Abstract
BACKGROUND:Thrombus formation and neointimal hyperplasia still limit the use of small-caliber expanded poly(tetrafluoroethylene) (ePTFE) vascular prosthesis with a diameter less than 6 mm for revascularization in the coronary or peripheral circulation. Bioactive surface heparin coating is one conceivable path for above-mentioned problems.
OBJECTIVE: To elevate the anticoagulant property of ePTFE, this study promoted the patency of a novel small-caliber ePTFE vascular graft by modifying its luminal surface with covalently crosslinked poly(vinyl alcohol)/ p-diazonium diphenyl amine polymer/heparin gel (PVA/PA/Hep gel) and examined the hemocompatibility of the graft.
DESIGN, TIME AND SETTING: Observational experiments were performed at the Nanomedicine and Biosensor Lab, Biomedical Engineering Center, Harbin Institute of Technology from May 2006 to June 2007.
MATERIALS: The ePTFE vascular grafts (diameter of 4 mm), Nafion (Naf) and Poly(vinyl alcohol) (Aldrich, USA), heparin (MW 12 000-14 000) (Calbiochem, USA), p-diazonium diphenyl amine polymer (PA) (this lab, China) were used in this study.
METHODS: ①The vascular graft surface was firstly modified with Nafion. ②Following the impregnation of the mixture of PVA/PA/Hep, covalent crosslinking between polyvinyl alcohol and heparin was performed using crosslinker PA under ultraviolet radiation.
MAIN OUTCOME MEASURES: ①Contact angles, ②Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR), ③Activated partial thromboplastin time (APTT) and prothrombin time (PT), ④hemolysis test, ⑤platelet adhesion test and ⑥thrombosin inactivation test.
RESULTS: ①The water contact angle of the vascular graft surface was greatly decreased after modifying. ATR-FTIR revealed the disappearance of diazonium groups at 2 172 cm-1 and 2 224 cm-1. Vascular prosthesis after modifying had prolonged APTT and PT, low percent hemolysis and low amount of platelet adhesion. Modified vascular prosthesis had inhibitory effect on thrombosin activity and good coating stability.
CONCLUSION: Converage of PVA/PA/Hep has good antithrombotic function and low percent hemolysis, resulting in improving hemocompatibility of vascular prosthesis.






INTRODUCTION

Medium- to large-diameter expanded poly (tetrafluoroethylene) (ePTFE) vascular graft had been used in clinic for many years, but small diameter artificial grafts with less than 5-6 mm inner diameter had not yet been realized in clinical settings [1-2]. The failure in the early phase of implantation is mainly due to occlusion derived from thrombus formation. Taken together, the addition of enhanced antithrombogenic and tissue-regenerative potentials into a graft surface design was essential for a small-caliber artificial graft [3-7].
Heparin is known to prevent thrombus build-up on an intraluminal surface where it has been coated or bonded, such as intraluminal surface of vascular graft. Nevertheless, heparinization of artificial surfaces based on ionic binding [8] and covalent binding [9] of heparin had been used to prevent thrombus formation with limited success [10]. The ionically bound heparin is gradually replaced in blood by proteins, while the covalent binding of heparin decrease the anticoagulant activity of heparin often by a chemical activation of heparin or by a too tight attachment of heparin molecules to the surface [11]. Poly(vinyl alcohol) (PVA) hydrogel obtained by chemical or radiation crosslinking methods are soft, highly water-swollen materials. PVA hydrogel is recognized as the least bioadhesive among the polymeric biomaterials. PVA hydrogel has been extensively used in biomedical area due to high biocompatibility.
The present paper includes a method of coating an ePTFE vascular prosthesis to prevent thrombus formation. The method includes the steps of providing a stable poly(vinyl alcohol)/heparin (PVA/Hep) dispersion and applying the same to the ePTFE vascular prosthesis to effectuate impregnation and impart anti-thrombogenic properties thereto. The ePTFE vascular prosthesis impregnated with the gel of PVA/Hep has an extraluminal and an intraluminal surface. To stably deposit the PVA/Hep hydrogels into the interstices of the graft fabrics, the photocrosslinker, diazoresin, was introduced into the PVA/Hep hydrogels. Under ultraviolet irradiation, the PVA/Hep hydrogel was covalently crosslinked via photoreaction, resulting in a higher stability.

MATERIALS AND METHODS

Materials
The GORE-TEX ePTFE vascular grafts (W.L Gore & Associates, Inc., Flagstaff, AZ) were 4 mm in internal diameter. Heparin sodium salt (Hep, porcine intestinal mucosa, MW: 12 000-14 000) was obtained from Calbiochem. Nafion (5% w/v mixture in water and lower aliphatic alcohols) and PVA (MW: 85 000-146 000) were used in this study. The photosensitive crosslinker, p-diazonium diphenyl amine polymer (PA) was synthesized according to the method reported in the article [12]. Human α-thrombin and antithrombase

(AT) Ⅲ were purchased from Haematologic Technologies, Inc. S-2238 was purchased from Chromogenix.

Methods
Preparation of the PVA/PA/Hep solution
A PVA solution was prepared by adding 10 g of PVA to 90 mL water. The resulted solution was gentlely heated for a few minutes and stirred overnight. The PVA/PA solution was prepared by adding 63 mg of PA to 21 mL of the viscous PVA solution. Subsequently, 150 mg of heparin in 9 mL deionized water was added to the 21 mL PVA/PA solution.

Preparation of the PVA/PA/Hep impregnated ePTFE vascular graft
The ePTFE graft was firstly cleaned by soaking in ethanol for half an hour, and then dried in an oven at 60 ℃ for 1 hour. Then, it was consecutively coated with a 0.1wt% Nafion/ethanol solution and an aqueous PA solution (1 g/L), and allowed for 15-minutes incubation followed by three washings with deionized water. Subsequently, the PVA/PA/Hep solution was impregnated into the porous vascular graft using a Luer-Lok syringe. During this process the PVA/PA/Hep solution was extruded from the pores on the sides of the graft. One hour after incubation, the graft was drained and the air was pushed through the lumen to remove excess solution. The graft was oriented vertically for 24 hours at 25 ℃ in the air. The coating weight was calculated from the dry-weight difference before and after impregnation.

Static water contact angles
Static water contact angles of the graft surface were detected by employing drops of pure deionized water using the contact angle and surface tension meter (KAM101, KSV). The procedures were performed according to the article reported by Liu [13].

Attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR)
The ATR-FTIR spectra were collected using a Pike Technologies horizontal ATR unit with a ZnSe crystal on a Varian Excalibur 3100 spectrometer (USA). The procedures were performed according to the article reported by Liu [14].

Blood coagulation
The in vitro coagulation times, including activated partial thromboplastin time (APTT) and prothrombin time (PT) were determined using a semi-automatic blood coagulation analyzer (Steellex LG-PAPER-I, China). The procedures were conducted according to the article reported by Liu [14].

Percent hemolysis
The procedures were conducted according to the article reported by Liu [14].

Platelet adhesion in vitro
The procedures of platelet adhesion assay were performed according to the article reported by Liu [14].

Chromogenic assay for heparin activity
The activity of immobilized heparin was defined by its capacity to inactivate thrombin by activated antithrombin. Thrombin activity was assessed using the chromogenic substrate S-2238. The procedures were conducted according to the article reported by Liu [14].

RESULTS

Coating weight
The coating weight of the impregnated graft was determined gravimetrically to be 625 mg/g and surface concentration of heparin on modified ePTFE was 20 mg/g according to its primary mass fraction was 0.5%.

Static water contact angle
Static contact angles were measured before and after 30 minutes ultraviolet irradiation. As expected, static contact angles for the bare ePTFE graft were extremely high (140°±1°) and decreased greatly after PVA/PA/Hep gel impregnation (37°± 2°) and cross-linking (47°±5°). After 30 minutes in situ photopolymerization, the static contact angle slightly increased from 37°to 47°due to the decrease amount of hydrophilic groups (-COO- and -OH).

ATR-FTIR
ATR-FTIR was employed to detect the heparinized vascular prosthesis. After impregnation, -COO- and -COSO2- characteristic absorption peak appeared at 1 581 and 840 cm-1 (Figure 1). This confirmed that heparin had been coated onto the surface of ePTFE. There was an -OH characteristic peak at 3 318 cm-1 in both heparin and PVA before and after ultraviolet irradiation. For the specimen before irradiation, two quite strong vibration bands at 2 172 and 2 224 cm-1 could be assigned to the asymmetric and symmetric stretching modes of diazonium, respectively.

Blood coagulation
APTT and PT are standard tests for respectively analyzing the effects of materials on intrinsic and extrinsic coagulation pathways. When the vascular graft was exposed to the blood for 45 s, APTT of ePTFE impregnated by PVA/PA/Hep gel was 48.1 s, which was longer than bare ePTFE (42.3 s). When the exposure time was 30 minutes, the APTT value for the ePTFE impregnated by PVA/PA/Hep gel was more than 120 s, which was much longer than bare graft. To get the PT, the same procedure was carried out. When the contacting time was 45 s, there was no obvious change for the PT value between the modified graft and the bare one. When the contacting time was 30 minutes, there was great change for the PT values from 14.3 s before modification to more than 70 s after modification. This demonstrated that the contact period with the blood had great effects on the PT values.

Percent hemolysis
No big differences in hemolysis were observed between the bare ePTFE vascular graft (0.395%) and the PVA/PA/Hep gel modified ePTFE vascular graft (0.159%). Both of them were much lower than the permissible hemolysis level of 5%.

Platelet adhesion in vitro
Scanning electron microscopy (SEM) analysis after incubating with fresh platelet enriched plasma showed no significant difference between bare ePTFE and PVA/PA/Hep modified ePTFE (Figure 2). No platelets were seen on the graft surface.

Chromogenic assay for heparin activity and stability test
Kinetic data showed that thrombin incubated with heparinized graft decayed faster than incubated with bare graft over time (Figure 3). The stability of PVA/PA/Hep coating was also shown in Figure 4. After being stored in phosphate buffered solution (PBS) (pH 7.4) for 17 days, the heparinized vascular graft still kept its ability of inactivating thrombin, which meant the PVA/PA/Hep coating was very stable in PBS.

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DISCUSSION

The present study focused on surface modification of artificial vascular graft. It is very difficult to introduce hydrophilic chemicals into PTFE because its surface is inert. To improve its wettability [14-15], Nafion was used as the basecoat because of the Teflon-like backbone structure, which facilitating it impregnation into the PTFE interstices easily. It preserved the bulk properties and needed no pretreatment. The sulfonic head groups in Nafion create "anchor" sites for subsequent ionic adsorption of diazoresin. This procedure allowed the PVA/Hep gels to uniformly penetrate into the interstices of the graft fabric using impregnation technique. Biocompatibility studies on Nafion had shown no acute or chronic foreign body response. To stably deposit the PVA/Hep gels into the interstices of the graft fabrics, diazoresin was introduced into the PVA/Hep hydrogels. It is assumed that reactions occur between PVA and diazoresin after decomposition of the diazonium groups of diazoresin, resulting in the formation of stable phenyl ether bonds. It is also possible that the diazonium salt reacts with sulfate groups from heparin (Figure 4). Therefore, the hydrogels networks will likely be formed with covalently bound PVA and heparin.
The coating weight study showed the modified ePTFE was much heavier than the one before modification, indicating large amount of PVA/PA/Hep gel was coated onto the ePTFE surface. The contact angle measurements provided an important evidence for the successful impregnation. Static contact angles decreased greatly after PVA/PA/Hep gel impregnation. ATR-FTIR was used to characterize the heparin immobilization. After impregnation, the peak appearance at 1 581 cm-1 and 840 cm-1 confirmed the successful immobilization of heparin onto the surface of ePTFE vascular graft. The appearance before irradiation and subsequent disappearance after irradiation of the asymmetric and symmetric stretching modes of diazonium indicated the decomposition of the diazonium groups in the graft, which was consistent with the changes in static contact angle. Thus, we could conclude that heparin was photo-polymerized to PA in solid graft. Therefore, the surface modification of Hep/PA/PVA gel was carried out by forming ionic bonds between negative charged group -COO- in heparin and positive charged group -N≡N+ in PA. Furthermore, the cross-linking reactions between -N≡N+, -COOH and -OH facilitated membrane formation on the vascular graft and its stability maintenance.
APTT and PT assay showed that as the contact period prolonged, the cost of blood coagulation factors increased, which led to the prolongation of APTT and PT. Thus, the heparinized vascular graft can inhibit blood coagulation by inhibiting both the intrinsic and extrinsic pathways of coagulation. Percent hemolysis results suggested that PVA/PA/Hep gel modified ePTFE graft showed very slight hemolytic action. It is believed that an increased wettability as a result of the impregnation of the PVA/PA/Hep gel may lead to a decreased hemolysis due to a diminished or more reversible protein adsorption. The results for platelet adhesion demonstrated that modification of PVA/PA/Hep gel onto the vascular graft surface did not increase the number of platelets adhered to the vascular graft surface, which could prevent the start of the blood coagulation system, thus prevent the blood coagulation[16]. The chromogenic assay provided an additional evidence for the successful impregnation of crosslinked PVA/PA/Hep gel into the ePTFE vascular graft at molecular level. The heparin modified the graft surface still kept its ability to inactivate thrombin because only part of sulfate groups on heparin pentasaccharide reacted with diazonium groups of PA, while most sulfate groups were reserved and the structure of pentasaccharide binding with AT Ⅲ still existed. In a word, of the small caliber ePTFE vascular grafts modified, the PVA/PA/Hep gels have significantly enhanced patency.

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肝素/聚乙烯醇凝胶灌注修饰的小口径膨体聚四氟乙烯人工血管**☆

马 艳1，岳秀丽2，刘 萌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
国家自然科学基金资助项目（NSFC-50573015）*；新世纪优秀人才支持计划资助项目
摘要
背景：小口径（直径小于6 mm）膨体聚四氟乙烯人工血管由于血栓形成和内膜增生等原因，在冠状和外周血液循环中的应用受到了限制，而生物活性表面肝素涂层是解决上述问题的一条有效途径。
目的：为了提高膨体聚四氟乙烯人工血管的抗凝血性能，采用新型肝素涂层——共价交联的聚乙烯醇/重氮树脂/肝素凝胶修饰小口径膨体聚四氟乙烯人工血管以提高其通畅性，并观察其血液相容性。
设计、时间及地点：观察性实验，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。
材料：膨体聚四氟乙烯人工血管（直径4 mm），全氟磺酸和聚乙烯醇购于美国Aldrich公司，肝素购于Calbiochem公司（MW12 000~ 14 000），重氮树脂由实验室自己合成。
方法：①用全氟磺酸修饰膨体聚四氟乙烯表面。②用聚乙烯醇/重氮树脂/肝素凝胶进行灌注修饰，在紫外光照射的条件下，重氮树脂作为交联剂，将肝素和聚乙烯醇分子进行共价交联。
主要观察指标：①接触角。②衰减全反射-傅里叶变换红外光谱表征材料表面的化学结构。③活化部分凝血激酶时间、凝血酶原时间。④溶血试验。⑤血小板黏附试验。⑥凝血酶失活试验。
结果：①修饰后的人工血管的接触角降低，衰减全反射-傅立叶变换红外光谱显示在2 172 cm-1和2 224 cm-1处出现了重氮基团的特征峰位。②与未修饰的人工血管相比，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、很少数量的血小板黏附。③与未修饰的人工血管相比，修饰后的人工血管对凝血酶的活性有较强的抑制作用，且涂层稳定。
结论：聚乙烯醇/重氮树脂/肝素凝胶形成的肝素涂层不仅具有很好的抗血栓形成的性能，而且具有较低的溶血度，因此显著提高了人工血管的血液相容性。
关键词：膨体聚四氟乙烯人工血管；共价交联；抗凝血表面修饰；血液相容性
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)14-02773-04
马艳，岳秀丽，刘萌，刘绍琴，戴志飞.肝素/聚乙烯醇凝胶灌注修饰的小口径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(14):2773-2776
[www.zglckf.com/zglckf/ejournal/upfiles/12-14/14k-2773(ps).pdf]
(Edited by Mo XM/Qiu Y/Wang L)