Gulbins H, Pritisanac A, Petzold R, Goldemund A, Doser M, Dauner M, Meiser B, Reichart B, Daebritz S
Department of Cardiac Surgery, University Hospital Grosshadern, LMU Munich, Munich, Germany.
Thorac Cardiovasc Surg. 2005 Apr;53(2):96-102. doi: 10.1055/s-2004-830325.
The purpose of this study was to evaluate the effect of different adaptation phases on the shear-stress resistance of endothelial cells seeded artificially onto vascular prostheses and biological heart valves.
Human endothelial cells (EC), fibroblasts (FB), and smooth muscle cells (SMC) were isolated from vena saphena magna pieces and expanded in culture. Group A: 15 polyurethane vascular grafts (20 mm diameter) were seeded with FB and SMC (53 +/- 1.2 million cells), followed by EC seeding (39 +/- 0.9 million cells). Group B: eight stentless porcine valves (Freestyle, Medtronic, USA) were seeded with FB (68 +/- 1.5 million cells) and EC (42 +/- 1.1 million cells). Shear-stress testing was done under pulsatile flow (pulse rate: 80 pulses/min.). Adaptation phase: flow was set to 0.9 +/- 0.3 l/min (systolic pressure: 40 - 50 mm Hg). High flow was 3.2 +/- 0.6 l/min. (systolic pressure: 140 - 160 mm Hg) and lasted over four hours in all groups. The vascular grafts were divided into three groups (n = 5 each): group 1 (high flow immediately), group 2 (adaptation phase of 15 minutes), and group 3 (adaptation phase of 30 minutes). The valves either were given high flow immediately (n = 4) or had an adaptation phase of 30 minutes (n = 4). Specimens were obtained after cell seeding, before, and after perfusion.
A confluent EC layer was achieved on all grafts. After perfusion without adaptation, large defects within the cell layer were found. No FB and SMC were seen at the bottom of these defects. In group B, the defects were largest on the ventricular surface of the leaflets. After an adaptation phase of 15 minutes in group A, only a few defects within the EC layer were detected with a still confluent FB and SMC. After a 30-minute adaptation phase defects within the EC layer were very rare and no interruption of the underlying FB and SMC layer was seen. Immunohistochemical staining for factor VIII and CD31 proved the EC to be viable and staining for collagen IV and laminin revealed the formation of a basement membrane. After perfusion, the specimen also stained positive for eNOS.
An adaptation phase of 30 minutes proved to be sufficient to allow artificially seeded endothelial cells to adapt to shear stress. The formation of a basement membrane was of great importance for the maintenance of a confluent EC layer.
本研究的目的是评估不同适应阶段对人工接种到血管假体和生物心脏瓣膜上的内皮细胞抗剪切应力能力的影响。
从大隐静脉片段中分离出人内皮细胞(EC)、成纤维细胞(FB)和平滑肌细胞(SMC),并在培养中进行扩增。A组:15个聚氨酯血管移植物(直径20mm)接种FB和SMC(53±1.2百万个细胞),随后接种EC(39±0.9百万个细胞)。B组:8个无支架猪瓣膜(Freestyle,美敦力公司,美国)接种FB(68±1.5百万个细胞)和EC(42±1.1百万个细胞)。在脉动流(脉搏率:80次/分钟)下进行剪切应力测试。适应阶段:流量设定为0.9±0.3升/分钟(收缩压:40 - 50mmHg)。高流量为3.2±0.6升/分钟(收缩压:140 - 160mmHg),且在所有组中持续超过4小时。血管移植物分为三组(每组n = 5):第1组(立即给予高流量)、第2组(适应阶段为15分钟)和第3组(适应阶段为30分钟)。瓣膜要么立即给予高流量(n = 4),要么有30分钟的适应阶段(n = 4)。在细胞接种后、灌注前和灌注后获取标本。
所有移植物上均形成了融合的EC层。在无适应的灌注后,发现细胞层内有大的缺损。在这些缺损底部未见FB和SMC。在B组中,小叶心室表面的缺损最大。在A组中经过15分钟的适应阶段后,仅在EC层内检测到少数缺损,FB和SMC仍融合。经过30分钟的适应阶段后,EC层内的缺损非常罕见,且未见下层FB和SMC层的中断。因子VIII和CD31的免疫组织化学染色证明EC存活,IV型胶原和层粘连蛋白的染色显示基底膜形成。灌注后,标本eNOS染色也呈阳性。
30分钟的适应阶段被证明足以使人工接种的内皮细胞适应剪切应力。基底膜的形成对于维持融合的EC层非常重要。