REDUCTION OF PROSTHETIC VASCULAR GRAFT THROMBOGENICITY BY PHOSPHORYLCHOLINE CONTAINING LIPIDS AND POLYMERS. The thrombogenicity of arterial grafts can be modified by coating with new polymers that mimic haemocompatible blood cell membranes. Richard le Roy Bird. Master of Surgery Thesis for the University of London. Approved by the University of London for the Degree of Master of Surgery ProQuest Number: U064002 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U064002 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT 2 This thesis reviews the clinical problems associated with atheromatous arterial disease, its treatment with prosthetic bypass biomaterials, and considers the development and assessment of these materials. The thesis tests the hypothesis that coating existing biomaterials with biological membrane phospholipids, which mimic the good haemocompatibility of erythrocyte membranes, may be a step towards the solution of the problem of biomaterial thrombogenicity. Twelve phospholipids from the RBC membrane are evaluated as haemocompatible surface coatings by two in vitro assays; 1) material thrombelastography (MTEG), and 2) fibrinopeptide A generation. The technique of MTEG is presented in detail and important developments of this methodology are described. The underlying mechanisms of haemocompatibility of these phospholipids are examined by incubation of lipid liposomes with human plasma using individual clotting factor assays. Phospholipids and polymers containing phosphorylcholine (PC), namely sphingomyelin and dipalmitoylphosphatidylcholine (DPPC), very significantly reduce the activation of platelets and clotting factors. They are considerably better than the biomaterials Dacron and PTFE. The stability of DPPC, sphingomyelin and the PC-polymers during sterilisation with steam, ethylene oxide and irradiation are studied. Marked decomposition is documented using the techniques of chromatography, FTIR and MTEG. Despite radiation induced changes, sphingomyelin and some PC-polymers remain less thrombogenic than PTFE or Dacron to both platelets and clotting factors. Untreated Dacron vascular grafts are compared with grafts coated with sphingomyelin and two PC-polymers in a double blind randomised in vivo trial, using a modified sheep arteriovenous fistula preparation. The end points are indium-111 platelet imaging and uptake, platelet survival times, iodine-125 fibrinogen uptake and graft histology. Sphingomyelin significantly reduces platelet uptake by 66% and the cross sectional area of graft thrombus by 90% compared to the control grafts. All three materials prolong platelet survival, but do not reduce fibrinogen activation significantly. The original hypothesis is supported and further investigation of these materials should be undertaken. 3 Table of Contents A bstract.................................................................................................................................2 Table of Contents ..............................................................................................................3 List of Tables .......................................................................................................................8 List of Figures ................................................................................................................ 10 List of Photographs ...................................................................................................... 12 D edication ....................................................................................................................... 13 Statement of Originality ............................................................................................... 14 Acknowledgements ......................................................................................................... 15 Preface ......................................................................................................................... 17 Chapter 1 Background to the current investigation....................................................... 19 1.1 The clinical problem .................................................................................................. 20 1.1.1 Vascular disease ................................................................................................20 1.1.2 Current vascular prostheses .............................................................................22 1.1.2.1 D acron......................................................................................................... 23 1.1.2.2 P T F E ............................................................................................................23 1.1.2.3 Autologous grafts.........................................................................................24 1.1.3 Immunological and systemic effects of prosthetic materials ........................ 25 1.2 Biomaterials ..............................................................................................................29 1.2.1 H istoric ................................................................................................................ 29 1.2.2 Properties of an ideal biomaterial .................................................................31 1.2.3 Changing concepts in biomaterials .................................................................33 1.2.4 Problems of biomaterial evaluation ...............................................................34 13 Current approaches to graft thrombogenicity ..................................................... 35 1.3.1 Attempts to modify materials .......................................................................... 35 1.3.2 New materials ................................................................................................. 36 1.3.3 Endothelial cell seeding ................................................................................. 38 Chapter 2 The hypothesis under te st ...................................................................................... 41 2.1 Background to current approach of biomembrane m im icry ............................. 42 2.1.1 The concept .......................................................................................................42 2.1.2 The supporting evidence .............................................................................. 46 2.2 The hypothesis ......................................................................................................... 48 Chapter 3 In vitro assessment of materials ........................................................................ 49 3.1 Introduction ............................................................................................................. 50 3.2 Material Thrombelastography............................................................................... 52 3.2.1 Historical aspects of thrombelastography ....................................................... 52 3.2.2 Historical aspects of Material thrombelastography .......................................56 3.2.3 The current MTEG investigation ......................................................................58 3.2.3.1 TEG Technique .........................................................................................58 3.2.3.2 TEG A nalysis............................................................................................. 58 3.2.3.3 Materials and methods of preparation.....................................................59 Contents 4 3.2.4 Results ................................................................................................................ 63 3.2.4.1 Group 1 : the standards and controls.......................................................63 3.2.4.2 Group 2 : the phospholipids......................................................................65 3.2.4.3 Group 3 : the materials .........................................................................69 3.2.5 Discussion of MTEG results .............................................................................72 33 Parallel plate test chamber ...................................................................................... 79 3.3.1 Introduction ......................................................................................................... 79 3.3.2 Design and m ethods ...........................................................................................80 3.3.3 Analysis ................................................................................................................ 83 3.3.4 R esu lts...................................................................................................................83 3.3.5 Discussion ............................................................................................................86 3.4 The liposome uptake of clotting factors .................................................................88 3.4.1 Introduction ........................................................................................................