应用四枝化状聚乙二醇-乙烯砜基交联去细胞带瓣管道的实验研究

An Experimental Study of Decellularized Valved Conduits Cross-linked with 4-arm Branched Polyethylene Glycol-VS

目的利用四枝化状聚乙二醇-乙烯砜基(polyethylene glycol-VS,PEG-VS)交联去细胞主动脉带瓣管道,制备新型组织工程复合支架,研究其力学和生物学性能。方法采用胰酶法制备兔主动脉去细胞带瓣管道,利用四枝化状PEG-VS与兔去细胞带瓣管道交联构建复合支架,体外静态条件应用力学测试仪检测去细胞管道和复合支架力学性能。将纯种新西兰大白兔30只随机分为3组,每组10只,对照组:正常兔主动脉带瓣管道;去细胞组:去细胞带瓣管道;复合支架组:PEG复合支架。构建兔颈总动脉瓣膜移植模型,术后28 d分别用超声心动图检测3组移植管道通畅率,用HE染色、扫描电子显微镜观察微观形态和炎症细胞浸润,免疫荧光染色检测复合支架体内内皮化程度。结果体外力学测试结果示:PEG复合支架弹性模量及最大负荷力均较去细胞管道有明显提高(P弹性模量=3.1×10-9,P最大负荷力=1.1×10-6)。术后血管彩色多普勒超声心动图提示:复合支架组管腔通畅率高于对照组(P=0.054)和去细胞组(P=0.019),腔内血栓形成率、管腔形变率明显降低;HE染色、扫描电子显微镜检测显示:复合支架组体内内皮化程度增高,内皮细胞于支架上均匀附着;免疫荧光检测显示:支架表面内皮细胞标记物CD34阳性率高于对照组和去细胞组。结论利用四枝化状功能化PEG-VS改性去细胞主动脉瓣带瓣管道,可明显改善组织工程支架生物力学性能和组织相容性。

Objective To develop a tissue engineering scaffold by using 4-arm branched polyethylene glycol-VS（PEG-VS） cross-linked with decellularized valved conduits（DVC）,and to research on its mechanical and biological functions.Methods The valved aortic conduits of rabbits were taken and decellularized by trypsin method and then were cross-linked with 4-arm branched PEG-VS to construct the composite scaffolds（CS）.The functions of decellularized valved conduits and the composite scaffolds were tested by mechanics test system.Thirty New Zealand white rabbits were equally and randomly assigned to one of the three groups： the control group,the DVC group,and the CS group.Valved aortic conduits,decellularized valved conduits and composite scaffolds were transplanted into the common carotid artery of the above-mentioned three groups of rabbits respectively.Twenty-eight days after the operation,patency of the transplants was tested by Color Doppler ultrasound;micro-morphology and inflammatory infiltration were observed by hematoxylin eosin（HE） staining and scanning electron microscope（SEM）,and endothelialization of composite scaffolds was detected by immunofluorescent staining.Results A series of biomechanical analyses revealed that the composite scaffolds had highly similar mechanical properties as fresh tissue,and had superior elastic modulus（P=3.1×10-9） and tensile strength（P=1.1×10-6） compared with decellularized valved conduits.Color Doppler ultrasound revealed that the graft patency for the CS group was better than the control group（P=0.054） and the DVC group（P=0.019）,and the intra-aortic thrombosis rate and distortion rate decreased significantly.HE staining and SEM showed that the endothelialization of composite scaffolds in the CS group was significantly higher than the other two groups with the endothelial cells evenly distributed on the scaffolds.The immunofluorescent staining indicated that the positive rate of the endothelial cell marker CD34 was higher than the other two groups. Conclusion The composite scaffolds using 4-arm branched PEG-VS cross-linked with DVC have great mechanical and biological properties.