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Novel therapeutics in the prevention of flexor tendon adhesion formation PDF

231 Pages·2011·2.14 MB·English
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NOVEL THERAPEUTICS IN THE PREVENTION OF FLEXOR TENDON ADHESION FORMATION Benjamin Robert Klass A thesis submitted to University College London for the degree of Doctor of Medicine (Research) MD (Res) The RAFT Institute of Plastic Surgery, Mount Vernon Hospital, Middlesex The Royal Free Hospital, Hampstead, London 2011 1 Declaration I, Benjamin Robert Klass, confirm that the research forming the basis of this thesis is original and the ideas were developed in conjunction with my supervisors. I performed the majority of experiments myself with guidance and technical assistance from the scientific and laboratory staff in each of the institutes where the work was undertaken. Histological processing was performed by the RAFT histopathologist, Liz Clayton, and adhesion analysis was performed in a blinded fashion by a mixed group of scientists and surgeons from the RAFT institute. The institutes include The RAFT Institute, Mount Vernon Hospital and the Biological Services Department, The Royal Free Hospital. Where I have drawn on the work, ideas and results of others this has been appropriately acknowledged in the thesis. This work has been partly funded by Britannia Pharmaceuticals Ltd. (suppliers of Pumactant) however the research undertaken has been performed independently without any personal financial interest. Ben Klass 2 Abstract Tendon injuries of the hand are common with nearly one-third of a million digital flexor tendon injuries per year in the United States. Injuries in zone II of the flexor tendon are notoriously difficult to repair and the main complications are either tendon rupture or adhesion formation. Adhesions remain a problem despite many attempts at prevention using various chemicals and physical barrier techniques. The overall aim of this thesis was to further understand the biology of tendon adhesion formation and to develop novel treatments targeting this process. Uninjured flexor tendons were obtained from New Zealand White rabbits. Tenocytes derived from different parts of the flexor tendon-sheath complex were grown using standard tissue culture techniques. Each of the three different cell types (endotenon, epitenon and tendon sheath) was subjected to various assays (proliferation/toxicology, cell adhesion, and mRNA expression,) using TGF-β1 and our proposed treatments; epigallocatechin-3-gallate (EGCG), Resveratrol and Pumactant. A further study then compared the three treatments in vivo. New Zealand White rabbits (n=8 per group; 32 in total) were anaesthetised and the flexor digitorum profundus (FDP) of digits 2 and 4 of the forepaw was subjected to a partial tenotomy. The three treatments (compared with control groups) were infiltrated into the flexor sheath of immobilised tendons and the wound was then sutured closed. After two weeks the tendons were harvested and randomised to either mechanical or histological assessment of adhesion formation. The major findings from the in vitro study were as follows: TGF-β1 showed a statistically significant increase in collagen type I gene expression in epitenon cells at 24 and 48 hours and an increase in collagen type III in sheath cells between 6 and 24 hours. There was a statistically significant down-regulation of collagen type III in endotenon and epitenon cells at various time points. Resveratrol showed a statistically significant increase in collagen type I gene expression in epitenon cells with a corresponding down-regulation of fibronectin and PAI-1 in both epitenon and sheath cells. Resveratrol also up-regulated collagen type III at late time points in tendon 3 sheath cells. Pumactant also showed some therapeutic advantages at the gene expression level with a statistically significant increase in collagen type III in endotenon cells at late time points, corresponding with an overall down-regulation of PAI expression in the same cell type and sheath cells. The results from the in vivo study were that all three treatments showed a statistically significant reduction of tendon adhesion formation when compared to operated controls in both mechanical and histological assessments (p<0.05). However, Pumactant was the only treatment to demonstrate a statistically significant reduction in adhesion formation when compared to the H 0-group using both methods (p<0.05). 2 All three treatments displayed potential therapeutic advantages. However, Pumactant showed the most promising in vivo results and it would be worthwhile investigating this further with an in vivo model of tendon healing and if successful a pilot clinical trial. Hopefully this could act as a suitable adjunct to tendon repair in the future and improve the lives of patients with disabling tendon injuries. 4 Acknowledgments There are numerous people that I would like to thank without whom none of this research would have been possible. Firstly, I would like to thank my supervisors; Mr Addie Grobbelaar for his continued support, advice, direction, precious time and enthusiasm for this project and Dr Kerstin Rolfe for the laboratory skills training, technical support, editing and proof reading, understanding and constant friendly encouragement throughout the last few years. I was extremely grateful to have regular structured meetings with both of my supervisors and their constant guidance helped me to develop my scientific thoughts and plan experiments appropriately. At The Royal Free Hospital, thank you to Duncan Moore and Mark Neal (Biological Services Department) for their help in design, setting up, application for my project licence, and practical teaching for the in vivo experiments, and Professor Seifalian (Department of Surgery) for being the project licence holder for this work. Thank you to all the staff at RAFT including the CEO, Leonor Stjepic, the scientific group leaders; Julian Dye, Rachel Haywood and Claire Linge, the administration staff; Christine Miles, Amanda Bailey, Laura Ripley and Mary Pearmain. Thank you to Hilary and Stephanie who kindly helped me during the first few weeks of settling in at RAFT. Thanks to Liz Clayton who expertly processed and sectioned the tendons for histological analysis. Also thank you to all my Research Fellow colleagues (RAFTers!) with whom I shared many conversations and friendly banter, Oly Branford, Nick Sheppard, Dan Marsh, Sophie Dann, Richard Baker, Fulvio Urso- Baiarda, Fabrice Rogge, Mo Akhavani, Chris Baldwin, and Bill Townley. I would like to thank the staff at Britannia, Dr Nick Topley, Mr Derek Woodcock and Ms Tedd Fuell, who helped with the development and supply of Pumactant for this thesis. Our meetings were very amicable with sharing of important information and there was often useful debate regarding experimental design and strategies. I must also acknowledge that none of this work would have been possible without the financial support from The Restoration of Appearance and Function Trust, The Royal 5 College of Surgeons in partnership with the British Society for Surgery of the Hand, The Rosetrees Charity and Britannia Pharmaceuticals and to them I am truly grateful. Finally, a huge thank you to my family who have supported me through everything I have ever done and achieved. In particular my wife, Claire who has shown amazing encouragement and understanding and my children Archie and Jemima who I have watched grow and blossom since the dawn of this thesis. I dedicate this work to them. 6 Contents Page Number _____________________________________________________________________ Title Page 1 Declaration 2 Abstract 3 Acknowledgements 5 Contents 7 List of Figures 12 List of Tables 17 List of Abbreviations 19 _____________________________________________________________________ Chapter 1. GENERAL INTRODUCTION 22 _____________________________________________________________________ 1.1 Introduction 23 1.2 Histology and Structure of Tendons 24 1.3 Flexor Tendon Anatomy 25 1.3.1 Flexor Digitorum Superficialis 25 1.3.2 Flexor Digitorum Profundus 26 1.3.3 The Flexor Tendon Sheath 27 1.4 Tendon Nutrition 31 1.4.1 The Vinculae 32 1.4.2 Synovial Fluid 33 1.5 Tendon Healing 34 1.6 Animal models in the study of tendon healing and adhesion formation 36 1.7 Tendon Adhesion Formation 37 1.7.1 Tendon Adhesion Types 38 1.8 Methods used for the reduction of tendon adhesion formation 40 7 1.8.1 Pre-operative methods of adhesion reduction 40 1.8.2 Intra-operative methods of adhesion reduction 41 1.8.3 Post-operative methods of adhesion reduction 45 1.9 Rationale for proposed anti-adhesion treatments 47 1.9.1 Epigallocatechin-3-gallate (EGCG) 47 1.9.2 Resveratrol 48 1.9.3 Pumactant 50 1.10 Rationale for in vitro mRNA gene selection 52 1.10.1 Collagen type I and Collagen type III 52 1.10.2 Fibronectin 52 1.10.3 Plasminogen Activator Inhibitor 1 (PAI-1) and Tissue Plasminogen Activator (t-PA) 53 1.11 Rationale for Thesis 56 1.11.1 Thesis Hypothesis 56 1.11.2 Thesis Objectives 56 _____________________________________________________________________ Chapter 2. MATERIALS AND METHODS 58 _____________________________________________________________________ 2.1 Tissue Sampling and the Isolation of Cells 59 2.1.1 Tendon Sampling 59 2.2 Cell Culture Techniques 60 2.2.1 Cell Passage and Counting 60 2.2.2 Cryopreservation 60 2.3 In Vitro Flexor Tendon Cell Assays 61 2.3.1 Toxicology Assay 61 2.3.2 Cell Adhesion Assay 62 2.3.3 Quantitative Real-Time PCR 63 2.4 In Vivo Model of Flexor Tendon Adhesion Formation 70 2.4.1 Operative Procedure 70 2.4.2 Mechanical Assessment of Adhesion Strength 75 2.4.3 Histological Analysis 75 8 2.4.4 Data Analysis 80 _____________________________________________________________________ Chapter 3. THE IN VITRO FLEXOR TENDON CELL RESPONSE TO 81 TGF-β1 _____________________________________________________________________ 3.1 Introduction 82 3.1.1 Hypothesis 84 3.1.2 Objectives 84 3.2 Materials and Methods 85 3.2.1 Cell Culture 85 3.2.2 Proliferation (Toxicology) Assay 85 3.2.3 RNA Isolation and Real-Time Polymerase Chain Reaction (QRT-PCR) 85 3.2.4 Data Analysis 86 3.3 Results 86 3.3.1 Proliferation Assay 86 3.3.2 Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR) 90 3.4 Discussion 101 _____________________________________________________________________ Chapter 4. THE IN VITRO FLEXOR TENDON CELL RESPONSE TO 3 NOVEL ANTI-ADHESION TREATMENTS: EPIGALLOCATECHIN-3-GALLATE, RESVERATROL AND PUMACTANT 106 _____________________________________________________________________ 4.1 Introduction 107 4.1.1 Epigallocatechin-3-gallate (EGCG) 107 4.1.2 Resveratrol 107 4.1.3 Pumactant 108 4.1.4 Hypothesis 109 4.1.4 Objectives 109 9 4.2 Materials and Methods 110 4.2.1 Toxicology Assay 110 4.2.2 Cell Adhesion Assay 110 4.2.3 Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR) 110 4.3 Results 110 4.3.1 Toxicology Assay 110 4.3.2 Cell Adhesion Assay 118 4.3.3 Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR) 122 4.4 Discussion 147 4.4.1 EGCG 147 4.4.2 Resveratrol 147 4.4.3 Pumactant 151 4.5 Conclusions 152 _____________________________________________________________________ Chapter 5. THE IN VIVO FLEXOR TENDON CELL RESPONSE TO 3 NOVEL ANTI-ADHESION TREATMENTS: EPIGALLOCATECHIN-3-GALLATE, RESVERATROL AND PUMACTANT 154 _____________________________________________________________________ 5.1 Introduction 155 5.1.1 Hypothesis 155 5.1.2 Objectives 155 5.2 Materials and Methods 156 5.2.1 Mode of Anaesthesia 156 5.2.2 Preparation of Treatments 156 5.2.3 Operative Procedure 157 5.2.4 Mechanical Assessment of Adhesion Strength 157 5.2.5 Histological Analysis 158 5.2.6 Data Analysis 158 5.3 Results 159 5.3.1 Mechanical Assessment of Adhesion Strength 159 5.3.2 Histological Assessment 160 10

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