Table Of ContentNOVEL 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
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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
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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
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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).
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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.
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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
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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.
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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
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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
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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
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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
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