Abstract
BACKGROUND:Thrombogenesis is the most common cause of failure in the implantation of tissue engineered small-caliber vesselgrafts. And immobilizing heparin onto the surfaces of vascular scaffoldgrafts is often applied to improve their blood compatibility and patency.
OBJECTIVES: To investigate the small intestinal submucosa (SIS) surface after heparinization with hypothermic plasma technique, to improve the blood compatibility of SIS and to explore the possibility for the construction of small-caliber vascular grafts with modified SIS scaffolds in vivo.
DESIGN: Single exponent study.
SETTING: Department of Orthopaedics, the Six People's Hospital Affiliated to Shanghai Jiao Tong University; Shanghai Institute for Microsurgery of Extremities.
MATERIALS: This study was performed in the Shanghai Institute for Microsurgery of Extremities from January to October 2006. The jejuna were taken from farm pigs.
METHODS: The SIS surface of pigs were processed by argon plasma (20 mL/min) technique at different time periods (0, 2, 4, 6, 8, 10, 12, and 14 s), which were then immediately immerged in heparin sodium solution for 24 hours. Dogs were divided into two groups. The SIS films were sewn into a 3-mm diameter tube and implanted into the defect of a canine femoral by anastomosis as a vascular graft. The observation lasted for 6 weeks.
MAIN OUTCOME MEASURES: The surface morphologies of SIS were observed under scanning electron microscope (SEM). The antithrombogenicity of SIS films was tested by water contact angle, blood coagulation time and platelet adherence observation by SEM. The efficiency of the SIS graft was evaluated by the patency in the circulation of blood with colour doppler detection and histology.
RESULTS: Heparinized SIS showed great different surface morphology comparing with untreated SIS. Untreated SIS surface looked like wrinkled film, but on heparinized SIS surface spread with uniform micro-dots, which looked like a layer of heparin adhesion. Water contact angle decreased with the increase of plasma irradiation time. Prothrombin time (PT), partial thromboplastin time (APTT), and thrombin time (TT) of the SIS films modified with heparin were prolonged. Platelets adhered much more on untreated SIS film than on heparinized SIS film. Vascular graft from SIS embolized in the lumina completely at day 3 after anastomosis. Heparinized SIS graft kept patency for six weeks, and the inner surface of graft was covered with full endothelial cells.
CONCLUSION: Hydrophilicity and antithrombogenicity of heparinized SIS are increased obviously after hypothermia plasma treatment.


INTRODUCTION

Thrombogenesis is the most common cause of failure in the implantation of tissue engineered small-caliber vesselgrafts. And immobilizing heparin onto the surfaces of vascular scaffold grafts is often applied to improve their blood compatibility. The mechanism of immobilization can be categorized into physical adsorption and chemical integration. The binding force with physical method is usually too weak to keep the release slow and the action time long. The chemical treatment is much more stable than any other binding form of heparinization. But the bioactivity of heparin will be reduced if its conformation is affected. Plasma surface modification as a novel technology is characterized by its high effectiveness and efficiency,, no pollution, easy operationand its ability to improve the prorerties of material surfaces [2]. Many studies indicate that this method can combine more heparin to the surfaces of polymers than others[3]. In the present study, we investigated the method of plasma surface modification and heparinization with covalent linkage on the surfaces of SIS (small intestinal submucosa), and the antithrombogenic property of heparinized small intestinal submucosa (SIS) films.

MATERIALS AND METHODS

Preparation of SIS films
The jejuna were taken from farm pigs, frozen to transport, and cleaned within 2 hours. Then the mucosal and muscular tunicae of the jejuna were scraped off by a knife handle wrapped with bandages, while being rinsed by 40℃ water continuously. Then the SIS were harvested and subsequently processed as described by Abraham [4]. The SIS was frozen and dried and the samples were sterilized by 35kGyγ-ray at last.

Hypothermia plasma treatment
Place the SIS films in plasma processor, and treat under the conditions that: discharge power=90 W, Argon gas flow=20 mL/min, evacuation of background to 0.5 Pa, and treating time was 0, 2, 4, 6, 8, 10, 12, 14 s respectively in 8 groups, which were then immediately immerged in heparin sodium solution in vacuum immediately after the plasma
treatment and keep for 24 hours at 37 ℃. Then the films were vacuum-dried for use.

Surface characterization and test
The surfaces of treated SIS films were studied by GSM-5800 scanning electron microscope (Jeol corp. Japan), after dried and coated with platinum in vacuum. The contact angles were determined using sessile drop technique[5] by contact goniometer with the metering error less than 0.5°.

In vitro antithrombogenicity assessment
Platelet adhesion test
The anticoagulated whole blood was centrifuged at 1 000 r/min for 10 minutes to obtain a platelet-rich plasma (PRP). This PRP (100 μL) was placed on the SIS films (3×3 cm2) after preswelling with distilled water (2 mL) and kept at 37 ℃ for 30 and 60 minutes. Then the films were flushed by normal saline, stabilized in 2.5% glutaraldehyde fixing solution, dehydrated by alcohol, dealcoholized by acetoacetate, and dried at critical point of CO2 in sequence. And the morphous of adhered platelet on the films was observed and photographed under scanning electron microscope.

Determination of prothrombin time (PT), partial thromboplastin time (APTT) and thrombin time (TT) [6]
The blood was harvested from healthy donors, and was added sodium citrate (1:9) to prepare blood plasma. Put the films into sample tubes and seal them. Then incubate in waterbath at 37 ℃ for 30 minutes before the tests. The PT, APTT and TT were determined using the automated blood coagulation analyzer, testing more than 5 samples for each group and repeating 3 times for each sample to get the mean value.

In vivo antithrombogenicity assessment
Twenty purebred adult dogs were classified into 2 groups equally. During the experments, dogs were performed general intravenous anesthesia with 2.5% pentothal sodium, before the femoral arteries of both sides were dissected and cut off. Make the treated and untreated SIS films into vasiform scaffolds by interrupted and everted suture with 6-0 polyglycolic acid noninvasive suture lines, and anastomose them to the ends of the femoral arteries. Loose the vascular clamps to pour the blood into the scaffolds. Observe the immediate pulsation and blood flow for 3 hours before closing the incision layer by layer. The dogs were giving benzylpenicilllin potassium of 10 million units intramuscularly and heparin sodium of 3 000 units subcutaneously for 3 days postoperatively.

Detection by color Doppler ultrasound
The patency, inner diameters, wall thickness and blood current status of the scaffolds were detected at 3 and 6 weeks respectively after the operation.

Histological determination
The dogs were sacrificed 6 weeks after the operation, dissect and harvest the SIS conduits. Whether there were any mural thromboses on the inner walls was examined and further observed after HE stain.

RESULTS

Surface characterization of heparinized SIS films
The surfaces of SIS films showed regular islandlike and groovelike change in scanning electron microscope (SEM) photographs (Figure 1a, b) after immobilized heparin by hypothermia plasma. The water contact angles decreased obviously because of the modification, and were 70.6°, 60.0°, 72.7°when treated for 8 s, 10 s and 12 s respectively, showing that they reached minimal when treated for 10 s. While those of untreated films were about 105.3°.

In vitro antithrombogenicity properties
The coagulation time of plasm on untreated and variously treated SIS films is listed in Table 1, showing that when treated for 10 s, the SIS got the best antithrombogencity, and the APTT, PT and TT were prolonged most obviously.

The SEM photographs in platelet adhesion tests are present as Figure 2, showing a large amount of adhered platelets gathering and stretching out pseudopodia in untreated group while much fewer platelets without any activated or deformed ones in treated group.
The animal studies of implanting small-caliber SIS scaffolds met with the results that, 4 scaffolds were completely embolized within 3 hours postoperatively and the obliterated lumina full of thrombi could be observed; and the other 6 were also embolized 2 days after operation detected by color Doppler ultrasound (Figure 3). As for the 10 scaffolds made of heparized SIS, their patency was kept favourable in all of the detections including the 3 hours observation after blood flowage was relaunched, and postoperative color Doppler detections at 1, 3 and 6 weeks (Figure 4); The conduits, which were harvested 6 weeks after the operation, showed a complete coverage of endothelial cells without any thrombi in the lumina (Figure 5).

DISCUSSION

The energy is provided by hypothermia plasma generator, is not very high, the argon ions cannot go very deeply into the substrate and a big portion of their energy is transferred to the surface atoms via collisions with the materials[5].The energy is not high enough to ionize the atoms of giant molecules but can change them into free radicals by breaking the chemical bonds. The plasma treatment could alter the surface free energy and biocompatibility, However nothing happened to the performance of materials[6].
And SIS contains abundant hydroxy and carboxyl groups, which can produce lots of free radicals when provocated by hypothermia plasma. Therefore, effective covalent bonds can form on the surfaces when the treated SIS films are immersed in the solution of high-dencity heparin. Tan et al[7] found that ammonia plasma treated poly (vinyl chloride) can be well modified with heparin to reduce platelet adhesion effectively. According to the characterization of the present study, the surfaces of plasma treated SIS films were changed obviously, and the contact angles decreased markedly indicating the increasing of polarity and hydrophilicity, their free energy was increased, which was benefit to reduce the reaction between the SIS and blood constituents and thus to reduce the coagulation caused by platelets and coagulation factors effectively.
For most medical materials of high polymer, coagulation may happen to different degree on their surfaces when blood gets in touch with them. The collagen proteins of SIS films characterize them with favourable biocompatibility[8]. SIS alone as a vascular graft has shown some promising results, but requires rigorous anticoagulation therapy to prevent thrombosis. And as a modification method for material surface, the plasma technique can endow the materials with good blood compatibility of the surfaces, without any influence to their bulk properties[9]. PLGA films were treated by plasma, water contact angle decreased, hydropilicity increased, platelet adhesion decreased greatly, and platelet activity decreased[10]. Our study found the coagulation time reached its maximum when treated for 10 s, then the antithrombogenicity would decline if treated for longer time. The main reason of this phenomenon may be that with the raising of processing power, the density of plasma will increase to produce more free radicals; But the probability of collision quenching between the high-energy particles will increase at the same time[11]. Therefore, with a certain power of processing, a relative balance can be reached between the producing and collision quenching of the free radicals, and the best antithrombogenecity can be obtained.
SIS is a commonly used kind of scaffold materials for tissue engineering. Baltoyannis[12] prepared moderate-caliber conduits with canine SIS and implanted into the donor dogs, and found the patency could kept one year later. But when small-caliber tissue engineered vessels were prepared, thrombi were prone to occur because of the great resistance and low velocity of blood flow[13]. The SIS tissues used commonly at present are of extensive heterogenous sources, Zheng[14] investigated that prepared swine SIS films still contained some cellular DNA, and could induce inflammatory reactions with infiltration of lymphocytes when implanted subcutaneouly into rats. As a result, the SIS should be modified by effective technique to improve the blood compatibility, when adopted as vascular scaffolds. Sipehia[15] indicated that the ePTFE grafts for small diameter blood vessels replacement could acquire good blood compatibility and keep patent for long time when implanted to bridge the aortas of rabbits after ammonia plasma modification, and the inner lumina could covered completely with endothelial cells 1 month later while those of untreated control group were embolized in 1 week. In our study, the plama modified SIS scaffolds were put into blood circulation to contact platelets and coagulation factors directly under the stress condition of blood flow, with the results indicating that the small-caliber SIS conduits could keep durable patency and be covered completely by endothelial cells.

CONCLUSION

Heparin could be immobilized onto SIS films by hypothermia plasma initiation technique. The surface characterization and the correlate index of in vitro and in vivo antithrombogenecity indicated that the modified surfaces provided good and persistent antithrombogenicity, and showed potent blood compatibility. Accordingly, hypothermia plasma technique is an effective method of surface modification, and the heparinized SIS is suitable for small-caliber scaffolds in vascular tissue engineering.

Acknowledgement: This research was supported by Shanghai Science & Technology Committee, China, No. 044119722.

REFERENCES

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低温等离子体肝素化改性后小肠黏膜 下层的血液相容性及其构建小口径 血管的可行性*★

韩本松1，范存义1，刘生和1，莫秀梅2
1上海交通大学附属第六人民医院骨科，上海市四肢显微外科研究所，上海市 200233；2东华大学生物科学技术研究所，上海市 200051
韩本松★，男，1971年生，湖北省人，汉族，1996年上海交通大学医学院毕业，硕士，主治医师，主要从事显微血管外科和修复重建外科研究。
通讯作者：范存义，上海交通大学附属第六人民医院骨科，上海市四肢显微外科研究所，上海市 200233
上海市科学技术委员会科研计划项目（044119722）*
摘要
背景：栓塞是小口径血管植入后失败的主要原因，目前常常对血管支架材料进行抗凝改性，以期提高其血液相容性，从而提高血管的有效通畅性。
目的：观察低温等离子体肝素化改性后的内植物修复材料小肠黏膜下层的血液相容性，并探讨其体内构建小口径血管的可行性。
设计：单一样本实验。
单位：上海交通大学附属第六人民医院骨科、上海市四肢显微外科研究所。
材料：实验于2006-01/10在上海市四肢显微外科研究所实验室进行。小肠黏膜下层来自农场猪。
方法：①改性：将猪小肠黏膜下层用氩等离子体处理器照射处理，氩气流量20 mL/min，照射时间分别为0, 2, 4, 6, 8, 10, 12, 14 s，接着浸入肝素钠溶液24 h。②体内抗凝血实验：将20条狗分为2组，分别植入经过改性或未改性的小肠黏膜下层缝合成的3 mm口径血管支架，与股动脉直接吻合，观察6周。
主要观察指标：①血液相容性检测：通过扫描电镜观测表面形态，并通过液滴接触角、凝血时间及血小板黏附实验检测小肠黏膜下层改性前后的抗凝性。②体内抗凝血：通过彩色多普勒和组织学检测，评价血管支架直接在体内循环血流下的长期通畅性和形成血管的可行性。
结果：①改性小肠黏膜下层膜表面呈现出均匀的微结构改变，随着等离子体照射时间增加，表面液滴接触角降低；改性后凝血酶原时间、活化部分凝血活酶时间和凝血酶时间延长；血小板黏附减少。②植入体内后未改性小肠黏膜下层血管支架3 d内栓塞，改性组在6周内仍保持通畅，管腔内表面有完整内皮细胞覆盖。
结论：经低温等离子体肝素化改性后小肠黏膜下层的亲水性、抗凝性有明显提高。
关键词：小肠黏膜下层；改性；血管移植；低温等离子体；抗凝血性；生物材料
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)14-02753-04
韩本松，范存义，刘生和，莫秀梅.低温等离子体肝素化改性后小肠黏膜下层的血液相容性及其构建小口径血管的可行性[J].中国组织工程研究与临床康复，2008，12(14):2753-2756
[www.zglckf.com/zglckf/ejournal/upfiles/12-14/14k-2753(ps).pdf]
(Edited by Chi-Hsiao Yeh/Ji H/Wang L)