Improving small-diameter vascular grafts: from the application of an endothelial cell lining to the construction of a tissue-engineered blood vessel.

One of the main reasons why vascular reconstruction with synthetic small-diameter grafts has limited success is the absence of endothelial cells. To improve the outcome of nonvenous vascular bypass surgery, cell seeding of vascular grafts and other tissue-engineering techniques were developed. In this article, an overview is given of the artificial blood vessel as an alternative for venous vascular bypass surgery.

Application of a clinical grade CD34-mediated method for the enrichment of microvascular endothelial cells from fat tissue.

BACKGROUND: Microvascular endothelial cells (MVEC) derived from s.c. fat are seeded on vascular grafts to prevent early occlusion. We have demonstrated the presence of contaminating cells contributing to MVEC seeding-related intimal hyperplasia in MVEC isolates from fat tissue. We found that cell isolates additionally purified after the isolation process, were associated with a reduced thrombogenicity and development of intimal hyperplasia in vitro. A combination of 11Fibrau (F11)- and CD14-coated Dynabeads was used to deplete the contaminating cells, fibroblasts, and monocytes/macrophages. Unfortunately, clinical-grade F11 is not available, and thus cannot be used for clinical practice. CD34 selection with clinical-grade products is widely used for the isolation of hematopoietic progenitors, and endothelial cells (EC) express CD34 on their surfaces. The aims of this study were to test the effectiveness of two different CD34-selection techniques for purification of MVEC, and to compare the results with those of the F11/CD14-method. METHODS: Liposuction fat was enzymatically digested and centrifuged twice to remove adipocytes and collagenase. CD34 selection was performed using the commercially available methods from Nexell or Miltenyi. Both techniques were modified for our use. The purity after isolation and culture, and recovery were determined by flow-cytometry (CD31-expression) and compared with that of cells purified with the F11/CD14-method. RESULTS: Besides MVEC, the contaminating fibroblasts and macrophages/monocytes weakly expressed the CD34 Ag. Enrichment of MVEC was not successful with the Miltenyi method. Variations in neither the dose of Ab nor the use of direct selection and different separation programs improved the results. With the Nexell method, MVEC were enriched to 86%, a comparable purity to that obtained with the F11/CD14-method. However, a lower recovery was achieved with the Nexell method. CONCLUSION: Enrichment of MVEC could be achieved with a modified protocol of the clinical grade CD34(+) selection method from Nexell, but not with the CD34 method from Miltenyi.

Reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and intimal hyperplasia.

INTRODUCTION: fat derived microvascular endothelial cells (MVEC) seeded on prosthetic vascular grafts, improve patency in animals. Results in humans were disappointing, due to thrombogenicity and progressive intimal hyperplasia. Also in animals intimal hyperplasia was found. We postulate that contaminating cells present in the transplant are involved in the intimal hyperplasia. We developed a method to further purify human MVEC from 40-90%. Here we tested the effects of enrichment upon thrombogenicity and seeding-related intimal hyperplasia. METHODS: liposuction fat was enzymatically digested and centrifuged. To enrich MVEC, contaminating macrophages and fibroblasts were removed with dynabeads coated with macrophage- and fibroblast-specific antibodies. Thrombogenicity was assessed by measuring tissue factor and thrombomodulin activity, presence of endothelial nitric oxide synthase and via perfusion of the cells with whole blood. To investigate seeding-related intimal hyperplasia, PTFE grafts were seeded with the cells and cultured for 3 weeks. RESULTS: tissue factor activity of purified cells was reduced compared to nonpurified cells. Purified cells showed thrombomodulin activity and eNOS expression. Fragment 1+2 and Fibrinopeptide A generation after perfusion of purified cells were significantly lower than after perfusion of nonpurified cells, and only nonpurified cells were covered with platelets and fibrin. Prostheses seeded with nonpurified cells showed an EC monolayer above a multilayer of myofibroblasts, prostheses seeded with purified cells only showed a single EC monolayer. Mixing experiments with human umbilical cord EC (HUVEC) and fibroblasts showed that when more than 25% HUVEC were present a confluent EC layer was formed. When the amount of fibroblasts was 25% or less, no development of a subendothelial multilayer of myofibroblasts was found within 3 weeks. CONCLUSION: reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and might prevent seeding-related intimal hyperplasia.

Contaminants from the transplant contribute to intimal hyperplasia associated with microvascular endothelial cell seeding.

OBJECTIVES: seeding prosthetic grafts with fat-derived microvascular endothelial cells (MVEC) results not only in a non-thrombogenic EC layer, but also in intimal hyperplasia. Here we investigated incidence, composition, progression, and cause of this intimal hyperplasia. DESIGN: EPTFE grafts with MVEC were implanted as carotid interpositions in six dogs with 1 month, and in three dogs with 4, 8 and 12 months follow-up. Grafts seeded without cells, implanted in the contralateral carotid, served as a control. In another three dogs labelled cells were seeded to investigate the contribution of the seeded cells (2-3 weeks). MATERIALS AND METHODS: MVEC were isolated from the falciform ligament. Cells were pressure seeded on ePTFE grafts. Labelling was performed using retroviral gene transduction. The grafts were analysed with immunohistochemical techniques. RESULTS: after 1 month, all patent non-seeded grafts (5/6) showed fibrin and platelet deposition, and all patent seeded grafts (5/6) were covered with a confluent endothelial monolayer on top of a multilayer of myofibroblasts, elastin and collagen. After long term follow-up, all non-seeded grafts were occluded, all patent seeded grafts (4 and 12 months) were covered with an EC-layer with intimal hyperplasia underneath. The thickness of the intima did not progress after 1 month. Transduced cells were found in the endothelial monolayer, hyperplastic intima and luminal part of the prosthesis. CONCLUSIONS: MVEC seeding in dogs results in intimal hyperplasia in all patent grafts, which contains myofibroblasts. Contaminants from the transplant contribute to this intimal hyperplasia.