将人血管细胞接种到小直径聚氨酯血管移植物上。

Gulbins H, Pritisanac A, Dauner M, Petzold R, Goldemund A, Doser M, Meiser B, Reichart B

Department of Cardiac Surgery, University Hospital Grosshadern, LMU Munich, Germany.

Thorac Cardiovasc Surg. 2006 Mar;54(2):102-7. doi: 10.1055/s-2005-865916.

INTRODUCTION

Thrombogenicity of small diameter vascular prostheses might be reduced by complete coverage of the luminal surface with vascular cells. We investigated cell seeding on polyurethane vascular prostheses (PUVP).

METHODS

45 PUVP were divided into three groups of n = 15 each: Group A (diameter 20 mm, gamma-sterilized), Group B (diameter 4 mm, gamma-sterilized), and Group C (diameter 4 mm, ethylene oxid [Eto]-sterilized). Human smooth muscle cells (SMC), fibroblasts (FB), and endothelial cells (EC) were isolated from saphenous vein segments and expanded in culture. PUVPs were pre-seeded with a mixed culture of FBs and SMCs (mean 7.7 +/- 2.3 x 10(6) cells) followed by EC seeding (mean 4.4 +/- 0.9 x 10(6) cells). Seven days after cell seeding, PUVPs were perfused under a pulsatile flow. Flow definitions were as follows: adaption phase: low flow, resulting pressure: 60/30 mm Hg; high flow: resulting pressure: 160/50 mm Hg, lasting for 4 hours in all groups. Three subgroups were defined out of each group, differing in the perfusion strategy: high flow immediately, adaption phase of 15 minutes followed by high flow, and adaption phase of 30 minutes followed by high flow. Specimens were taken after each seeding procedure, prior to and after perfusion, and then examined using a scanning electron microscope (SEM) and immunohistochemical staining procedures.

RESULTS

Pre-seeding with the mixed culture revealed a better initial adhesion in Groups A and B compared to group C (76% vs. 41%). In Groups A and B, EC seeding (adhesion 72%) resulted in a confluent EC layer. Immunohistochemical stainings were positive for collagen IV, laminin, CD31, and factor VIII, but negative for eNOS. In Group C, only isolated cells were found after each seeding procedure, which rounded up and vanished during the next days. When perfused with high-flow immediately, Group A and B prostheses revealed small defects (< 10% of the surface) of all cell layers. After perfusion with an adaption phase of 15 minutes only few defects were found within the EC layer with an intact basement membrane. An adaption phase of 30 minutes resulted in a confluent cell layer without significant cell defects. After perfusion, the endothelial cells also stained positive for eNOS.

CONCLUSION

Seeding of a mixed culture consisting of FBs and SMC resulted in an excellent EC adhesion and resistance to shear stress. Cell attachment was better on gamma-sterilized PUVPs compared to Eto-sterilization. The cells obviously maintained their ability to adapt to shear stress.

引言

通过用血管细胞完全覆盖管腔表面，可能会降低小口径血管假体的血栓形成性。我们研究了聚氨酯血管假体（PUVP）上的细胞接种情况。

方法

将45个PUVP分为三组，每组n = 15：A组（直径20 mm，γ射线灭菌），B组（直径4 mm，γ射线灭菌），C组（直径4 mm，环氧乙烷[Eto]灭菌）。从大隐静脉段分离出人平滑肌细胞（SMC）、成纤维细胞（FB）和内皮细胞（EC），并在培养中扩增。PUVP先用FB和SMC的混合培养物预接种（平均7.7 +/- 2.3 x 10(6)个细胞），然后接种EC（平均4.4 +/- 0.9 x 10(6)个细胞）。细胞接种7天后，对PUVP进行脉动流灌注。流量定义如下：适应阶段：低流量，产生的压力：60/30 mmHg；高流量：产生的压力：160/50 mmHg，所有组持续4小时。每组再分为三个亚组，灌注策略不同：立即高流量、15分钟适应阶段后高流量、30分钟适应阶段后高流量。在每次接种程序后、灌注前后取标本，然后用扫描电子显微镜（SEM）和免疫组织化学染色程序进行检查。

结果

与C组相比，用混合培养物预接种在A组和B组中显示出更好的初始粘附（76%对41%）。在A组和B组中，接种EC（粘附率72%）导致形成汇合的EC层。免疫组织化学染色对IV型胶原、层粘连蛋白、CD31和因子VIII呈阳性，但对eNOS呈阴性。在C组中，每次接种程序后仅发现分离的细胞，这些细胞在接下来的几天内变圆并消失。当立即用高流量灌注时，A组和B组假体显示所有细胞层有小缺陷（<表面的10%）。在15分钟适应阶段灌注后，EC层内仅发现少量缺陷，基底膜完整。30分钟适应阶段导致细胞层汇合，无明显细胞缺陷。灌注后，内皮细胞对eNOS也呈阳性染色。