Jantzen Alexandra E, Lane Whitney O, Gage Shawn M, Haseltine Justin M, Galinat Lauren J, Jamiolkowski Ryan M, Lin Fu-Hsiung, Truskey George A, Achneck Hardean E
Department of Biomedical Engineering, Duke University, USA.
J Vis Exp. 2011 Sep 9(55):3197. doi: 10.3791/3197.
Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs). By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating. After administration of 100 USP/kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope. We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface. Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.
可植入式心血管装置由人工材料（如钛（Ti）、膨体聚四氟乙烯）制成，这些材料存在形成血栓栓塞的风险。我们开发了一种方法，用自体血源性人或猪内皮祖细胞（EPCs）内衬Ti管的内表面。通过将含有汇合的猪EPCs层的Ti管植入猪的下腔静脉（IVC），我们在大型动物模型的血栓形成环境中测试了细胞接种表面改善的生物相容性，并将其与未修饰的裸金属表面进行比较（图1）。该方法可用于在植入后几分钟内使装置内皮化，并在体内测试其抗血栓功能。从50千克约克郡猪获取外周血，培养其单核细胞部分以分离EPCs。将Ti管（内径9.4毫米）预先切成三个4.5厘米长的纵向段，并用热缩管重新组装。构建了一种接种装置，可使Ti管缓慢旋转。我们对猪进行剖腹手术，将肠和膀胱外置。采用锐性和钝性分离法，从右肾动脉近端的分叉处向远端将IVC骨骼化。然后将Ti管充满荧光标记的自体EPC悬浮液，并以10转/小时×30分钟的速度旋转，以实现细胞均匀包被。给予100 USP/千克肝素后，夹住IVC的两端和一条腰静脉。进行4厘米的静脉切开术，插入装置并用磷酸盐缓冲盐水填充。用4-0普理灵连续缝合关闭静脉切开术时，松开一个夹子以使IVC排气。手术结束时，用0-PDS（聚二氧六环酮缝线）缝合筋膜，用2-0薇乔缝线关闭皮下间隙，并用皮肤吻合器缝合皮肤。3至21天后，对猪实施安乐死，将装置整块取出并固定。拆开Ti管，用荧光显微镜对其内表面成像。我们发现裸金属Ti管完全闭塞，而接种EPCs的管保持通畅。此外，我们能够在与血液接触的内表面证明有一层汇合的EPCs。总之，我们的技术可用于在植入后几分钟内用自体EPCs使Ti管内皮化，以防止装置形成血栓。我们的手术方法允许以最小的失血量和EPCs接种表面破坏来测试这种改良装置改善的生物相容性。
