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Sarah Anne Underwood

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Sarah Anne Underwood THE PRODUCTION OF HUMAN MATERIAL FOR SKIN REPLACEMENT A thesis submitted to the University of London as part of the requirements for the degree of Doctor of Philosophy BY Sarah Anne Underwood 1999 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering UNIVERSITY COLLEGE LONDON Torrington Place LONDON WCIE 7JE ProQuest Number: 10015877 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 complete 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 10015877 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT This thesis describes the research to identify a process for the production of cell guidance scaffolds with potential use as human skin replacements. Fibronectin, a plasma protein with well-documented cell adhesion properties, can be extracted from the cryoprecipitate collected from human plasma donations. Purified solutions of fibronectin have been used to make mats with approximate dimensions 1 x 2 cm and consisting of orientated, protein fibrils with diameter 5-10 pm. These mats have demonstrated excellent in vitro and in vivo cell guidance properties. The aim of this research was to produce scaffolds with the same cell guidance / adhesion properties as the mats combined with a simple manufacturing method, little material wastage, reproducibility and potential for large scale production. Polyethylene glycol fractionation was used to produce fibronectin enriched precipitate from cryoprecipitate, with fibronectin and fibrinogen in a 65 : 35 percentage ratio. The precipitation of fibronectin and fibrinogen with a reduction in pH was examined and it was found that fibronectin / fibrinogen cables could be drawn up from a solution when protein was precipitated with 0.1 M citric acid, final pH 4.0-4.5. These cables, average diameter 200 pm, were found to consist of numerous micron-diameter fibrils giving the cable ultrastructural orientation. The cables were found to be hygroscopic and could be stabilised against solubilisation by severe dehydration. Cables which were twisted as they were drawn upwards from solution had an average ultimate tensile strength of 61 N/mm^, but were brittle. Although these cables could be made easily and were found to have good cell adhesion properties, drawing upwards from solution is not a technique applicable to large scale manufacture. Development of wet-spun fibres using traditional textile manufacturing techniques required that both the solution viscosity and protein concentration were increased. Urea was used as a solvent to increase protein concentration and sodium alginate was used to increase the viscosity of the solution and alter its flow behaviour. A variety of coagulation baths were tested in order to find the optimal pH and salt concentrations for fibre formation. Fibres spun on a pilot scale wet-spinning rig, diameter 500 pm, were found to have properties which varied with their composition and post spinning treatments. The fibres had a lower tensile strength, 28 N/mm^, than the drawn, twisted cables but had a higher elongation at break, 52.4 % compared to 3.5 %, making them easier to handle. Wet spinning has been shown to be a suitable and flexible process for the production of fibronectin / fibrinogen fibres. The production of fibres is considered with respect to process design, virus inactivation and GMP considerations. A test of the fibronectin material’s ability to support the attachment and alignment of human dermal fibroblasts is also described. ACKNOWLEDGEMENTS The interdisciplinary nature of tissue engineering is reflected by the broad spectrum of people I have had the privilege of working alongside during this project. The enthusiasm shown by all has been the key to this stimulating and enjoyable challenge. My thanks to: Prof. Peter Dunnill (UCLACBE), Dr. Robert Brown (UCL, Tissue Repair Unit) and Dr. Alex MacLeod (Protein Fractionation Centre, Edinburgh) for their supervision, encouragement and thought-provoking discussions. Dr. Andrew Afoke at the Division of Design and Mechanical Engineering, University of Westminster for his helpful advice and assistance with the tensile testing. Drs George East and Vic Rogers at the Department of Textile Industries, University of Leeds for their advice on wet spinning and an enjoyable and productive fortnight using their pilot scale equipment. The Development department at the Protein Fractionation Centre, Edinburgh for their assistance in preparing the fibronectin precipitate and several enjoyable weeks in Edinburgh Kirsty Smith at the Tissue Repair Unit for assistance with scanning electron microscopy and Stuart Pope at UCL for his photographic skills. I would also like to extend my appreciation to: My tea-drinking and cake-eating companions in Room 115 for 3 years of varied and amusing discussion. One day the self-filling cake-tin will become reality. The Tissue Repair Unit Team for adopting me into their department. Members of the University of London Orienteering Club for numerous opportunities to get away from it all, usually lost in a forest. Maggie, Ali, Lilia and Helen for being great frm. William for his patience, support and ‘outrageous’ humour and my family for their support, encouragement and excellent humour throughout. This work was supported by the BBSRC and a CASE studentship from the Protein Fractionation Centre, Scottish National Blood Transfusion Service, Edinburgh. TABLE OF CONTENTS 1.0 INTRODUCTION 18 Organisation of the thesis 18 1.1 Tissue Engineering 19 1.1.1 Skin replacement technologies 22 1.1.2 Future developments within tissue engineering 27 1.2 Plasma fractionation 30 1.2.1 Plasma fractionation with polyethylene glycol 31 1.3 Fibronectin 33 1.3.1 History and Isolation 3 3 1.3.2 Primary structure and conformation 3 4 1.3.3 Effect of environmental change on the fibronectin molecule 36 1.3.4 Fibronectin in plasma 40 1.3.5 Biological properties of fibronectin 41 1.4 Fibrinogen 43 1.4.1 Structure 43 1.4.2 Fibrinogen’s role in blood clotting 44 1.4.3 Other interactions between fibronectin and fibrinogen 46 1.5 Wound healing and contact guidance 47 1.5.1 Wound healing and the role played by fibronectin 47 and fibrinogen 1.5.2 The fibronectin receptor 48 1.5.3 Cellular interactions with fibronectin and / or fibrinogen 49 substrates 1.5.4 Cell movement and contact guidance 51 1.6 Recombinant cell adhesion substrates 54 1.7 Protein precipitation 55 1.7.1 Precipitation by salt addition 55 1.7.2 Isoelectric protein precipitation 56 1.7.3 Precipitation using organic solvents 57 1.7.4 Precipitation using non-ionic polymers 5 7 1.7.5 Other less frequently used forms of precipitation 57 1.8 Fibre wet spinning 58 1.8.1 Wet spinning 58 1.8.2 Fibre orientation 58 1.8.3 Protein wet spinning 59 1.9 Fibronectin mats as contact guidance materials 62 1.10 Project aims 65 2.0 MA TERIALS AND METHODS 66 2.1 Precipitate preparation 66 2.2 Methods of solution characterisation 66 2.2.1 Protein assay 67 2.2.2 Fibronectin ELISA 67 2.2.3 Fibrinogen assay 69 2.2.4 Polyethylene glycol assay 69 2.2.5 Albumin assay 69 2.2.6 Viscometry 69 2.2.7 Gel electrophoresis 71 2.2.8 Dialysis 72 2.3 Methods of material manufacture 72 2.3.1 Fibronectin mat manufacture 75 2.3.2 Cable manufacture 76 2.3.3 Small scale fibre spinning 76 2.3.4 Pilot scale fibre spinning 77 2.4 Mechanism of precipitation and material characterisation 78 2.4.1 Solubility curves 78 2.4.2 Tensile testing 79 2.4.3 Moisture absorption - dry weight determination 80 2.4.4 Stability 81 2.4.5 Yield and denier measurement 81 2.4.6 Birefringence 81 2.4.7 Scanning electron microscopy 81 2.4.8 Image analysis 82 2.5 Fibronectin cables on backing materials 82 2.5 .1 Human dermal fibroblast cell culture 82 2.5.2 Preparation of collagen sponges / fibronectin cables 82 6 3.0 THE PRODUCTION AND CHARACTERISATiON OF FIBRONECTIN FOR FIBRE PREPARATION 84 3.1 Introduction 84 3.2 Assay development 84 3.2.1 ELISA for fibronectin 85 3.2.2 Fibrinogen assay 91 3.2.3 Total protein assay 93 3.3 Solution production and characterisation 95 3.3.1 Mass balancing the production process 95 3.3.2 Solution characterisation 96 3.3.3 Fibronectin ; Fibrinogen batch variations 99 3.4 Affinity chromatography purification of fibronectin 103 3.5 Fibronectin mat making 106 3.6 Solution storage 108 3.7 Summary 109 4.0 DEVELOPMENT OF DRAWN CABLES AND THEIR PHYSICAL PROPERTIES 111 4.1 Introduction 111 4.2 Addition of acid / salt to protein solutions 111 4.3 Solubility curves 116 4.3.1 pH vs viscosity 123 4.3.2 Fibronectin / fibrinogen composition of precipitates 125 4.4 Optimisation of precipitation 126 4.4.1 Acid and protein concentration 126 4.5 Physical properties 131 4.5.1 Yield measurements and dimensions 131 4.5.2 Tensile strength measurement 133 4.5.3 Solubility 137 4.5.4 Moisture absorption 139 4.5.5 Birefringence 142 4.6 Summary 142 5.0 DEVELOPMENT OF A WET SP!NN!NG TECHNIQUE FOR FIBRONECTIN FIBRES 144 5.1 Introduction 144 5.2 Concentration / viscosity for fibre production 144 5.2.1 Concentration 145 5.2.2 Viscosity of concentrated solutions 148 5.2.3 Addition of viscosity enhancers 150 5.3 Coagulation
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