Seeding of Human Vascular Cells onto Small Diameter Polyurethane Vascular Grafts

Abstract

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, γ-sterilized), Group B (diameter 4 mm, γ-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 × 10⁶ cells) followed by EC seeding (mean 4.4 ± 0.9 × 10⁶ 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 γ-sterilized PUVPs compared to Eto-sterilization. The cells obviously maintained their ability to adapt to shear stress.

Key words

Endothelialization - polyurethane vascular prostheses - artificial vessel - graft tissue engineering

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