肝素末端结合修饰猪肝去细胞化支架材料的抗凝性能研究

Anticoagulant property of the decellularized porcine liver biological material scaffolds heparinized by end-point attachment

目的通过对猪肝去细胞化材料进行肝素末端结合修饰，改善其作为肝脏组织工程支架材料的抗凝血性能．方法实验用巴马香猪3只，猪龄3～4个月，体质量15kg。取猪肝脏，采用1％十二烷基磺酸钠＋1％曲拉通X-100门静脉灌注获得去细胞化支架材料。对支架材料进行末端连接（EPA）肝素化修饰，之后，检测了它们的肝素化效果及抗凝血性能。结果去细胞化材料经肝素化修饰后结合上了肝素。材料中肝素总含量为（16．00±0．01）μg／mg，并逐渐释放。材料抗凝血性能指标与未经处理的去细胞化材料相比有明显改善，凝血酶时间（TT）、凝血酶原时间（PT）、活化部分凝血活酶时间（AFIT）均超过检测仪器最大值；复钙时间延长达（350．3±29．1）s，具有良好的抗凝性能：溶血实验表明．修饰材料符合生物材料的溶血性要求，血液相容性好。结论在体外研究中，EPA肝素化修饰的去细胞化材料获得良好的抗凝血性能．

Objective To enhance the anticoagulant properties of the heparin modified decellularized porcine liver biological materials as the scaffold for tissue engineering research. Methods Three Bama miniature pigs were enrolled, which were aged 3 - 4 months with body weight of 15 kg. The pig livers were taken, and decellularized scaffolds were prepared by portal perfusion with 1% sodium dodecyl sulfate ＋ 1% Triton X-100. The decellularized porcine liver scaffolds were immobilized with heparin by end-point attachment（EPA）, and the effect of heparinization and anticoagulant property were tested. Results The scaffolds were immobilized with heparin and the content of heparin in the scaffolds was （16.00 ± 0.01） μg/mg, which was gradually released. The heparinized scaffolds demonstrated better anticoagulant property than the untreated scaffolds. The thrombin time（TT）, prothrombin time （PT） and activated partial thromboplastin time （APTT） of the scaffolds immobilized with heparin by EPA exceeded the maximum measurement capability of the instrument. The re-calcification time of the scaffolds was extended to （350.3 ± 29.1） seconds. The hemolytic test showed good blood compatibility, and the heparinized materials met with the requirements for biomaterials. Conclusion It is demonstrated that the decellularized biological materials for tissue engineering acquired stronger anticoagulant ability in vitro via EPA heparinization technique.