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The Effect of Cell-Based Therapy on LASIK-Like Corneal Wounds

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The Effect of Cell-Based Therapy on LASIK-Like Corneal Wounds The effect of cell-based therapy on LASIK-like corneal wounds Thesis Submitted to Cardiff University for the degree of Doctor of Philosophy December 2018 Camilla Phillips Structural Biophysics Research Group School of Optometry and Vision Sciences Cardiff University i ii Abstract The aim of this study was to determine if oral mucosal lamina propria progenitor cell (OMLP-PC)-based therapy to the stromal beds of LASIK-like wounded corneas could improve wound healing. After epithelial and/or stromal injury, a wound healing cascade is triggered that includes keratocyte apoptosis and proliferation in the underlying stroma. Activation of keratocytes leads to their transformation into myofibroblasts, which are responsible for matrix remodelling. Unfortunately, LASIK-like wound healing can result in incomplete closure at the flap interface, reduced biomechanical strength and decreased transparency. The hypothesis of this study was that a therapeutic application of OMLP-PCs, known to facilitate non-scarring healing, to the corneal wound bed, would reduce keratocyte activation and consequent myofibroblastic transformation to augment matrix remodelling and restore transparency. To test this hypothesis, a corneal organ culture model was developed to monitor LASIK-like wound healing outcomes. The efficacy of a cell-based therapy, namely OMLP-PCs, on LASIK-like wound healing was evaluated and optimised over four weeks. The wound healing outcomes of the organ cultured porcine corneas included a significant reduction of percentage α-sma immunopositive cells (p<0.01) from ~45% (untreated wounded) to ~25% (OMLP-PC-treated) during weeks 2-3 post wounding. Peak percentage proliferation was 52.2% ±3.7 at 1 week in OMLP-PC treated wounds, earlier compared to 42.2% ±3.3 at 2 weeks in untreated control corneas. OMLP-PCs did not alter stromal percentage apoptosis. Coherence (degree of collagen lamellae alignment) at 4 weeks after wound healing was significantly higher in OMLP-PC treated wounded corneas (p<0.05), compared to that in untreated corneas. By 4 weeks corneal transparency in OMLP-PC treated wounded corneas was significantly higher than in untreated corneas (p<0.01). To conclude, an OMLP-PC therapeutic application to LASIK-like wounds enhanced stromal cell proliferation and reduced myofibroblast expression, but increased collagen organisation with consequent improved epithelial-stromal flap wound closure and increased corneal transparency. iii Acknowledgements The path to completion of this PhD has been a long and difficult one, and so naturally there are many people to thank for their endless help and support. First and foremost, I would like to thank my Supervisors Professor. Keith Meek and Dr. Julie Albon. I am very grateful to Keith for his support, guidance and, above all patience (this PhD has been a frustratingly long time in the making!). A special thank you goes to Julie for all her help and understanding in both PhD and personal matters and the long chats (which very often veered off course!). My advisor Dr. Tony Redmond was also incredibly understanding and helpful during my long (and frequently interrupted) path to completion of this thesis. And of course, no PhD in this School would be possible without the fantastic Sue Hobbs- thank you so much! In pursuit of this PhD I have received help from many people. Firstly, a thank you to Nick White, who we sadly lost in April 2019, for all his help and patient training on various microscopes- I hope you knew how grateful I was. Thank you to Prof. Phil Stephens, as well as Katrina Duggan, Sarah Youde and Emma Board-Davies at Cardiff School of Dentistry provided the cells integral to this study and the invaluable expertise in cell culture. James Galloway, who worked over a summer helping with culture and imaging. A number of statisticians from Cardiff University were consulted in the post-grad statistics clinic over the years- thank you all for your patience! All members of the Structural Biophysics group in the School of Optometry and Vision Sciences are a fantastic set of people who are always ready to lend a hand in anything you need. Professor Andrew Quantock, Dr Justyn Regini, Dr Sally Hayes, Basha, Petar (thank you for your patience with all things computer!!) and Jacek. In particular Dr Phil Lewis and Dr Rob Young with their utmost patience and eagerness to help with TEM (I’m sorry my samples were so squishy and large!). In the Post Grad room many, many, many people have come and gone in my time there; Nada, Tom, Ben, Ellie, Greg, Alina, Elena, Frankie and Stacey (I am SO sorry if I’ve missed anyone out!), a special thanks to lovely Saleha, who shared the ups and downs of juggling parenthood and research (you felt my pain!). Most importantly Dr Erin Dooley and Dr Sian Morgan, whose work lead the way for me and my PhD. Their generous tutelage through so iv many different techniques was incredible! An extra thanks to Sian for her help and encouragement in the last few months in particular- Thank you. This PhD would certainly not have been possible without the support of my friends. The mums at school (Lydia especially) who have helped with childcare and been so supportive! The awesome Christine and Tash, who were there to support me from the beginning. The rather fabulous ladies of Ardwyn Walk; Jennie, Amy, Sian and Karina who have helped with childcare, venting via messaging and always with a glass of wine (or two?!). Also Lovely Lottie- we both embarked on PhD’s, got married, had children, moved houses all at the same time (slightly crazy!) and no one else can really understand just how hard this has been. Thank you for being there for me (albeit 240 miles north)- I wish you lived closer. The last round of “thank yous” goes to my family. The amount of support from you has been phenomenal. A huge thank you to my Parents- in- Law Rob and Sheila for looking after the children, doing school runs and picking up poorly little ones when I couldn’t. For my parents, Wiggy and Alan, who tirelessly and repeatedly made the 4hr journey westwards from Norfolk to help with everything- the children, the house, the shopping, the laundry and you always brought wine and h’uile picante, thank you isn’t really enough! You are such supportive parents and fantastic grandparents- I am so lucky. Thank you so much. And finally, to my little family. My gorgeous husband Tim- this has been such a long “journey” and the last few months have been especially hard. Thank you so much for being my rock and a brilliant father to the sprogs. The biggest thank you though, is reserved for Emilia and Toby, my two (rather beautiful) children who made their appearance during this PhD. You are quite simply the best. And, of course, George. I love you all so much and I am so sorry this has taken me away from you… I’m all yours now!!! v Dedication To Emilia and Toby. Love From Mummy x In Memory of Nick White. You will be greatly missed. vi Table of Contents Chapter 1: Introduction ..................................................................................... 1 1.1. Corneal Structure .................................................................................................. 2 1.1.1. Epithelium ........................................................................................................ 4 1.1.2. Bowman’s Layer .............................................................................................. 5 1.1.3. Stroma .............................................................................................................. 6 1.1.4. Pre Descemet’s Membrane/ Dua’s Layer......................................................... 6 1.1.5. Descemet’s Membrane ..................................................................................... 6 1.1.6. Endothelium ..................................................................................................... 7 1.1.7. Species Difference............................................................................................ 7 1.2. Corneal Ultrastructure .......................................................................................... 8 1.2.1. Collagen ................................................................................................................ 9 1.2.2. Keratocytes ......................................................................................................... 12 1.2.3. Proteoglycans ...................................................................................................... 12 1.3. Corneal Transparency ........................................................................................ 13 1.4. Refractive Surgery ............................................................................................... 15 1.4.1. Refractive Conditions ......................................................................................... 16 1.4.2. PRK ..................................................................................................................... 19 1.4.3. LASIK ................................................................................................................. 20 1.4.4. LASEK ................................................................................................................ 21 1.4.5. LASIK Post-operative Complications ................................................................ 22 1.5. Wound Healing .................................................................................................... 24 1.6. Corneal Wound Healing ....................................................................................
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