was performed at least three times.

Detecting static water contact angles
Static contact angles were detected using the contact angle and surface tension meter (KAM101, KSV). Measurements are reported as average degree±standard deviation. For each sample, at least five measurements on different surface sites were averaged.

Characterization of the surface morphology
The scanning electron microscopy (SEM) (X-650 Scanning Electron Micro Analyzer) was used to examine the surface morphologies and Au was coated onto the surface to get a conducting surface.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)
The ATR-FTIR spectra were collected using a Pike Technologies horizontal ATR unit on a Varian Excalibur 3100 spectrometer (USA). Data were collected by averaging over 128 scans at 4 cm-1 resolution between 4 000 cm-1 and 600 cm-1. The blank ATR crystal (ZnSe) was used as reference backgrounds.

Blood coagulation
Activated partial thrombin time (APTT) and prothrombin time (PT) were determined using a semi-automatic blood coagulation analyzer (Steellex LG-PAPER-I, China). The procedures were carried out according to the literature reported by Liu et al [12].

Percentage of hemolysis
The procedures were performed according to the literature reported by Liu et al [12].

Platelet adhesion in vitro
The procedures were conducted according to the literature reported by Liu et al [12].

Chromogenic assay for heparin activity
The activity of immobilized heparin was defined by chromogenic assay. The heparinized sample (0.5 cm×0.5 cm) was first incubated in Hepes buffer (20 mmol/L Hepes, 100 mmol/L NaCl, 0.5 mg/mL bovine serum albumin) at 37 ℃ then incubated with 60 nmol/L AT III for 5 minutes, followed by adding 30 nmol/L human thrombin. At timed intervals, aliquots were removed and Tris buffer was added (50 mmol/L Tris, 175 mmol/L NaCl, 0.05 mg/mL bovine serum albumin, 20 mmol/L ethylenediamine tetraacetic acid), followed by adding chromogenic substrate S-2238 (5 mmol/L final concentration). Residual thrombin was determined by Varian Cary 4000 spectrophotometer and a standard curve.

Stability test
In stability tests, samples were incubated in PBS buffer at 4 ℃ for specified time periods. All the solutions and containers were sterilized before use.

RESULTS

Coating weight
Total coating weight was determined gravimetrically to be 47.97 mg/g of ePTFE vascular graft and heparin surface concentration was calculated to be 10.39 mg/g of the graft according to the mass fraction of heparin.

Static water contact angle
The water contact angles on the unmodified ePTFE surface were very high (125±1)°. After modification, the contact angle decreased to (84±2)°.

Characterization of the surface morphology
Figure 1 shows the appearance of microporous ePTFE interior surface. Before H/A gel impregnation, the graft surface was porous in structure with macro (10-25 μm) and micro (1-3 μm) pores. After impregnation, macropores became smaller while micropores were bigger, which means the size of pores became more uniform. This may be caused by the impregnation procedure, which led to the expansion and then shrinkage of the vascular graft.

ATR-FTIR
ATR-FTIR analysis was used to characterize the surfaces compositions of ePTFE vascular grafts (Figure 2). The bare ePTFE surface had shown substantial amount of fluoride as indicated by CF2 and CF3 stretching vibrations at 1 147 cm-1 and 1 209 cm-1, respectively (Figure 2a). The symmetric stretching vibration of the highly polar -SO3- group was observed at 1058 cm-1. Assignment of the 975 cm-1 band to the symmetric stretching of C-O-C vibration was obvious (Figure 2b). In Figure 2c, the appearance of the distinct transmission band at about 1 626 cm-1, attributable to -NH-CO-, suggests the covalent crosslinking between heparin and alginate was successful. Moreover, the spectrum of the Hep/Alg modified surface showed a wide peak between 3 100-3 500 cm-1 (OH groups) and other functional groups.

Blood coagulation
The anticoagulant properties of graft samples were measured by APTT and PT assays. Results are shown in Table 1.

Percentage of hemolysis
No big difference in hemolysis was observed between the bare ePTFE vascular graft (0.503%) and the H/A gel modified ePTFE vascular graft (0.065%). Both of them were much lower than the permissible hemolysis level of 5%.

Platelet adhesion in vitro
The SEM analysis showed clear differences among the samples (Figure 3). The bare ePTFE showed a weak platelet adhesion and the adhered platelets were aggregated, distorted with pseudopodia. In comparison, the H/A gel on the graft resulted in a distinct reduction of platelet adhesion, and adhering platelets maintained their rounded shape with no formation of pseudopodia.

Chromogenic assay for heparin activity and stability test
Kinetic data showed that thrombin incubated with heparinized graft decayed faster than incubated with the bare one over time (Figure 4). The stability of H/A coating was also shown in Figure 4. After immersed in pH 7.4 PBS for 17 days, the heparinized vascular graft still kept its activity.

DISCUSSION

Here we proposed a new technique to modify small diameter. ePTFE vascular prosthesis with covalently crosslinked H/A gel [13] to increase its hemocompatibility[14] as shown in Figure 5. Briefly, the vascular graft surface was first modified with Nafion. Its sulfonic head groups within pendant chains create "anchor" sites for subsequent ionic adsorption of Chitosan/Nafion/Chitosan multilayer[15-16]. Following the impregnation of the mixture of heparin and alginate, covalent crosslinking between polysaccharide molecules was performed using ethylenediamine and EDC.

The contact angle, surface morphology and ATR-FTIR results indicated the successful modification of H/A gel onto ePTFE vascular graft. The contact angle measurements provide an important evidence for the successful impregnation of crosslinked alginate and heparin gel into the ePTFE vascular graft. After modification, the hydrophilicity of the H/A gel modified graft was enhanced greatly, resulting in a decreased hemolysis. Surface analysis ATR-FTIR revealed the presence of new functional groups on the modified graft surfaces.
APTT and PT results indicated that the H/A gel modified vascular graft was less thrombogenic than the bare graft and the anticoagulant activity of heparin would increase with the contacting time because the surface heparin molecules involved in the crosslinking reaction still kept their anticoagulate activity. Therefore, this impregnation technique provides a novel anticoagulate system of heparin for ePTFE vascular graft.
An apparent decrease of hemolysis was seen after the H/A gel modification, which suggested that H/A gel modified ePTFE graft was not considered to have any significant hemolytic side effects and thus showed very slight hemolytic action. It is believed that an increased wettability may lead to a decreased hemolysis.
As concerning the lower platelet binding onto our H/A surface modification, one can speculate that the impregnation procedure we used to coat the ePTFE surface lead to a significant loss of platelet adhesive sites.
Chromogenic assay confirmed that heparin immobilized on the ePTFE graft could inhibit thrombin activity by activation of AT III effectively, and the H/A coating had good stability.
Therefore, the H/A modified ePTFE vascular graft has great potential in applications utilizing small-diameter vascular grafts.

REFERENCES

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共价交联的肝素/海藻酸盐水凝胶表面修饰膨体聚四氟乙烯人工血管**☆

马 艳1，岳秀丽2，刘 萌1，彭 涛1，刘绍琴1，戴志飞1
1哈尔滨工业大学生物医学工程中心纳米医药与生物传感器实验室，黑龙江省哈尔滨市 150080；2 哈尔滨工业大学市政与环境工程学院，黑龙江省哈尔滨市 150001
马 艳☆，女，1983年生，河北省唐山市人，汉族，哈尔滨工业大学在读博士，主要从事生物医用材料的研究。
通讯作者：戴志飞，教授，博士生导师，哈尔滨工业大学生物医学工程中心纳米医药与生物传感器实验室，黑龙江省哈尔滨市 150080
国家自然科学基金（NSFC-50573015）*；新世纪优秀人才支持计划资助项目*
摘要
背景：虽然膨体聚四氟乙烯人工血管植入体具有易于缝合、质地柔软和抗压迫等诸多优良性能，但由于血栓形成等原因，使这些材料的应用受限。为了解决前述问题，目前的工作主要集中在对现有人工血管材料表面修饰与改性上，最终使其达到血管植入的要求。
目的：用共价交联的肝素-海藻酸钠水凝胶对小口径膨体聚四氟乙烯人工血管进行表面修饰和改性，考察其血液相容性和组织相容性。
设计：观察性实验。
单位：哈尔滨工业大学生物医学工程中心，纳米医药与生物传感器实验室。
材料：实验所用直径4 mm的膨体聚四氟乙烯人工血管为W.L Gore & Associates, Inc.产品，海藻酸钠和1-乙基-3-3-二甲基氨丙基碳化二亚胺购自美国Sigma公司，肝素购于Calbiochem公司，全氟磺酸和壳聚糖购于美国Aldrich公司。人α-凝血酶和抗凝血酶III购于Haematologic Technologies，S-2238购于Chromogenix。
方法：实验于2006-05/2007-06在哈尔滨工业大学生物医学工程中心的纳米医药与生物传感器实验室完成。首先用全氟磺酸修饰膨体聚四氟乙烯表面，然后用肝素-海藻酸钠凝胶进行灌注修饰，以乙二胺为交联剂，1-乙基-3-3-二甲基氨丙基碳化二亚胺为引发剂，将多糖分子进行共价交联。用接触角表征了涂层前后人工血管表面亲水性能的变化，扫描电镜表征了材料表面形貌及血小板黏附，衰减全反射-傅立叶变换红外光谱表征了材料表面的化学结构，然后用活化部分凝血激酶时间、凝血酶原时间、溶血试验以及凝血酶失活试验表征了涂层后人工血管表面的血液相容性。
主要观察指标：①接触角。②用扫描电镜表征材料表面形貌及血小板黏附情况。③衰减全反射-傅立叶变换红外光谱。④活化部分凝血激酶时间、凝血酶原时间。⑤溶血度。⑥凝血酶失活试验。
结果：① 修饰后的人工血管，衰减全反射-傅立叶变换红外光谱结果显示在1 626 cm-1处出现了-CO-NH-基团的峰位。②修饰后人工血管的接触角由（125±1）°降低为（84±2）°。③修饰后的人工血管，具有较长的活化部分凝血激酶时间和凝血酶原时间、较低的溶血度0.065%、较少数量的血小板黏附。④凝血酶失活实验结果显示，凝胶灌注修饰后的人工血管，对凝血酶的活性有较强的抑制作用，因此具有血栓形成的性能且稳定性好。
结论：肝素-海藻酸钠凝胶修饰的膨体聚四氟乙烯具有良好血液相容性及组织相容性，可应用于小口径人工血管。
关键词：膨体聚四氟乙烯人工血管；肝素；抗凝血；表面修饰
中图分类号: R318 文献标识码: A 文章编号: 1673-8225(2008)10-01954-04
马艳，岳秀丽，刘萌，彭涛，刘绍琴，戴志飞.共价交联的肝素/海藻酸盐水凝胶表面修饰膨体聚四氟乙烯人工血管[J].中国组织工程研究与临床康复，2008，12(10):1954-1957
[www.zglckf.com/zglckf/ejournal/upfiles/08-10/10k-1954(ps).pdf]
（Edited by Dubruel P/Song LP/Wang L）