Rashid, ST; (2007) Development of a hybrid vascular bypass graft using a tissue engineering approach. Doctoral thesis , UCL (University College London).

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Abstract

Introduction: A third of patients needing arterial bypass grafts lack sufficient autologous vessels. Prosthetic alternatives - principally PTFE and Dacron - have poor patency rates because of compliance mismatch with elastic arteries and inherent surface thrombogenicity. The aim of this research was to develop - for the first time - a hybrid tissue-engineered bypass graft consisting of an elastic scaffold of compliant poly(carbonate-urea)urethane (CPU), incorporated with human smooth muscle cells (SMC) and endothelial cells(EC).;Methods: 1) Methods of human vascular SMC and EC extraction were assessed for both saphenous vein and umbilical cord vessels. 2) Extracted cells were assessed by immunostaining and for SMC the ability to contract collagen gels. 3) Coating CPU with various biomolecules (to make the surface bioactive), cell seeding density and attachment period were assessed for their impact on SMC attachment. 4) Cell growth on CPU was investigated by retroviral transduction of the GFP (Green Fluorescent Protein) gene and assays of cell viability and nucleic acid content. 5) A bioreactor and pulsatile flow circuit was developed for long-term culture of cells on CPU. 6) The impact of shear stress pre-conditioning on cell retention on the hybrid bypass graft was investigated under arterial flow conditions.;Results: 1) SMCs were reliably extracted from umbilical cord and saphenous vein. ECs were only reliably extracted from umbilical cord. 2) Cord SMCs grew faster than saphenous vein SMCs (doubling time of 3.4+0.6 days against 5.6+1.9 days p = 0.0227): all SMCs stained for alpha-actin and contracted collagen gels 3) SMC attachment to CPU was significantly enhanced by Fibronectin-like Engineered Polymer Protein Plus FEPP+ (from 20.7+4.6% to 31.5 5.9%: pO.Ol), higher cell density but not longer attachment period. 4) Transducing SMCs with GFP successfully allowed live cell imaging on CPU and assays of both viable cells and nucleic acid confirmed cell growth on CPU. 5) The flow circuit successfully allowed long-term sterile culture of cells on CPU. 6) Retention of SMCs and ECs on CPU was improved by a period of shear stress pre-conditioning: from 56.7+7.0% to 76.2+6.5% SMC retention and from 45.6+2.3% to 67.4+4.0% EC retention (p<0.03).;Conclusion: Pre-lining CPU with FEPP+ enhances SMC attachment. GFP-transduction allows study of SMC growth on CPU. Pre-conditioning enhances retention of SMCs and ECs onto CPU, probably because the mechanical stimuli orientate the cells and increase the release of matrix proteins and attachment factors. The stage is now set for developing a hybrid graft for in vivo studies.

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