Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Poormasjedi-Meibod, Malihe-Sadat
Doctor of Philosophy - PhD
University of British Columbia
It is well established that more than 15 million patients develop pathological scarring each year following elective operations, surgical procedures, deep trauma, thermal and electrical injuries. These scars, that cause major functional, cosmetic, psychological, and social consequences for the patients, impose a significant financial burden on health care systems. The current treatment modalities for these pathological conditions vary from topical application and intralesional injection of anti-scarring agents to surgical revisions and radiotherapy. The limited efficacy of these therapeutics for prevention of scar formation raised a great need for innovation within the wound care industry. Recently Kynurenine (Kyn), a tryptophan metabolite, has been identified as a potent anti-fibrotic agent. Kyn prevents scar formation by enhancing the expression of ECM degrading enzymes, matrix metalloproteinases (MMPs), and suppressing the expression of collagen. Although daily topical application of Kyn-cream improved the wound healing outcome in animal models, this method of drug delivery is not clinically practical in situations where dressings need to be kept on for 3-5 days. In this dissertation, it is hypothesized that topical application of a slow and controlled releasing Kyn or its metabolites from nanofiber dressing at the wound site improves and/or prevents dermal fibrosis by modulating the expression of the key ECM components involved in dermal fibrotic conditions. To test this hypothesis, three specific objectives were employed: (1) Evaluating and comparing the anti-fibrotic effects of Kynurenic acid (KynA) and Kyn, (2) Developing, characterizing and optimizing the nanofibrous dressings as a slow releasing drug delivery system for Kyn and KynA, (3) Examining the functionality of the developed anti-fibrotic dressings in open wounds in animal models. The findings of these specific objectives of this work demonstrated that topical application of the developed polymeric dressings, which slowly release Kyn/KynA over the course of 4 days, effectively reduces dermal fibrosis by modulating the key ECM components such as MMPs, collagen and fibronectin. The findings of this study support our hypothesis that development of an anti-fibrogenic dressing is feasible and as such its application would overcome the difficulties associated with development of hypertrophic scarring frequently seen in millions of patients worldwide.
DEVELOPMENT AND APPLICATION OF ANTI-FIBROGENIC DRESSINGS by Malihe-Sadat Poormasjedi-Meibod B.Sc., University of Tehran, 2007 M.Sc., University of Tehran, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (EXPERIMENTAL MEDICINE) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2015 © Malihe-Sadat Poormasjedi-Meibod, 2015 ii Abstract It is well established that more than 15 million patients develop pathological scarring each year following elective operations, surgical procedures, deep trauma, thermal and electrical injuries. These scars, that cause major functional, cosmetic, psychological, and social consequences for the patients, impose a significant financial burden on health care systems. The current treatment modalities for these pathological conditions vary from topical application and intralesional injection of anti-scarring agents to surgical revisions and radiotherapy. The limited efficacy of these therapeutics for prevention of scar formation raised a great need for innovation within the wound care industry. Recently Kynurenine (Kyn), a tryptophan metabolite, has been identified as a potent anti-fibrotic agent. Kyn prevents scar formation by enhancing the expression of ECM degrading enzymes, matrix metalloproteinases (MMPs), and suppressing the expression of collagen. Although daily topical application of Kyn-cream improved the wound healing outcome in animal models, this method of drug delivery is not clinically practical in situations where dressings need to be kept on for 3-5 days. In this dissertation, it is hypothesized that topical application of a slow and controlled releasing Kyn or its metabolites from nanofiber dressing at the wound site improves and/or prevents dermal fibrosis by modulating the expression of the key ECM components involved in dermal fibrotic conditions. To test this hypothesis, three specific objectives were employed: (1) Evaluating and comparing the anti-fibrotic effects of Kynurenic acid (KynA) and Kyn, iii (2) Developing, characterizing and optimizing the nanofibrous dressings as a slow releasing drug delivery system for Kyn and KynA, (3) Examining the functionality of the developed anti-fibrotic dressings in open wounds in animal models. The findings of these specific objectives of this work demonstrated that topical application of the developed polymeric dressings, which slowly release Kyn/KynA over the course of 4 days, effectively reduces dermal fibrosis by modulating the key ECM components such as MMPs, collagen and fibronectin. The findings of this study support our hypothesis that development of an anti-fibrogenic dressing is feasible and as such its application would overcome the difficulties associated with development of hypertrophic scarring frequently seen in millions of patients worldwide. iv Preface The work presented in this thesis has already been published in peer-reviewed journals, or submitted and under review by peer-reviewed journals. As the first author of all of the publications shown below I was responsible for designing and performing the experiments, data analysis and manuscript preparation. Distribution of the work is as follows: Chapter 2: Complete and unmodified original publication. Malihe-Sadat Poormasjedi-Meibod, Ryan Hartwell, Ruhangiz Taghi Kilani, Aziz Ghahary. Anti-scarring properties of different tryptophan derivatives. PLoS One, 2014. 9(3): p.e91955. In this work Malihe-Sadat Poormasjedi-Meibod designed and performed all the experiments, alone or in collaboration with the co-authors. In addition she was responsible for writing the manuscript and performing the subsequent revisions. Ryan Hartwell assisted with animal surgery and immunostaining. Ruhangiz Taghi Kilani performed the lasting effect study. Aziz Ghahary supervised the project and assisted with manuscript review and editing. Chapter 3: Malihe-Sadat Poormasjedi-Meibod, Victor Leung, Sanam Salimi Elizei, Raza B. Jalili, Frank Ko, Aziz Ghahary. Kynurenine Modulates MMP-1 and Type-I Collagen Expression via Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts [Submitted]. Malihe-Sadat Poormasjedi-Meibod was the project lead and performed all the experiments, alone or in collaboration with the co-authors, in addition to writing the first draft of the manuscript and performing the subsequent revisions. Victor Leung executed the electrospinning process, scanning electron microscopy in addition to manuscript editing. Sanam v Salimi Elizei assisted with animal surgery and immunocytochemistry. Raza B. Jalili assisted with animal surgery and manuscript review. Frank Ko oversaw the engineering aspects of the manuscript. Aziz Ghahary supervised the project and assisted with manuscript review and editing. Chapter 4: Malihe-Sadat Poormasjedi-Meibod, Saman Pakyari, John K. Jackson, Sanam Salimi Elizei, Aziz Ghahary. Development and Application of Anti-scarring Nano-fibrous Wound Dressings to Prevent the Emergence of Skin Fibrosis [Submitted]. Malihe-Sadat Poormasjedi-Meibod was the project lead and performed all the experiments, alone or in collaboration with the co-authors, in addition to writing the first draft of the manuscript and performing the subsequent revisions. Saman Pakyari assisted with animal surgery, tissue sectioning and immunostainings. John K. Jackson assisted with experimental design, performing the drug release assays and manuscript review. Sanam Salimi Elizei assisted with animal surgery. Aziz Ghahary supervised the project and assisted with manuscript review and editing. Dr. Aziz Ghahary was the Principle Investigator for the research project presented here. The financial support for this thesis was provided by NSERC and CIHR grants held by Dr. Ghahary, in addition to support from the BC Professional Fire Fighters Burn Fund. All methods and procedures, as well as the use of animals and tissue specimens obtained from animals and humans, are approved by both Human and Animal Ethics Committee of the University of British Columbia (Protocol numbers; H05-0103, A10-0147 and A10-1372). vi Table of Contents Abstract ........................................................................................................................... ii Preface ............................................................................................................................ iv Table of Contents ........................................................................................................... vi List of Tables .................................................................................................................. ix List of Figures ................................................................................................................. x List of acronyms and abbreviations ........................................................................... xii Acknowledgements ..................................................................................................... xiv 1 Introduction ............................................................................................................... 1 1.1 Wound healing biology ..................................................................................... 1 1.2 Cytokines and growth factors in wound healing ............................................ 3 1.3 Wound healing pathology ................................................................................. 5 1.4 Current treatment modalities for hypertrophic scars ..................................... 8 1.5 Kyn pathway ..................................................................................................... 10 1.5.1 Role of Kyn pathway in scarring .................................................................. 10 1.5.2 Role of Kyn pathway in central nervous system (CNS) ............................... 11 1.5.3 Role of Kyn pathway in immune system ...................................................... 14 1.6 Aryl hydrocarbon receptor (AHR) .................................................................. 15 1.7 Advancements in the wound management products ................................... 18 1.7.1 Electrospinning process ............................................................................... 19 1.7.2 Poly(vinyl alcohol) ........................................................................................ 21 1.7.3 Poly(lactic-co-glycolic acid) .......................................................................... 22 1.7.4 Poly(methyl methacrylate) ........................................................................... 23 1.8 Hypothesis and objectives .............................................................................. 24 2 Anti-scarring properties of different tryptophan derivatives .............................. 26 2.1 Introduction ...................................................................................................... 26 2.2 Materials and methods .................................................................................... 29 2.2.1 Ethics statement .......................................................................................... 29 2.2.2 Cell culture ................................................................................................... 29 2.2.3 RNA extraction and quantitative real time PCR (Q-PCR) ............................ 30 2.2.4 Preparation of cell lysates and Western blotting .......................................... 31 2.2.5 MMP activity assay ...................................................................................... 32 2.2.6 Collagen accumulation assay by Sirius Red staining .................................. 32 2.2.7 Lasting effect study ...................................................................................... 32 2.2.8 Cell proliferation assay ................................................................................ 33 2.2.9 Live/dead® viability/cytotoxicity assay ......................................................... 33 2.2.10 In vitro wound healing scratch assay ........................................................... 33 2.2.11 Hypertrophic scar animal model and treatments ......................................... 34 vii 2.2.12 Tissue processing and determination of scar elevation index (SEI) and epidermal thickness index (ETI) ................................................................ 34 2.2.13 Collagen staining and tissue cellularity ........................................................ 35 2.2.14 MMP-1, type-I collagen, and fibronectin expression in wounds .................. 36 2.2.15 Statistical analysis ....................................................................................... 36 2.3 Results .............................................................................................................. 36 2.4 Discussion ........................................................................................................ 52 3 Kynurenine modulates MMP-1 and type-I collagen expression via aryl hydrocarbon receptor activation in dermal fibroblasts ............................................ 61 3.1 Introduction ...................................................................................................... 61 3.2 Materials and methods .................................................................................... 63 3.2.1 Ethics statement .......................................................................................... 63 3.2.2 Cell culture ................................................................................................... 63 3.2.3 AHR immunocytochemistry and blockade of AHR activity .......................... 63 3.2.4 Preparation of Kyn-containing electro-spun PVA fiber mats ........................ 64 3.2.5 In vitro drug release ..................................................................................... 65 3.2.6 In vitro cytocompatibility assay .................................................................... 65 3.2.7 Effects of Kyn on expression of MMP-1 and type-I collagen in fibroblasts .. 66 3.2.8 Wound creation and treatment scheme ....................................................... 67 3.2.9 Histological analyses and immuno-staining ................................................. 67 3.2.10 MMP-1, type-I collagen, and α-SMA expression in the wounds .................. 68 3.2.11 Statistics ...................................................................................................... 68 3.3 Results .............................................................................................................. 69 3.3.1 Kyn modulates the expression of MMP-1 and type-I collagen via AHR activation in dermal fibroblasts .................................................................. 69 3.3.2 Characterization of nanofibers and in vitro drug release ............................. 72 3.3.3 The Kyn-loaded mats are cytocompatible ................................................... 74 3.3.4 Nanofiber-released Kyn modulates the expression of different ECM components in vitro ................................................................................... 74 3.3.5 Kyn-loaded dressings improved wound healing outcome ........................... 75 3.3.6 Kyn suppresses α-SMA expression and myofibroblasts ............................. 79 3.4 Discussion ........................................................................................................ 81 4 Development and application of anti-scarring nano-fibrous wound dressings to prevent the emergence of skin fibrosis ...................................................................... 88 4.1 Introduction ...................................................................................................... 88 4.2 Method and materials ...................................................................................... 91 4.2.1 Materials ...................................................................................................... 91 4.2.2 Electrospinning ............................................................................................ 91 4.2.3 Characterization of electrospun fibers ......................................................... 92 4.2.4 Water contact angle measurements ............................................................ 92 4.2.5 Determination of the water absorbency of the electrospun mats ................ 92 4.2.6 KynA release assay ..................................................................................... 92 4.2.7 Ethics statement .......................................................................................... 93 viii 4.2.8 Cell culture ................................................................................................... 93 4.2.9 In vitro cytocompatibility assay .................................................................... 94 4.2.10 Cell proliferation assay ................................................................................ 94 4.2.11 In vitro anti-scarring activity determination .................................................. 95 4.2.12 Wound creation and treatment .................................................................... 96 4.2.13 MMP-1, fibronectin, and type-I collagen expression in the wounds ............ 96 4.2.14 Statistics ...................................................................................................... 97 4.3 Results and discussion ................................................................................... 97 4.3.1 Fiber formation and characterization ........................................................... 97 4.3.2 Contact angle and water uptake measurements ....................................... 101 4.3.3 Release characteristics of KynA ................................................................ 102 4.3.4 Cytocomptatibility ....................................................................................... 106 4.3.5 Nanofiber releasabled KynA modulates the fibroblast proliferation and the expression of type-I collagen, fibronectin, and MMP-1 in vitro ................ 108 4.3.6 Nanofiber releasabled KynA modulates the expression of type-I collagen, fibronectin, and MMP-1 in vivo ................................................................ 111 4.4 Summary and conclusion ............................................................................. 115 5 Conclusions and suggestions for future work .................................................. 116 5.1 Suggestions for future work ......................................................................... 125 References ................................................................................................................... 128 ix List of Tables Table 1.1. The main growth factors and cytokines participating in the wound healing process, their cellular source and role in the wound healing. ..................................... 4 Table 1.2. Treatment modalities for HSC and their mechanism of action ......................... 9 Table 1.3. Physical and mechanical characteristics of poly(lactic-co-glycolic acid) ........ 23 Table 1.4. Physical and mechanical characteristics of poly(methyl methacrylate) ......... 24 Table 3.1: List of primers used in this study. ................................................................... 68 Table 4.1. The electrospinning conditions used in this study. ......................................... 91 Table 4.2. List of primers used in this study. ................................................................... 97 Table 4.3. Average diameter of electrospun PMMA fibers with increasing concentrations of PEG. ..................................................................................................................... 99 x List of Figures Figure 1.1 Phases of repair in acute wound healing ......................................................... 3 Figure 1.2 Hypertrpphic scarring ....................................................................................... 7 Figure 1.3 Keloids ............................................................................................................. 8 Figure 1.4 Kyn pathway .................................................................................................. 12 Figure 1.5 The molecular mechanism of actovation of gene expression by the AHR .... 18 Figure 1.6 Schematic of the electrospinning process ..................................................... 21 Figure 1.7 Schematic of poly(vinal alcohol) .................................................................... 21 Figure 1.8 Schematic of synthesis of poly(lactic-co-glycolic acid) .................................. 23 Figure 1.9 Schematic of synthesis of poly(methyl methacrylate) .................................... 24 Figure 1.10 Cartoon representation of the electrospun medicated wound dressing containing anti-fibrotic agent(s) ................................................................................. 26 Figure 2.1 Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines ............................................................................................................... 40 Figure 2.2 Reduction of soluble collagen level in KynA and Kyn treated fibroblast-conditioned medium .................................................................................................. 41 Figure 2.3 Stimulatory effect of kynurenines on MMP-1 expression ............................... 42 Figure 2.4 Stimulatory effect of kynurenines on MMP-3 secretion by fibroblasts ........... 43 Figure 2.5 Stimulatory effect of kynurenines on MMP activity and kynureninesâ lasting effect on MMP-1 expression ..................................................................................... 45 Figure 2.6 Effect of kynurenines on fibroblast and keratinocyte proliferation rate and viability ...................................................................................................................... 48 Figure 2.7 Effect of kynurenines on fibroblast and keratinocyte migration ..................... 49 Figure 2.8 Clinical appearance and histological evaluation of wounds in rabbit ear model ........................................................................................................................ 51 Figure 2.9 Effect of Kyn and KynA topical application on collagen deposition, tissue cellularity, and ECM expression ................................................................................ 53 Figure 3.1 Kyn activates AHR ......................................................................................... 71 Figure 3.2 TMF antagonizes Kyn-dependent AHR-mediated MMP-1 up-regulation and type-I collagen down-regulation ................................................................................ 73 Figure 3.3 Scanning electron microscope photographs of electrospun nanofibers and Kyn release profiles before and after post-spinning modifications ............................ 76 xi Figure 3.4 Effect of nanofiber released Kyn on fibroblast and keratinocyte proliferation and viability ............................................................................................................... 77 Figure 3.5 Nanofiber released Kyn modulates the expression of different ECM components .............................................................................................................. 78 Figure 3.6 Effect of Kyn-incorporated dressings on tissue cellularity and CD3+ inflammatory cells ..................................................................................................... 80 Figure 3.7 Effect of Kyn-PVA/PLGA dressing on the α-SMA+ myofibroblasts and different ECM components expression at day 15 post-wounding ............................. 82 Figure 4.1 Scanning electron microscopy images of electrospun PMMA fibers as a finction of the increasing concentrations of PEG .................................................... 100 Figure 4.2 Contact angle and water uptake measurements of the nano-fibrous mats . 103 Figure 4.3 Cumulative release profiles of KynA from electrospun PMMA fibers containing increasing concentrations of PEG ........................................................................... 105 Figure 4.4 Cumulative release profiles of KynA from electrospun PMMA-PEG fibers .. 107 Figure 4.5 Live/dead® viablity/cytotoxicity assay ......................................................... 109 Figure 4.6 Evaluation of KynA-incorporated electrospun dressing with anti-scarring activity in vitro ......................................................................................................... 113 Figure 4.7 Effect of Kyn/PMMA-PEG dressings on ECM components expression at day 15 post-wounding .................................................................................................... 114 xii List of Acronyms and Abbreviations 3-HAA 3-hydroxyanthranilic acid 3HK 3-hydroxykynurenine AHR aryl hydrocarbon receptor AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BaP benzo(a)pyrene BBB blood brain barrier BCA bicinchoninic acid bFGF basic fibroblast growth factor bHLH basic helix-loop-helix BPE bovine pituitary extract CD3 cluster of differentiation 3 cDNA complementary DNA CNS central nervous system DMEM Dulbeccoâs Modified Eagleâs Medium DMF dimethylformamide DNase deoxyribonuclease DRE dioxin responsive element ECL enhanced chemiluminescence ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor ERK extracellular-signal-regulated kinase EthD-1 ethidium homodimer ETI epidermal thickness index FBS fetal bovine serum FGF-2 fibroblast growth factor-2 GAPDH glyceraldehyde 3-phosphate dehydrogenase GCN2 general control nonderepressible 2 GM-CSF granulocyte macrophage colony stimulating factor H&E haematoxylin-eosin HPF high power field HPLC high-performance liquid chromatography HRP horseradish peroxidase HSC hypertrophic scar hsp heat shock protein ICC immunocytochemistry IDO indoleamine 2,3-dioxygenase IL interleukin KSFM keratinocyte serum-free medium xiii Kyn kynurenine KynA kynurenic acid MAPK mitogen-activated protein kinase MEK MAPK/Erk kinase MMP matrix metalloproteinase mRNA messenger ribonucleic acid MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide Mw molecular weight nAChR nicotinic acetylcholine receptor NF nanofiber NK natural killer NMDA N-methyl-D-aspartate PAS period/aryl hydrocarbon receptor/single minded PBS phosphate buffered saline PDGF platelet-derived growth factor PEG poly(ethylene glycol) PEO poly(ethylene oxide) PLGA poly(lactic-co-glycolic acid) PMMA poly(methylmethacrylate) PVA polyvinyl alcohol PVDF polyvinylidene difluoride QA quinolinic acid Q-PCR quantitative polymerase chain reaction RNA ribonucleic acid SDS sodium dodecyl sulfate SEI scar elevation index SEM standard error of the mean TCDD 2,3,7,8-tetrachlorodibenzodioxin TDO tryptophan 2,3-dioxygenase TF transcription factor TGF-β transforming growth factor beta Th T helper THF tetrahydrofuran TIMP Tissue inhibitor of metalloproteinase TMF 6,2â²,4â²-trimethoxyflavone TNF-α tumor necrosis factor alpha Trp tryptophan VEGF vascular endothelial growth factor XAP2 hepatitis B virus X-associated protein α-SMA alpha smooth muscle actin xiv Acknowledgements My sincere gratitude to Dr. Aziz Ghahary for being an exceptional mentor and supervisor during the course of this project. I am thankful to the members of my advisory committee, Dr. Emma Guns and Dr. Frank Ko, for their valuable guidance, encouragement, support, and contributions. A special thank you to John K. Jackson in the Faculty of Pharmaceutical Sciences at UBC for his great comments on controlled drug delivery. My deep gratitude to Dr. Vincent Duronio for his kind support and encouragement during the past 5 years. I also thank the amazing group of people in the BC Professional Fire Fightersâ Burn and Wound Healing Research Laboratory at UBC. Their friendship, support, and wisdom have improved the quality of my work and life. I am grateful to Sanam Salimi Elizei, Dr. Layla Nabai, Dr. Ryan Hartwell, Dr. Saman Pakyari, Dr. Ruhangiz Taghi Kilani, Dr. Reza Jalili, Dr. Yunyuan Li, Dr. Mohsen Khosravi, Ali Farrokhi, Dr. Yun Zhang, and Dr. Azadeh Hosseini-Tabatabai. I acknowledge the funding agencies Vanier Canada Graduate Scholarships Program, the CIHR-Skin Research Training Program, and CIHR-Transplantation Research Training Program, Four Year Fellowship for their financial support. Finally and most importantly, I thank my family and my two brothers for their continued love, support, and strength throughout my life. 1 1 Introduction 1.1 Wound healing biology The wound healing process (Baum & Arpey, 2005; Gurtner, Werner, Barrandon, & Longaker, 2008; Singer & Clark, 1999) in skin involves three overlapping but well-defined phases (Figure 1.1, (J. Li, Chen, & Kirsner, 2007)): 1) Haemostasis and inflammation. Heamostasis is achieved by coagulation cascade activation, platelet activation/degranulation and platelet plug formation. Subsequently the provisional fibrin-fibronectin matrix forms, which becomes the primary scaffold for cell adhesion and migration (Clark et al., 1982). Cytokines and chemokines released by activated platelets and the product of bacterial degradation recruit neutrophils and macrophages to the wound site. These cells are responsible for removing the dead tissue, fighting off the infection and secreting cytokines and growth factors (Eming, Krieg, & Davidson, 2007; Jones, Edwards, & Thomas, 2004; Koh & DiPietro, 2011; Werner & Grose, 2003). 2) New tissue formation. This phase is characterized by cell proliferation/migration and extracellular matrix (ECM) deposition. Keratinocytes at the wound edge and remaining adnexal structures in the dermis proliferate and migrate over the injured dermis to restore the integrity of the epidermis. Fibrin-fibronectin matrix is replaced by granulation tissue through angiogenesis and ECM deposition, mainly fibronectin and type-III collagen, by activated fibroblasts. Some of the activated fibroblasts differentiate into myofibroblasts, which play a key role in ECM contraction and 2 wound closure (Desmouliere, Chaponnier, & Gabbiani, 2005; Greaves, Ashcroft, Baguneid, & Bayat, 2013; Tonnesen, Feng, & Clark, 2000). 3) Remodeling phase. During this stage the entire repair processes, activated after tissue injury, wind down or stop. Tissue cellularity decreases through the apoptosis induction in myofibroblasts, macrophages and endothelial cells. Deposited ECM is remodeled and the type-III collagen is replaced by type-I collagen. Matrix metalloproteinases (MMPs), secreted by fibroblasts, macrophages and endothelial cells, are the main players in tissue remodeling and collagen fiber organization. In most injuries the wound healing process leads to the replacement of a once functional tissue with a scar. Scar tissue lacks epidermal appendages (hair follicles and sebaceous glands), consists mainly of collagen and contains few cells (Desmouliere, Redard, Darby, & Gabbiani, 1995; Toriseva & Kahari, 2009; Velnar, Bailey, & Smrkolj, 2009). 3 Figure 1.1. Phases of repair in acute wound healing (adopted from Li et al. 2007 (J. Li et al., 2007)). 1.2 Cytokines and growth factors in wound healing The complex process of wound healing is orchestrated by an equally complex array of growth factors and cytokines, which regulate the growth, proliferation, differentiation, and migration of target cells. The main cytokines involved in wound healing are the transforming growth factor beta (TGF-β) family, fibroblast growth factor (FGF) family, epidermal growth factor (EGF) family, platelet- derived growth factor (PDGF), vascular endothelial growth factor (VEGF), granulocyte macrophage colony stimulating factor (GM-CSF), and interleukin (IL) family (Barrientos, Stojadinovic, Golinko, Brem, & Tomic-Canic, 4 2008; Faler, Macsata, Plummer, Mishra, & Sidawy, 2006; Hosgood, 1993; McGee et al., 1988; Mutsaers, Bishop, McGrouther, & Laurent, 1997; Schultz, Rotatori, & Clark, 1991; Werner & Grose, 2003). Table 1.1 summarizes the major growth factors and cytokines involved in the wound healing process. Table 1.1. The main growth factors and cytokines participating in the wound healing process, their cellular source and role in the wound healing. Growth factors and cytokine Producing cells Function in the wound healing Transforming growth factor beta (TGF-β) Platelets Keratinocytes Macrophages Lymphocytes Fibroblasts Inflammation Granulation tissue formation Re-epithelialization Matrix formation and remodeling Basic fibroblast growth factor (bFGF) Keratinocytes Mast Cells Fibroblasts Endothelial cells Smooth muscle cells Granulation tissue formation Re-epithelialization Matrix formation and remodeling Epidermal growth factor (EGF) Platelets Macrophages Fibroblasts Inflammation Granulation tissue formation Re-epithelialization Matrix formation and remodeling Platelet-derived growth factor (PDGF) Platelets Keratinocytes Macrophages Endothelial cells Fibroblasts Inflammation Granulation tissue formation Re-epithelialization Matrix formation and remodeling Vascular endothelial growth factor (VEGF) Platelets Neutrophils Macrophages Endothelial cells Smooth muscle cells Fibroblasts Granulation tissue formation Granulocyte macrophage-colony stimulating factor (GM-CSF) Keratinocytes Inflammation Re-epithelialization Interleukin-1 (IL-1) Neutrophils Monocytes Macrophages Keratinocytes Inflammation Re-epithelialization Interleukin-6 (IL-6) Neutrophils Macrophages Inflammation Re-epithelialization 5 1.3 Wound healing pathology Scar formation is an expected outcome of wound healing; however, excessive scarring emerges as a result of exaggerated wound healing process and may occur after any injury of the deep dermis, such as burns, surgery, lacerations, aberrations, piercing and vaccination (Bayat, McGrouther, & Ferguson, 2003; J. Li et al., 2007). Hypertrophic scars (HSCs) and keloids are the main forms of excessive scarring in skin. These scars impose immense psychological, functional and cosmetic burdens for patient and the economical systems (Bock, Schmid-Ott, Malewski, & Mrowietz, 2006; Brown, McKenna, Siddhi, McGrouther, & Bayat, 2008). HSCs, which are clinically red, raised, itchy and inelastic, remain in the borders of the primary wound, usually regress by time and may produce scar contractures (Figure 1.2.) (Brissett & Sherris, 2001; Mutsaers et al., 1997; Peacock, Madden, & Trier, 1970). The HSC occurrence rate vary from 40% to 70% following surgery and up to 90% after burn injuries. HSCs commonly emerge in deep second and third degree burns and at the border of skin grafts where the defect is slowly closed by contraction and re-epithelialization (Lewis & Sun, 1990; Li-Tsang, Lau, & Chan, 2005; Spurr & Shakespeare, 1990). Anatomically HSCs mainly develop at locations with high tension such as ankles, knees, presternum, shoulders and neck (J. C. Murray, 1994; Niessen, Spauwen, Schalkwijk, & Kon, 1999). These scars are characterized by high tissue cellularity, abundant ECM accumulation at the wound site and tissue contraction, which subsequently distort the normal tissue structure and function (Armour, Scott, & Tredget, 2007a; Tredget, 1999). 6 Histologically, HSCs have thicker dermis and epithermal layer compared to the normal skin and minimal amount of distinct collagen fibers, primarily type III collagen, and fiber bundles. Also the presence of nodular structures, containing α-smooth muscle actin (α-SMA) positive myofibroblasts, fine randomly oriented collagen fibrils and small blood vessels (Blackburn & Cosman, 1966; Ehrlich et al., 1994), characterize these scars. HSCs have equal sex distribution and highest incidence among younger individuals (20-30 years of age). Several mechanisms are involved in HSC formation including affected hemostasis, exaggerated inflammation, prolonged re-epithelialization, excessive ECM production, deficiencies in ECM degradation, augmented neovascularization and impaired apoptosis (Arakawa et al., 1996; Deitch, Wheelahan, Rose, Clothier, & Cotter, 1983; Kischer et al., 1989; Mutsaers et al., 1997; Niessen et al., 1999; L. Q. Zhang, Laato, Muona, Kalimo, & Peltonen, 1994). Keloid formation is another excessive scarring condition that results in the emergence of red and itchy scars that infiltrate into the surrounding normal tissue and exceed the margins of the initial injury. Clinically keloids appear as firm, mildly tender tumors with thinned epithelium and occasional presence of ulceration at their surface. Anatomically keloids are more prominent at shoulders, earlobes, upper arms, cheeks and anterior chest (Leventhal, Furr, & Reiter, 2006; Niessen et al., 1999). Histologically (Figure 1.3.), keloids are characterized by increased fibroblast proliferation and density, presence of primarily disorganized, thick type-I and III collagen bundles and lack of nodular structures 7 or excess myofibroblasts (Blackburn & Cosman, 1966; Bran, Goessler, Hormann, Riedel, & Sadick, 2009; Ehrlich et al., 1994). Most keloids emerge within a year of the trauma; however, a small percentage of keloids develop spontaneously in some patients without a known antecedent trauma. Figure 1.2. Hypertrophic scarring. (A) post-burn hypertrophic scarring, (B) hypertrophic scarring in a fibrotic rabbit ear model (Courtesy of Dr. Ghaharyâs slide collection). Keloids often happen in younger individuals (10-30 years of age), have higher prevalence among darker skinned individuals (incident rate of 6% to 16% A B 8 among African population) and shows a positive familial history. These scars continue to evolve over time, are difficult to revise by surgery and usually recur after the excision (Kiprono et al., 2015; Niessen et al., 1999; Slemp & Kirschner, 2006). Figure 1.3. Keloids. Keloid formation following anthelix plasty (A) or piercing of the ear lobe (B). Hematoxylin and eosin staining of a keloid (C). Presence of enlarged collagen fibers in the dermis (D) (Bran et al., 2009). 1.4 Current treatment modalities for hypertrophic scars Current treatment modalities vary from pressure garments, topical dermal creams to surgical revisions (Armour et al., 2007a; Berman, Viera, Amini, Huo, & Jones, 2008; Berman, Villa, & Ramirez, 2004; Gauglitz, 2013; Gauglitz, Korting, Pavicic, Ruzicka, & Jeschke, 2011; Juckett & Hartman-Adams, 2009; Y. Li, Kilani, Rahmani-Neishaboor, Jalili, & Ghahary, 2014; McCollum et al., 2011; Occleston et al., 2011; Reish & Eriksson, 2008; Sherris, Larrabee, & Murakami, 1995; Tredget, 1999; Tziotzios, Profyris, & Sterling, 2012; Viera, Amini, Valins, & Berman, 2010; Wolfram, Tzankov, Pulzl, & Piza-Katzer, 2009). Table 1.2. lists the current treatments and their mechanism of action. A B C D 9 Table 1.2. Treatment modalities for HSC and their mechanism of action HSC treatment Mode of action Pressure garment 1. Reduced blood flow, ischemic cell damage and myofibroblast apoptosis. 2. Reduced α2-macroglobulin level and subsequent increased collagenase activity and collagen breakdown. 3. Decreased scar hydration with subsequent reduction in neovascularization and ECM production. Corticosteroid injection 1. Decreased inflammation. 2. Decreased fibroblast proliferation and fibroblast-mediated contraction. 3. Increased fibroblast and mast cells apoptosis. 4. Decreased collagen synthesis. 5. Inhibition of TGF-β1 and TGF-β2. 6. Vasoconstriction, reduced α2-macroglobulin level and increased collagenase activity and collagen breakdown. Laser therapy 1. Photothermolysis of scar vasculature and thermal necrosis. 2. Increased fibroblast apoptosis. 3. Decreased TGF-β level. Silicon gel and sheeting 1. Increased hydration of stratum corneum. 2. Increased skin temperature. 3. Increased fibroblast apoptosis and decreased fibroblast-mediated contraction. 4. Decreased TGF-β level. Onion extract 1. Suppressed inflammation. 2. Decreased collagen synthesis and improved collagen organization. 3. Decreased fibroblast proliferation. 4. Increased collagenase expression. Bleomycin injection 1. Decreased TGF-β level. 2. Decreased collagen synthesis. Radiation therapy 1. Increased fibroblast apoptosis. 2. Decreased collagen synthesis. Cryosurgery 1. Decreased vascularity. 2. Increased fibroblast apoptosis. Interferon injection 1. Decreased fibroblast proliferation and fibroblast-mediated contraction. 2. Increased fibroblast apoptosis. 3. Decreased collagen and fibronectin synthesis. 4. Increased collagenase production 5. Reduced Tissue inhibitor of metalloproteinase-1 (TIMP-1) activity. 6. Decreased TGF-β level. 5-Fluorouracil injection 1. Decreased fibroblast proliferation. 2. Increased fibroblast apoptosis. 3. Decreased collagen synthesis. Imiquimod 5% cream 1. Increased interferon-α synthesis and subsequent induction of collagen breakdown. 2. Increased fibroblast apoptosis. Avotermin (Recombinant TGF-β3) 1. Decreases collagen synthesis. 2. Inhibits alpha-smooth-muscle actin (α-SMA) expression. 3. Induced MMP9 expression. 10 HSC treatment Mode of action Kynurenine 1. Decreased collagen expression. 2. Increased MMP expression. 1.5 Kyn pathway Tryptophan (Trp) is an essential amino acid that circulates in blood in either free form (10%) or bound to albumin (90%) (McMenamy, 1965). Kyn pathway (Figure 1.4.) is the main route of tryptophan metabolism in the body and up to 99% of the dietary Trp is metabolized through this pathway (Wolf, 1974). Through the Kyn pathway, Trp is oxidized to form N-formylkynurenine either by tryptophan 2, 3 dioxygenase (TDO), indoleamine 2, 3 dioxygenase-1 (IDO-1) or indoleamine 2, 3 dioxygenase-2 (IDO-2) (Ball, Yuasa, Austin, Weiser, & Hunt, 2009; Grohmann, Fallarino, & Puccetti, 2003; Watanabe, Fujiwara, Yoshida, & Hayaishi, 1980). N-formylkynurenine is metabolized to Kyn, which is the first stable intermediate compound in the pathway. Kyn is subsequently metabolized in two distinct routes to 3-hydroxykynurenine (3HK) and Quinolinic acid (QA) or to Kynurenic acid (KynA) (Bender & McCreanor, 1982). The role of Kyn pathway has been implicated in a variety of physiological and pathological processes including scarring, central nervous system (CNS), and immunity. 1.5.1 Role of Kyn pathway in scarring Recent studies (Chavez-Munoz et al., 2012; Y. Li et al., 2014) revealed the anti-scarring effects of local IDO expression and subsequent Kyn accumulation in a fibrotic rabbit ear model. Chavez-Munoz et al. reported that IDO expression not only protects the transplanted xenogeneic skin substitute 11 from immune rejection, but also prevents scar formation by reducing the scar elevation index, epithermal thickness index and tissue cellularity (Chavez-Munoz et al., 2012). Further studies by Li et al. indicated that topical application of a Kyn-containing cream in a fibrotic rabbit ear model improved the scar formation relative to that of controls. They also demonstrated that Kyn anti-scarring effects are mediated by an increase in MMP-1 and MMP-3 expression and inhibition of collagen production in dermal fibroblasts (Y. Li et al., 2014). 1.5.2 Role of Kyn pathway in central nervous system (CNS) Trp is actively transported across the blood brain barrier (BBB) by a competitive and non-specific L-type amino acid transporter (Fukui, Schwarcz, Rapoport, Takada, & Smith, 1991). Astrocytes, neurons, oligodendrocytes, endothelial cells, microglia and infiltrating macrophages metabolize Trp along the Kyn pathway (Guillemin et al., 2007; Guillemin et al., 2001; Guillemin et al., 2000; Guillemin, Smythe, Takikawa, & Brew, 2005; Heyes et al., 1996; Vecsei, Szalardy, Fulop, & Toldi, 2013). Deregulation of Kyn pathway and subsequent hypo- or hyperfunction of neuroactive metabolites have been reported in association with variety of neurodegenerative and psychiatric disorders, including Alzheimerâs disease, schizophrenia, amyotrophic lateral sclerosis (ALS), depression and AIDS dementia complex (Beal et al., 1986; Guillemin et al., 2003; Kegel et al., 2014; Moroni, 1999; Nemeth, Toldi, & Vecsei, 2006; Schwarcz, Bruno, Muchowski, & Wu, 2012; Vecsei et al., 2013). Several neuroactive compounds are formed in the Kyn pathway having neurotoxic or neuroprotective effects. 12 Figure 1.4. Kyn pathway (Rahman et al., 2009). 13 1) 3-HK, metabolized from Kyn by kynurenine-3-hydroxylase, generates reactive oxygen species such as superoxide and hydrogen peroxide. Produced reactive oxygen species cause oxidative stress and subsequent cellular damage and cell death (Nakagami, Saito, & Katsuki, 1996; Okuda, Nishiyama, Saito, & Katsuki, 1998). 2) QA, one of the Kyn pathway end products metabolized by macrophages and microglia (Espey, Chernyshev, Reinhard, Namboodiri, & Colton, 1997; Guillemin et al., 2005; Heyes et al., 1996; Savman, Heyes, Svedin, & Karlsson, 2013), is an excitotoxin in the CNS. QA is a weak but specific competitive agonist of N-methyl-D-aspartic acid (NMDA) receptor. QA-dependent over-activation of the NMDA receptor results in the accumulation of intracellular Ca2+ and subsequent excitotoxic neuronal cell death by apoptosis. QA also increases the synaptosomal glutamate level, via inducing the glutamate release and inhibiting the glutamate uptake by astrocytes. Generation of reactive oxygen species, such as hydrogen peroxide, superoxide and hydroxyl radicals, and lipid peroxidation enhance the neurotoxic potentials of QA in the CNS (Behan, McDonald, Darlington, & Stone, 1999; Perez-De La Cruz, Carrillo-Mora, & Santamaria, 2012; Rios & Santamaria, 1991; Stone & Perkins, 1981). Elevated level of QA and QA-induced neuronal degeneration has been reported in variety of neurodegenerative disorders including Alzheimer's disease, AIDS dementia complex and Huntingtonâs disease (Beal et al., 1986; Guillemin et al., 2003; Schwarcz et al., 2012). 14 3) KynA, directly metabolized from Kyn by neurons and astrocytes, act as a neuroprotective agent in the CNS (Guillemin et al., 2001; Guillemin et al., 2000; Vecsei et al., 2013). KynA is a competitive antagonist of glutamate receptors including NMDA receptors, kainate receptors and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The neuroprotective effects of KynA are also mediated by inhibition of α7 nicotinic acetylcholine presynaptic receptors (nAChRs) and subsequent inhibition of glutamate release (Birch, Grossman, & Hayes, 1988; Hilmas et al., 2001; Prescott, Weeks, Staley, & Partin, 2006; Stone, 1990). In addition, KynA is an antioxidant, which can scavenge reactive oxygen species such as superoxide anion and hydroxyl radicals (Lugo-Huitron et al., 2011; Stone, 2000; H. Q. Wu et al., 2010). KynA dependent inhibition of NMDA receptor-mediated excitotoxicity represents a promising clinical approach for prevention or treatment of neurodegenerative diseases (Vecsei et al., 2013). Halogenated derivatives of KynA, such as 7-chlorokynurenic acid and 5,7-dichlorokynurenic acid, are currently under preclinical investigation or Phase I clinical trial to be used as neuroprotective agents (Fulop et al., 2009; Stone, 2000; Vecsei et al., 2013). 1.5.3 Role of Kyn pathway in immune system Liver is the main site of Trp metabolism under physiological conditions. Inflammation and infection induce the expression of IDO in extra-hepatic tissues, including lungs, kidneys, brain, placenta, blood and spleen, and shifts the Trp metabolism away from the hepatic site (Heyes et al., 1993; Manuelpillai et al., 15 2005; Muller et al., 2008). IDO expression and Kyn pathway has been implicated in suppression of immune responses and development of immune tolerance (Mellor & Munn, 1999, 2004; Munn & Mellor, 2004). The primary mechanism accounted for IDO-induced immune suppression was through Trp starvation and accumulation of the proapoptotic kynurenines (Grohmann et al., 2003; Mellor & Munn, 1999). IDO-induced Trp depletion activates general control nonderepressible 2 (GCN2) pathway, which subsequently induces cell cycle arrest and apoptosis in macrophages and activated T-cells (Munn et al., 2005; Poormasjedi-Meibod, Jalili, Hosseini-Tabatabaei, Hartwell, & Ghahary, 2013). Trp metabolites, such as Kyn, 3-hydroxyanthranilic acid (3-HAA), and QA, dampens the inflammation by selective apoptosis induction in Th1 but not Th2 cells via caspase-8 activation (Fallarino et al., 2002) impairment of memory T-cells formation (Dai & Dai, 2008) and inhibition of cell proliferation in T lymphocytes, B-cells and natural killer (NK) cells (Frumento et al., 2002). In addition, recent observations pointed to the pivotal role of Kyn in suppressing the dendritic cells immunogenicity and induction of regulatory T-cells differentiation (Mezrich et al., 2010; Nguyen et al., 2010). Regardless of extensive research about pathophysiological effects of Kynurenines, the signaling pathway associated with these molecules has not been elucidated until recently. 1.6 Aryl hydrocarbon receptor (AHR) In 2011 Opitz et al. reported the role of aryl hydrocarbon receptor (AHR) signaling pathway in the biological effects of Kyn (Opitz et al., 2011). Subsequent 16 studies using different subsets of immune cells elucidated the pivotal role of this signaling pathway in the immunomodulatory effects of Kyn (Mezrich et al., 2010; Nguyen et al., 2010). AHR is a ligand-activated transcription factor (TF) that regulates the expression of wide range of genes in different species and tissues. This receptor is a member of the basic helix-loop-helix (bHLH)/PAS (Period [Per]-aryl hydrocarbon receptor nuclear translocator [ARNT]-Single minded [Sim]) family of transcription factors. The inactive form of AHR mainly resides in the cytoplasm in association with a molecular chaperone complex (Hsp90/XAP2/p23) (Hankinson, 1995; Petrulis, Hord, & Perdew, 2000). Upon ligand binding the receptor undergoes a conformational change that exposes a nuclear localization sequence and subsequently the receptor translocates to the nucleus (Davarinos & Pollenz, 1999; Ikuta, Eguchi, Tachibana, Yoneda, & Kawajiri, 1998). Inside the nucleus AHR heterodimerizes with ARNT and interacts with dioxin responsive element (DRE) located at the promoter of target genes and modulate their expression (Beischlag, Luis Morales, Hollingshead, & Perdew, 2008) (Figure 1.5, (Denison & Nagy, 2003)). The nuclear AHR complexes that fail to dimerize with ARNT are exported back to the cytoplasm, ubiqutinilated and degraded by proteasomes (Ma, 2007; Song & Pollenz, 2002). In addition to binding to the gene promoter, AHR can modulate transcription in a DNA-binding independent manner through cross talk with other signaling pathways and transcription factors (Haarmann-Stemmann, Bothe, & Abel, 2009; Puga, Ma, & Marlowe, 2009). Primary studies on the AHR signaling pathway focused on its role in the induction of phase-I and II drug metabolizing enzymes (e.g. cytochromes P450 such as 17 CYP1A-1) (Nebert, Dalton, Okey, & Gonzalez, 2004; Whitlock, 1999). However, the creation of AHR null mice indicated the pivotal role of this receptor in multiple aspects of growth, development, differentiation, and physiology (Gonzalez & Fernandez-Salguero, 1998; Hillegass, Murphy, Villano, & White, 2006; Puga, Xia, & Elferink, 2002) via modulating the expression of cytokines, TFs, ECM components and regulators of ECM metabolism and cell adhesion (Andreasen, Mathew, & Tanguay, 2006; Ishida et al., 2010; Kung, Murphy, & White, 2009; Ono et al., 2013). These studies resulted in the expansion of AHR ligand inventory from exogenous environmental contaminants, such as polyaromatic hydrocarbons (PAH) and halogenated aromatic hydrocarbons (HAH) (Barouki, Coumoul, & Fernandez-Salguero, 2007; Denison & Nagy, 2003; Mimura & Fujii-Kuriyama, 2003), to diverse variety of naturally occurring dietary molecules, such as curcumin and ceratinoids (Ashida, Nishiumi, & Fukuda, 2008; Ciolino, Daschner, Wang, & Yeh, 1998; Denison & Nagy, 2003) and endogenous AHR ligands including tryptophan metabolites such as Kyn (Mezrich et al., 2010; Nguyen et al., 2010) and KynA (DiNatale et al., 2010). 18 Figure 1.5. The molecular mechanism of activation of gene expression by the AHR. AHR, aryl hydrocarbon receptor; Arnt, AHR nuclear translocator; CYP1A-1, cytochrome P4501A-1; DRE, dioxin responsive element; hsp90, heat shock protein 90; mRNA, Messenger RNA; XAP2, hepatitis B virus X-associated protein (Denison & Nagy, 2003). 1.7 Advancements in the wound management products Promotion of rapid wound healing with the best aesthetic and functional outcome is the main objective in the wound care industry. The primary function of a wound dressing is to promote the wound healing process by providing the optimum healing environment (Sarabahi, 2012). In order to improve the healing process, an ideal wound dressing should be able to balance the wound moisture, decrease loss of electrolytes and protein from the wound, allow adequate gas exchange and minimize pain and infection (Benbow, 2010; Selig et al., 2012). 19 Development of nanofiber based wound dressings expanded the function of these wound care products from absorbing the wound exudate and protecting the wound from infection to delivering controlled amount of drugs to the wound (Boateng, Matthews, Stevens, & Eccleston, 2008; Ignatova, Rashkov, & Manolova, 2013; G. H. Kim et al., 2011; Thakur, Florek, Kohn, & Michniak, 2008). 1.7.1 Electrospinning process The electrospinning process, first patented by Formhals in 1934, is an efficient technique to produce nanofibrous scaffolds from polymeric solutions. Electrospinning has several advantages over conventional techniques for nanofiber manufacturing because of its consistency in fiber production, technical simplicity, and ease of manipulation and scale up. Also the flexibility of the polymer choice, synthetic or natural and biodegradable or non-biodegradable, made it possible to fine-tune the properties of the nanofibers to their application (Bhardwaj & Kundu, 2010; Teo & Ramakrishna, 2006; Valizadeh & Mussa Farkhani, 2014). Nanofibers produced by electrospinning process have many unique properties such as very large ratio of surface area to volume, pore size within nano range, flexibility in chemical/physical surface modification/functionalities, and unique physical characteristics (superior tensile strength and stiffness). The unique properties of nanofibers expanded their application from protective textiles, filtration and nanocomposite materials to tissue scaffolds, drug delivery and wound dressings (Khil, Bhattarai, Kim, Kim, & Lee, 2005; Pham, Sharma, & Mikos, 2006; Sill & von Recum, 2008; Venugopal et al., 2008). Application of electrospinning process for wound dressing 20 manufacturing is very attractive. The small pore size of the manufactured mats (500 nm to 1 µm) protects the wound from bacterial invasion. High surface area and void volume is effective in wound exudate absorption and drug delivery (Boateng et al., 2008; Choi, Kim, & Yoo, 2015; Ignatova et al., 2013; G. H. Kim et al., 2011; Thakur et al., 2008). In a typical electrospinning process (Figure 1.6.) a high voltage is applied to the solution, which leads to the formation of repulsive forces between charged particles within the solution. At a critical voltage, typically more than 5kV, the repulsive force between opposite charges within the solution overcomes the solutionâs surface tension and a jet erupts from the tip of the spinneret. As the jet accelerates toward the collector it undergoes a bending instability, further stretching and elongation process, which result in formation of a very long and thin jet. Solvent evaporation along the way results in formation of polymer fibers with the average diameter range of 100â500 nm. The morphology and diameter of the electrospun fibers are affected by several parameters including (Beachley & Wen, 2009; Cengiz-Callioglu, Jirsak, & Dayik, 2013; Deitzel, Kleinmeyer, Harris, & Tan, 2001; Henriques, Vidinha, Botequim, Borges, & Silva, 2009; Leach, Feng, Tuck, & Corey, 2011; Sill & von Recum, 2008; Teo & Ramakrishna, 2006): 1) The spinning process parameters such as applied voltage, syringe pump rate, nozzle-collector distance and spinning environment (humidity, air flow, surrounding gas). 21 2) The spinning solution properties such as polymer solution concentration, molecular weight, viscosity, elasticity, conductivity, surface tension and solvent vapor pressure. Figure 1.6. Schematic of electrospinning process. 1.7.2 Poly(vinyl alcohol) Poly(vinyl alcohol) (PVA) is a water soluble linear polymer made by dissolving polyvinyl acetate in an alcohol and subsequent alkaline treatment of the solution (Figure 1.7.). The degree of acetate group hydrolysis determines the physical, chemical and mechanical properties of the PVA. PVA solubility in water decreases by increasing the degree of hydrolysis and PVA polymerization. Figure 1.7. Schematic of synthesis of poly(vinyl alcohol). 22 PVA has been used extensively in the biomedical field because of its high water solubility, biocompatibility, chemical resistance and low protein adsorption. PVA needs to be cross-linked, either physically or chemically, to reduce its solubility and increase its stability in biological fluids. The physical and chemical characteristics of the polymer are tailored by adjusting the degree of cross-linking which subsequently determine its biological application (Kitade et al., 2002; Maria Soledad Peresin, 2014; Pluta & Karolewicz, 2001; Samiullah Khan, 2014; Stammen, Williams, Ku, & Guldberg, 2001). 1.7.3 Poly(lactic-co-glycolic acid) Poly(lactic-co-glycolic acid) (PLGA) is a FDA approved copolymer of glycolic acid and lactic acid synthetized by the ring-opening co-polymerization process. Successive monomers are linked together by ester bonds in the PLGA polymer. This ester bond is hydrolyzed in water and results in the polymer degradation and formation of the original monomers, lactic acid and glycolic acid (Figure 1.8.). Physicochemical and mechanical characteristics and degradation rate of PLGA polymer are easily tunable by controlling the ratio of lactide to glycol and polymer molecular weight. The higher the content of lactide units, the lower the hydrophilicity and water absorbance capacity of the polymer; hence, the slower the degradation rate. In past two decades PLGA has been used extensively for controlled drug delivery and tissue-engineering applications (Shive & Anderson, 1997; X. S. Wu & Wang, 2001). 23 Figure 1.8. Schematic of synthesis of poly(lactic-co-glycolic acid). Table 1.3. Physical and mechanical characteristics of poly(lactic-co-glycolic acid) Physical and mechanical properties Value Young Modulus 2 GPa Elongation at Break 3-10% Degradation rate 1-6 months Crystallinity Amorphous 1.7.4 Poly(methyl methacrylate) Poly(methyl methacrylate) (PMMA) was discovered by Rowland Hill and John Crawford in early 1930s at Imperial Chemical Industries (ICI) in England. It is a colorless, clear polymer available on the market in sheet, rod and tube forms. PMMA is the polymer of the esters of methyl methacrylate, with chemical formula (CH2=C[CH3]CO2CH3)n. It is a linear polymer, which is derived from methyl methacrylate by free-­ââradical polymerization (Figure 1.9.). PMMA has high mechanical strength, low elongation at break and high Youngâs modulus (Table 1.4.). This polymer has low moisture and water absorbing capacity; therefore, the materials made from PMMA are biocompatible, physiologically harmless and have good dimensional stability. PMMA has versatile applications including optics (lenses and glasses), vehicles (rear lights and indicators), 24 electrical engineering (lamp covers and control buttons), office equipment (writing and drawing instruments) and medicine (bone cement and packaging for pills and capsules) (Evans & Nelson, 1993; Kong & Jang, 2008; Majid, Lindberg, Gunterberg, & Siddiki, 1985). Figure 1.9. Schematic of synthesis of poly(methyl methacrylate. Table 1.4. Physical and mechanical characteristics of poly(methyl methacrylate) Physical and mechanical properties Value Density 1.15-1.19 g/cm3 Water absorption 0.3-2% Moisture absorption at equilibrium 0.3-0.33% Tensile Strength, Ultimate 47-­ââ79 MPa Elongation at Break 1-­ââ30% Young Modulus 2.2-­ââ3.8 GPa Melting Point 130 °C 1.8 Hypothesis and objectives Regardless of the source or site of injury, wound healing is a dynamic process that strikes a fine balance between synthesis and degradation of ECM. Over-healing processes in skin are disfiguring and devastating, resulting in bulky, itchy and inelastic scars that are detrimental for millions of burn and trauma patients. Unfortunately, current treatment modalities for dermal fibrosis still 25 remain unsatisfactory and despite the widespread use of these treatments, many patients still develop devastating scarring. Localized application of agents that suppress matrix accumulation, either by induction/acceleration of ECM degradation or inhibition of ECM deposition, can be used as an effective approach to prevent dermal fibrosis. It has been shown in a previous study (Y. Li et al., 2014) that Kyn, a Trp metabolite, functions as a potent stimulator of MMPs (MMP-1 and MMP-3) and effectively inhibits type-I collagen expression in dermal fibroblasts. The antiscarring effects of Kyn were further evaluated in a fibrotic rabbit ear model where the wounds received daily application of a Kyn-containing dermal cream (Y. Li et al., 2014). Although we anticipate that topical application of Kyn-containing dermal cream right after re-epithelialization markedly improves the formation of hypertrophic scarring, daily application of this cream is not feasible for clinical translation in burn patients where the dressings are changed every 3-5 days. Further, the use of topical cream may not prevent or significantly improve this fibrotic condition because the initial signal in developing the fibrotic condition would be much earlier prior to wound closure. For these reasons, a series of systematic experiments were conducted to address the following hypothesis and specific aims of the current study. Hypothesis: It is our hypothesis that a slow releasing nanofiber dressing can be developed from which either Kyn or its metabolites are released at the wound site where it improves and/or prevents dermal fibrosis by modulating the expression of the key ECM components such as MMPs, collagens (type-I and - 26 III) and fibronectin involved in dermal fibrotic conditions. The following objectives describe our approach in addressing this hypothesis. Objective 1 To evaluate the antifibrotic effects of other metabolites in the Kyn pathway in order to find the most active intermediate(s). Objective 2 To identify and formulate a combination of materials to serve as a nanofiber shell for Kyn and KynA encapsulation. Objective 3 To evaluate the anti-fibrotic efficacy of the fabricated wound dressings in open wounds in animal models. Figure 1.10. Cartoon representation of the electrospun medicated wound dressing containing anti-fibrotic agent(s). 2 Anti-scarring properties of different tryptophan derivatives1 2.1 Introduction Wound healing in skin is a highly regulated process the outcome of which is defined by a fine balance between extracellular matrix (ECM) deposition and 1 A version of chapter 2 has been published. Poormasjedi-Meibod, M-S., Hartwell, R., Taghi Kilani, R., and Ghahary, A. (2014) Anti-Scarring Properties of Different Tryptophan Derivatives. PLoS ONE 9(3): e91955. doi:10.1371/journal.pone.0091955. 27 degradation. Abnormal matrix deposition and remodeling, leading to ECM accumulation at the wound site, play a pivotal role in hypertrophic scar (HSC) formation (Armour et al., 2007a; Tredget, 1999). A study by Ghahary et al. (Ghahary, Shen, Scott, Gong, & Tredget, 1993) revealed higher levels of collagen (type-I and -III), and fibronectin expression in HSC tissue compared with normal skin. They also showed that collagenase messenger ribonucleic acid (mRNA) expression and activity in the fibroblast-conditioned medium is significantly reduced in HSC fibroblasts compared with the normal ones (Ghahary et al., 1996). Recent studies (Hayashi, Ikeda, Kitamura, Hamasaki, & Hatamochi, 2012; Mead, Wong, Cordeiro, Anderson, & Khaw, 2003; Rahmani-Neishaboor, Yau, Jalili, Kilani, & Ghahary, 2010) demonstrate that localized application of agents that are targeted to suppress matrix accumulation provide an approach to specifically reduce scarring. Indoleamine 2,3-dioxygenase (IDO), a cytosolic enzyme which catalyses the first and rate-limiting step in the tryptophan catabolism to N-formylkynurenine, is widely known for its immuno-regulatory properties (Mellor & Munn, 2004; Munn et al., 2002; Uyttenhove et al., 2003). A previous study by our group revealed that local IDO expression not only protects the transplanted xenogeneic skin substitute from immune rejection but also reduces scarring in a rabbit ear model (Chavez-Munoz et al., 2012). Further studies by our group revealed that the antiscarring properties of IDO, which are dependent on kynurenine (Kyn) accumulation, are in part mediated by increasing the expression of matrix metalloproteinases (MMPs) by dermal fibroblasts. This initial result that Kyn 28 could reduce dermal fibrosis was further confirmed with topical application of a Kyn cream that improved healing quality in a fibrotic animal model (Y. Li et al., 2014). Although Kyn demonstrated efficacy in preventing dermal fibrosis, possible adverse effects may result if higher than normal levels are achieved systemically as a therapeutic. Circulatory Kyn is actively transported across the blood brain barrier (BBB) by the large neutral amino acid carriers (Fukui et al., 1991). Kyn and tryptophan compete for the same carrier to be transported via the BBB; as such, elevated levels of Kyn in circulation may lead to a significant depletion in the tryptophan pool of the brain. Tryptophan deficiency is associated with dysregulation of serotonin (5-hydroxytryptamine) metabolism, which contributes to many psychiatric disorders (Baldwin & Rudge, 1995; Olivier, 2004). The transported Kyn is mainly metabolized to 3OH-kynurenine, 3OH-anthranilic acid, and quinolinic acid in the brain (Bender & McCreanor, 1982). These metabolites either directly cause apoptotic or necrotic neuronal death (Moroni, 1999; Nakagami et al., 1996; Okuda et al., 1998) or induce excitotoxic neuronal death via binding to N-methyl-D-aspartate (NMDA) receptors in the brain (Beal et al., 1986; Stone & Perkins, 1981). The potential adverse effects of Kyn prompted our search for an antifibrogenic compound with fewer potential risks. Contrary to other Kyn metabolites, kynurenic acid (KynA), which poorly crosses the BBB (Fukui et al., 1991), has neuroprotective properties and reduces the excitotoxin-induced neuronal death by antagonising the ionotropic glutamate receptors (Moroni, 1999) and the a7 nicotinic receptors (Hilmas et al., 2001). As a 29 potentially fitting candidate replacement for Kyn the purpose of this study was to evaluate the antifibrotic properties of KynA both in vitro and in a fibrotic rabbit ear model. Given that molecular toxicity can in many ways be stereospecific, we also evaluated the effect of L- and D-isomers of Kyn (L-Kyn and D-Kyn, respectively) on ECM expression in order to find the active enantiomer of the drug. Application of one active enantiomer only instead of the racemic form of the drug provides the possibility of reducing the administered dosage and clinically significant side-effects while having the desired biological effect (Baumann, Zullino, & Eap, 2002; Kasprzyk-Hordern, 2010). The results of this study provide evidence for the first time that KynA is a potent antifibrogenic replacement for Kyn both in vitro and in a fibrotic animal model. 2.2 Materials and methods 2.2.1 Ethics statement All methods and procedures, as well as the use of animals and tissue specimens derived from animals and humans, whether or not obtained solely for the purpose of this study are approved by both Human and Animal Ethics Committees of the University of British Columbia. Written consent from informed donors was received prior to conducting any sampling of tissue specimens. 2.2.2 Cell culture Foreskin samples were obtained from healthy patients undergoing elective circumcision. Human primary keratinocytes and fibroblasts were harvested from foreskin samples as described previously (M. Li, Moeen Rezakhanlou, Chavez-Munoz, Lai, & Ghahary, 2009). Fibroblasts were cultured in Dulbeccoâs Modified 30 Eagleâs Medium (DMEM, GIBCO, Grand Island, NY) with 10% fetal bovine serum (FBS). Keratinocytes were cultured in keratinocyte serum-free medium (KSFM, Invitrogen Life Technologies, Carlsbad, CA) supplement with bovine pituitary extract (25 ng/ml, BPE) and epidermal growth factor (0.2 ng/ml, EGF, GIBCO). Keratinocytes and fibroblasts at passage 4â7 were used in all experiments in this study. 2.2.3 RNA extraction and quantitative real time PCR (Q-PCR) In order to determine the effect of kynurenines on ECM component gene expression, dermal fibroblasts were cultured and treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 µg/ml) of KynA, Kyn, L-Kyn, or D-Kyn for 24 h. Total RNA was isolated using the GeneJET RNA Purification Kit (Fermentas International Inc, Thermo Fisher Scientific, Ottawa, ON) according to the manufacturerâs instructions. Total RNA (5 µg) was reverse transcribed into cDNA using a SuperScriptâ¢ II First-Strand cDNA Synthesis kit (Invitrogen). Q-PCR was carried out on the Applied Biosystems® 7500 Fast Real-Time PCR System, using the SYBR® Green PCR Master Mix kit (Applied Biosystems, Warrington, UK). The following cycling conditions were used for Q-PCR: 95 °C/15 min with 40 cycles of 95 °C/1 min, 55 °C/ 30 s, and 72 °C/30 s. The following primers were used for Q-PCR reactions: MMP-1 5â²-CTCAGGATGACATTGATGGC-3â² and 5â²-CCCCGAATCGTAGTTATAGC-3â², MMP-3 5â²-TGGCATTCAGTCCCTCTATRGG-3â², and 5â²-CAAAGCAGGATCACAGTT-3â², MMP-3 5â²-TTCCGCCTGTCTCAAGATGATAT-3â² and 5â²-AAAGGACAAAGCAGGATCACAGTT-3â², Col-1a1 5â²- 31 CTGGAATGAAGGGACACA-3â² and 5â²-CCATTGGCACCTTTAGCA-3â², and fibronectin 5â²-GATAAATCAACAGTGGGAGC-3â² and 5â²- CCCAGATCATGGAGTCTTTA-3â². Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene since its expression does not change in the response to the treatment. GAPDH 5â²-GACAAGCTTCCCGTTCTCAG-3â² and 5â²-CAATGACCCCTTCATTGACC-3â² (Invitrogen). 2.2.4 Preparation of cell lysates and Western blotting Dermal fibroblasts were cultured and treated as described above for 48 h. The whole cell proteins were separated by running the samples on 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membranes were blocked and probed for MMP-1, type-I collagen, and fibronectin using rabbit-anti-MMP-1 (1:2000 dilution, Abcam, Cambridge, MA), mouse-anti-collagen-I (1:100, Developmental Studies Hybridoma Bank), and rabbit-anti-fibronectin (1:1000, Santa Cruz Biotechnology, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ab (1:3000 dilution, Bio-Rad) and HRP-conjugated goat anti-mouse Ab (1:3000 dilution, Bio-Rad) were used as the secondary antibodies. β-actin was used as the loading control. Conditioned medium (30 µl) from the untreated and treated dermal fibroblasts were subjected to Western blotting for detection of secreted MMP-3 protein. Mouse-anti-MMP-3 antibody (1:2000, R&D Systems, Minneapolis, MN) and HRP-conjugated goat anti-mouse Ab were used as the primary and secondary antibodies, respectively. 32 2.2.5 MMP activity assay The MMP activity in the conditioned medium of control and treated fibroblast was determined using the SensoLyteâ¢ Plus 520 Generic MMP Assay Kit (Anaspec) according to the manufacturerâs instructions following 48 h of cell treatment with 100 µg/ml of KynA, Kyn, L-Kyn, or D-Kyn. The kit is designed to detect the activity of a variety of MMPs, including MMP-1, 2, 3, 7, 8, 9, 12, 13, and 14. 2.2.6 Collagen accumulation assay by Sirius Red staining To evaluate the effect of different tryptophan derivatives on soluble collagen level in the fibroblast conditioned medium cells were treated with increasing concentrations (50 and 150 µg/ml) of Kyn or KynA. Following 96 h of incubation, Sirius Red collagen detection kit (Chondrex, Inc.) was used to measure the amount of collagen in the cell culture medium, following the manufacturerâs instructions. Cells were harvested and counted after 96 h of incubation. The amount of soluble collagen was normalized based on the cell number. 2.2.7 Lasting effect study To determine the effect of a single treatment Kyn and KynA on MMP expression over time, fibroblasts were cultured and treated with 100 µg/ml of KynA or Kyn for 48 h. Cells were washed with phosphate buffered saline (PBS) and cultured in fresh medium without treatment. Cells were harvested immediately and 12, 24, and 48 hours after treatment removal, and the presence of MMP-1 was evaluated in cell lysate by Western blotting. 33 2.2.8 Cell proliferation assay To evaluate the effect of different tryptophan derivatives on cell proliferation rate, fibroblasts and keratinocytes were seeded on 6-well plates (30 Ã 103 and 20 Ã 103 cells/well, respectively) and treated with KynA or Kyn (100 µg/ml). Cells were harvested, and total number of the cells was counted after 36, 72, and 108 h of treatment. 2.2.9 Live/dead® viability/cytotoxicity assay Cells were plated in 6-well plates (250 Ã 103 cells/well), cultured, and treated with increasing concentrations (50 and 150 µg/ml) of Kyn or KynA. After 3 days of incubation cell viability was evaluated by flow cytometry using the live/dead® viability/cytotoxicity assay kit for mammalian cells (Invitrogen). In this assay ethidium homodimer (EthD-1), a red fluorescent nucleic acid dye, stains dead cells and those undergo apoptosis, whereas clacein AM is converted to a green fluorescent compound by active intracellular esterase in live cells. The percentage of dead cells plus cells with damaged (porous) cellular membrane was quantitatively evaluated based on the protocol suggested by the manufacturer (Molecular Probes, Invitrogen, Mississauga, ON). 2.2.10 In vitro wound healing scratch assay Fibroblasts and keratinocytes were seeded on 12-well plates. At 95% confluency a scratch wound was made across each well with a 200-µl pipette tip and the cells were washed with PBS. The cells were incubated in fresh medium containing either KynA or Kyn (100 µg/ml). Photographs were taken immediately and 12 and 24 h after treatment, and the total number of cells migrated into the 34 open wound area was counted manually. Cell migration in treated samples was evaluated by quantifying the total number of cells migrated to the open wound area and compared with those of control. 2.2.11 Hypertrophic scar animal model and treatments The fibrotic rabbit ear model established by Morris et al. (Morris et al., 1997) was used as the cutaneous scarring model. Briefly, four female New Zealand white rabbits were anesthetized and prepared for wounding. Four full thickness wounds (8 mm) were created down to the bare cartilage on the ventral surface of each ear. The wounds were covered for 3 days using a Tegaderm dressing (3M, St. Paul, MN). Treatments were started following the initiation of granulation tissue formation. Wounds were either left untreated to heal by secondary intention or were treated with cream alone or cream containing either KynA or Kyn (500 µg/ml). Treatments were applied once a day for 35 consecutive days. Animals were sacrificed on day 35 post-wounding and tissue was harvested for histology. 2.2.12 Tissue processing and determination of scar elevation index (SEI) and epidermal thickness index (ETI) Scars were harvested with a 0.5 cm margin of surrounding unwounded tissue and bisected through the maximum point of scar hypertrophy on visualization and palpation. Half of the samples were fixed in 10% buffered formalin solution and embedded in paraffin. Sections (5 mm) were rehydrated and subjected to haematoxylin-eosin (H&E) staining. 35 The SEI, ratio of the scarâs newly formed dermal area to the area of unwounded dermis, represents the degree of dermal hypertrophy of each scar. A SEI value greater than 1.0 depicts a raised or hypertrophic dermis. The ETI was used to express the degree of scar epidermal hypertrophy. The entire cross section of the scar, which corresponded to a total of five fields (400Ã), was measured for epidermal thickness. The five fields of uninjured skin from both sides of the scar were also evaluated to determine the epidermal thickness in normal skin. The ratio between the averaged epidermal height in scar tissue and the averaged epidermal height in the normal uninjured skin was measured to determine the ETI. The ETI value greater than 1.0 depicts epidermis hypertrophy. 2.2.13 Collagen staining and tissue cellularity In order to determine the amount of collagen deposition, paraffin embedded sections were stained for collagen with Massonâs Trichrome as described previously (Rahmani-Neishaboor et al., 2010). Keratin and muscle fibers are stained red, collagen fibers are stained blue, and cell cytoplasm and nuclei are stained light pink and dark brown, respectively. To evaluate the effect of topical KynA and Kyn on dermal cellularity, five randomly chosen fields of dermis were photographed under 200Ã magnification. Photos were subsequently coded and randomized; overall dermal cellularity was quantified by manually counting the number of nuclei per high-power field (hpf) by a blinded observer. The counts from the five fields were averaged and used for comparisons between wounds (n = 4 for each treatment or control group). 36 2.2.14 MMP-1, type-I collagen, and fibronectin expression in wounds Total RNA was extracted from half of the scar tissue using Trizol reagent according to the manufacturerâs instructions (Invitrogen). Following DNase treatment and cDNA synthesis expression of MMP-1, type-I collagen and fibronectin were evaluated in samples using Q-PCR as described above. The following primers were used for Q-PCR reactions: MMP-1 5â²- TCTGGCCACATCTGCCTAATGG-3â² and 5â²-AGGGAAGCCAAAGGAGCTGTG-3â², Col-1a1 5â²-TGTTCAGCTTTGTGGACCTCC-3â² and 5â²-TTCGCCTTCACTGTACCGGAC-3â², and fibronectin 5â²-AGCAGCTTTGTGGTCTCGTGG-3â² and 5â-TTCGGCCAGGAAGCAAGTCTG-3â² (Invitrogen). GAPDH was used as the reference gene. 2.2.15 Statistical analysis Data were expressed as Mean Â± standard error of the mean (SEM) of three or more independent observations. Statistical significance was calculated using a two-tailed unpaired Studentâs t-test or a one-way analysis of variance with post hoc test in case of multiple comparisons. P-values < 0.05 were considered statistically significant in this study. 2.3 Results The effect of tryptophan derivatives on the expression of different ECM components (type-I collagen and fibronectin) and key ECM degrading enzymes (MMP-1 and MMP-3) was evaluated using Q-PCR and Western blotting. Cells treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 µg/ml) of KynA, 37 Kyn, L- Kyn, or D-Kyn were harvested after either 24 or 48 h for mRNA and protein analysis, respectively. The result shown in Figure 2.1. panels A and B indicates a dose-dependent decrease in type-I collagen and fibronectin mRNA expression, respectively, in fibroblasts treated with increasing concentrations of KynA, Kyn, or L-Kyn relative to that of untreated control. Fibroblast treatment with KynA resulted in a significant decrease in type-I collagen and fibronectin mRNA expression at concentrations as low as 6.25 µg/ml, and this reduction remained significant up to 150 µg/ml. Kyn and L-Kyn with concentrations over 25 µg/ml significantly decreased the type-I collagen and fibronectin mRNA expression by fibroblasts. As is shown in this figure, KynA has the most profound inhibitory effect on fibronectin and type-I collagen mRNA expression among the tested kynurenines. The protein expression analysis similarly showed that KynA, Kyn, and L-Kyn suppress the production of type-I collagen and fibronectin in a concentration-dependent fashion (Figure 2.1. C). Figures 2.1. D and E depict the quantitative analysis of the data shown in Figures 2.1. C for type-I collagen and fibronectin protein expression, respectively. The difference between control and either KynA, Kyn, or L-Kyn treated fibroblast in fibronectin or type-I collagen expression was significant as low as 25 µg/ml and remained significant up to 150 µg/ml tested. KynA, Kyn, and L-Kyn demonstrate a comparable suppressive effect on type-I collagen and fibronectin protein expression. Moreover, the presented data generated by Q-PCR and Western blotting revealed that D-Kyn 38 does not have any significant inhibitory effect on type-I collagen or fibronectin expression at the mRNA or protein level. The effect of KynA and Kyn on the soluble collagen level was evaluated using a Sirius Red collagen detection kit. Sirius red dye which binds to the [Gly-X-Y]n helical structure on fibrillar collagen can detect collagen type I, II, III, and IV in cell-conditioned medium. Consistent with previous data generated by Q-PCR and Western blot analysis, KynA and Kyn significantly reduce the production of soluble collagen in treated fibroblasts compared with the control in a dose-dependent manner (Figure 2.2.). The results obtained from the Q-PCR analysis and Western blotting demonstrate that while KynA, Kyn, or L-Kyn treatment increases the MMP-1 mRNA and protein levels in a dose-dependent manner, D-Kyn does not have any noticeable stimulatory effect on MMP-1 expression by dermal fibroblasts (Figure 2.3. A and B, respectively). Figure 2.3. C depicts the quantitative analysis of the data shown in Figure 2.3. B for MMP-1 protein expression. KynA has the highest stimulatory effect on the MMP-1 expression as compared with other tryptophan metabolites examined. Also consistent with Q-PCR results, D-Kyn fails to significantly stimulate MMP-1 expression in dermal fibroblasts. Notably, in comparison with the results of MMP-1 mRNA and protein expression, the level of MMP-3 mRNA remained relatively unchanged (Figure 2.4. A). The Q-PCR data obtained for MMP-3 mRNA expression was confirmed using a second set of primers. However, the MMP-3 protein, released into treated fibroblast conditioned medium, significantly increased in a dose- 39 dependent manner (Figure 2.4. B). Figure 2.4. C depicts the quantitative analysis of the data shown in Figure 2.3. B for MMP-3 secretion. As it is shown in this figure, again KynA has the highest stimulatory effect on the MMP-3 secretion as compared with other tryptophan metabolites examined and D-Kyn fails to stimulate MMP-3 secretion by fibroblasts. 40 Figure 2.1. Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines. Type-I collagen and fibronectin expression at the mRNA and protein level in cultured fibroblasts treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 µg/ml) of KynA, Kyn, L-Kyn or D-Kyn. (A) and (B) Relative type-I collagen and fibronectin mRNA expression in treated fibroblasts, respectively. 41 GAPDH was used as the reference gene. (C) Evaluation of type-I collagen and fibronectin expression at the protein level using Western blotting. (D) and (E) The Mean ± SEM ratio of type-I collagen and fibronectin density to β-actin at the protein level, respectively. β-actin was used as protein loading control. Figure 2.2. Reduction of soluble collagen level in KynA and Kyn treated fibroblast-conditioned medium. To determine the effect of kynurenines on soluble collagen production by fibroblasts, cells were treated with KynA and Kyn (50 and 150 µg/ml). Following 96 h of incubation the amount of collagen in the cell culture medium was measured using a Sirius Red collagen detection kit. Results are expressed as the amount (µg) of soluble collagen per 105 cells (*P-value < 0.05 and **P-value < 0.01, n = 4). 42 Figure 2.3. Stimulatory effect of kynurenines on MMP-1 expression. (A) Dermal fibroblasts were treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 µg/ml) of KynA, Kyn, L-Kyn, or D-Kyn. Following 24 h of treatment, cells were collected and MMP-1 expression was determined by Q-PCR after RNA extraction and cDNA synthesis. (B) Evaluation of MMP-1 expression at the protein level by Western blotting after 48 h of treatment. (C) The Mean ± SEM ratio of MMP-1 to β-actin density at the protein level. β-actin and GAPDH were used as loading controls for Western blotting and Q-PCR, respectively. 43 Figure 2.4. Stimulatory effect of kynurenines on MMP-3 secretion by fibroblasts. (A) Evaluation of MMP-3 mRNA expression in fibroblasts treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 µg/ml) of KynA, Kyn, L-Kyn, or D-Kyn following 24 h of treatment. GAPDH was used as loading control for Q-PCR. (B) Evaluation of MMP-3 presence in the fibroblast conditioned medium using Western blotting after 48 h of treatment. (C) The Mean ± SEM ratio of MMP-3 density at the protein level. 44 Since the enzymatic activity of MMPs is of primary importance when investigating an antifibrogenic drug, a protease activity assay was performed using treated fibroblast-conditioned medium. Fibroblasts were first treated with different kynurenines (100 µg/ml) for 48 h prior to being subjected to the SensoLyte Plusâ¢ 520 generic MMP assay kit. Consistent with previous data generated by Western blot analysis, fibroblast treatment with Kyn, L-Kyn, or KynA significantly increased the MMP activity in comparison with cells treated with either D-Kyn or untreated cells (Figure 2.5. A). After considering the comparable effects of KynA, Kyn, and L-Kyn on ECM expression, KynA and Kyn were selected for further studies. To determine the lasting effect of KynA and Kyn on MMP-1 expression in fibroblasts, these cells were treated with 100 µg/ml of the drug. Following 48 h of treatment, the medium was changed and cells were harvested at 0, 12, 24, or 48 hours post-treatment removal. The result showed a marked increase in MMP-1 expression by fibroblasts in response to either KynA or Kyn treatment at 48 h after treatment. Following the removal of Kyn and KynA, the MMP-1 expression remained significantly higher than the untreated cells for another 24 h (Figure 2.5. B). Interestingly, while the MMP-1 protein expression gradually reduced to normal levels within 48 h after Kyn removal, the MMP-1 expression in response to KynA remained higher than controls (Figure 2.5. B). Figure 2.5. C represents the quantitative analysis of data in Figure 2.5. B. This finding shows that KynA has a longer lasting effect on expression of MMP-1 relative to Kyn in treated fibroblasts. 45 Figure 2.5. Stimulatory effect of kynurenines on MMP activity and kynureninesâ lasting effect on MMP-1 expression. (A) The effect of kynurenines on MMP activity. To determine MMP activity in the fibroblasts conditioned medium, cells were treated with 100 µg/ml of KynA, Kyn, L-Kyn, or D-Kyn for 48 h, and MMP activity was evaluated 46 using SensoLyteâ¢ Plus 520 generic MMP Assay Kit (**P-value < 0.001, n = 4). (B) Kynureninesâ lasting effect on the MMP-1 expression. To determine the lasting effect of kynurenines on MMP-1 expression, fibroblasts were treated with KynA or Kyn (100 µg/ml) for 48 h. The medium was replaced and cells were harvested immediately and 12, 24, and 48 h after treatment removal. The MMP-1 expression in dermal fibroblasts was evaluated using Western blotting. (C) MMP-1/β-actin expression ratio was calculated in treated fibroblasts. Data are Mean ± SEM of four independent experiments (*P-value < 0.05 and **P-value < 0.01, n = 4). To evaluate the effect of Kyn and KynA on dermal cell proliferation rate, cells were treated with Kyn and KynA (100 µg/ml) and the total cell number was counted after 36, 72, and 108 h of incubation. As shown in 2.6. A, Kyn and KynA treated fibroblasts demonstrate a significant reduction in the total cell number as compared with the cells cultured in DMEM+2% FBS at all tested time points. As it is shown in Figure 2.6. B, while the total number of the cells is comparable between KynA-treated keratinocytes (179.17 ± 16.84) and the control (208.68 ± 10.98), Kyn significantly reduces the total number of the keratinocytes (113.88 ± 7.19) at 108 h post-treatment. The effect of increasing concentrations of Kyn and KynA on dermal cell survival was determined by flow cytometry using a viability cytotoxicity assay in which live and dead cells are stained green and red, respectively. Fibroblasts and keratinocytes exposure to increasing concentrations (50 and 150 µg/ml) of kynurenines did not significantly increase the number of dead cells. This finding indicates that Kyn and KynA treatment does not compromise the viability of either keratinocytes or fibroblasts at the tested concentrations (Figure 2.6. C). 47 To examine the effect of Kyn and KynA on dermal cell migration, wound healing scratch assay was done on fibroblasts and keratinocytes treated with Kyn or KynA (100 µg/ml). Images of cells were taken at 0, 12, and 24 h post-treatment, and the total number of the cells migrated into the open scratch area was compared between the treated and untreated cells (Figure 2.7. A and B). As is shown in Figure 2.7. C, while Kyn-treated fibroblasts demonstrate comparable migration to the control, KynA significantly reduces the number of migrating fibroblasts into the wound area compared with the control at 12 (46.4 ± 14.58 vs. 78 ± 17.14) and 24 h (66.5 ± 11.67 vs. 116 ± 16.79) post-treatment. Interestingly, both Kyn and KynA significantly increased (488 ± 65.87 and 452 ± 73.52, respectively) the number of migratory keratinocytes into the wound area compared with the controls (358 ± 50.13) at 24 h after treatment (Figure 2.7. D). To validate the physiological antifibrogenic effects of topically applied KynA and Kyn, creams containing the compounds were applied on a fibrotic rabbit ear model. Four full thickness wounds were generated per ear, and 3 days later wounds received daily application of either cream only or cream containing KynA or Kyn (500 µg/ml). Wounds in the control group remained untreated and healed by secondary intention. Clinical and histological examination of wounds demonstrated a significant decrease in scar elevation in Kyn or KynA treated wounds compared with those of control and cream treated wounds (Figure 2.8. A). As is shown in Figure 2.8. B, dermal and epidermal layers are markedly thinner in wounds receiving topical application of either Kyn or KynA compared with the untreated wounds and wounds treated with cream only. 48 Figure 2.6. Effect of kynurenines on fibroblast and keratinocyte proliferation rate and viability. To determine the effect of different kynurenines on dermal cell proliferation rate, (A) fibroblast and (B) keratinocytes were treated with KynA or Kyn (100 µg/ml). Cells were harvested and total cell number was counted after 36, 72, and 108 h of incubation (*P-value < 0.05 and **P-value < 0.01, n = 6). (C) Determination of cellular viability by FACs analysis using live/dead® viability/cytotoxicity assay kit. Fibroblasts and keratinocytes were either cultured in DMEM+2% FBS or DMEM+2% FBS supplemented with increasing concentrations of KynA or Kyn (50 and 150 µg/ml). The viability of cells was determined by FACS analysis following 3 days of incubation. 49 Figure 2.7. Effect of kynurenines on fibroblast and keratinocyte migration. Images of human (A) fibroblasts and (B) keratinocytes taken immediately and 12 and 24 h after addition of KynA or Kyn (100 µg/ml) in an in vitro wound scratch assay. (C) Reduction of fibroblast migration in the presence of KynA after 12 and 24 h of treatment. (D) Enhancement of keratinocyte migration in the presence of KynA and Kyn. Cell migration was evaluated by manually counting the total number of cells migrated from the edges of the wound into the denuded area (*P-value < 0.05 and **P-value < 0.01, n = 4). 50 H&E stained tissue sections were examined to determine the SEI and ETI. The SEI was decreased significantly in wounds treated with topical Kyn (SEI of 1.15 ± 0.24) or KynA (SEI of 1.13 ± 0.13) in comparison with the wounds treated with cream only (SEI of 1.6 ± 0.13) or untreated control wounds (SEI of 1.61 ± 0.12). The SEI decrease corresponds to a reduction in scar hypertrophy of 28.56% and 29.75% in Kyn and KynA treated wounds, respectively (Figure 2.8. C). Epidermis is significantly thicker in untreated wounds (ETI of 1.57 ± 0.16) or wounds treated with cream only (ETI of 1.72 Â±.0.2) in comparison with uninjured skin. Epidermal hypertrophy decreased markedly in wounds that received Kyn (ETI of 1.08 ± 0.09) compared with the wounds treated with cream only or untreated control wounds (Figure 2.8. D). Topical KynA slightly reduced the ETI compared with the control untreated wounds, but this reduction did not reach the statistical significance (ETI of 1.32 ± 0.07). These scars exhibited a reduction in ETI of 31.30% or 16.19% when treated with Kyn or KynA cream, respectively. Collagen deposition was also evaluated using Massonâs Trichrome-staining in which collagen is stained blue and cell nuclei are stained dark brown (Figure 2.9. A). The effect of topical KynA and Kyn on dermal cellularity was evaluated by counting the number of nuclei per 20 of the high-power field from each group of wounds (Figure 2.9. B). The total cell count was the highest in untreated wounds and wounds treated with cream only with an average of 314.5 ± 8.66 and 362.34 ± 30.4 cells/hpf, respectively. Dermal cellularity was markedly reduced in wounds treated with Kyn or KynA, 237.38 ± 11.6 and 221.38 51 ± 21.7 cells/hpf, respectively, and corresponds to a respective 24.5% and 29.6% reduction in dermal cellularity (Figure 2.9. B). Modulation Figure 2.8. Clinical appearance and histological evaluation of wounds in rabbit ear model. (A) The clinical appearance of wounds that either received nothing (control), cream only (cream), or cream containing Kyn or KynA on day 35 post wounding. (B) Tissue samples were subjected to H&E staining to determine the dermal and epidermal hypertrophy. (C) Scar elevation index and (D) epidermal thickness index were evaluated quantitatively. Uninjured rabbit ear skin was used as the normal sample (*P-value < 0.05 and **P-value < 0.01, n = 4). A 52 of ECM expression in response to topical Kyn and KynA application was evaluated using Q-PCR. The result demonstrated that wound treatment with either KynA or Kyn leads to a significant increase in the expression of MMP-1 and decrease in the type-I collagen mRNA expression in comparison with untreated wounds or wounds treated with cream only (Figure 2.9. C and D, respectively). In general the Kyn and KynA have a comparable effect on type-I collagen and collagenase (MMP-1) expression in vivo. Topical application of KynA and Kyn markedly reduces fibronectin mRNA compared with wounds treated with cream only or control wounds (Figure 2.9. E). Also, as is shown in this figure, KynA is more effective in suppressing the fibronectin mRNA in comparison with Kyn (78% vs. 45% in fibronectin mRNA reduction relative to control wounds). 2.4 Discussion Fibrosis is a pathological scarring process associated with exaggerated ECM production, abnormalities in ECM degradation, and high tissue cellularity. Ideally, antifibrotic strategies aim to reduce the ECM accumulation via suppressing ECM biosynthesis or promoting matrix degradation. Our previous study regarding the antifibrotic effects of local IDO expression (Chavez-Munoz et al., 2012) led to identification of Kyn as an antiscarring agent (Y. Li et al., 2014). 53 Figure 2.9. Effect of Kyn and KynA topical application on collagen deposition, tissue cellularity, and ECM expression. (A) Evaluation of collagen deposition in tissue samples using Massonâs Trichrome staining at day 35 post wounding. In this staining collagen fibers are stained blue, keratin and muscle fibers are stained red, and cell 54 cytoplasm and nuclei are stained light pink and dark brown, respectively. (B) Quantification and statistical analysis of tissue cellularity. Q-PCR analysis of relative (C) MMP-1, (D) type-I collagen, and (E) fibronectin mRNA expression in tissue samples (*P-value < 0.05 and **P-value < 0.01, n = 4). Topical application of Kyn as an antifibrotic therapy is very attractive; however, the possible adverse effects of Kyn administration (Fukui et al., 1991; Nakagami et al., 1996; Okuda et al., 1998) prompted us to investigate comparable antifibrogenic compounds. KynA, the end product of Kynâs deamination via Kyn aminotransferase, was our candidate of choice considering its neuroprotective effects and relatively low toxicity. Moreover, the enantiomers L- and D-Kyn were investigated in order to identify the stereospecificity of the drug. We compared the biological activity of KynA and different Kyn enantiomers in terms of: 1) induction of ECM production and degradation, 2) the lasting effect of Kyn and KynA on MMP-1 expression in dermal fibroblasts, and 3) on the rate of dermal cell proliferation, viability, and migration. Moreover, we compared the antifibrotic effect of KynA to Kyn in a fibrotic rabbit ear model. Excessive collagen accumulation is one of the main features of HSCs. Local modulation of ECM expression has shown a promising impact on the treatment of fibrotic diseases (Chavez-Munoz et al., 2012; Iimuro et al., 2003; Rahmani-Neishaboor et al., 2010). Previous studies by our group (Ghahary et al., 1996; Ghahary, Shen, Scott, Gong, et al., 1993) demonstrated higher levels of collagen (type-I and âIII) expression in HSC samples. MMP-1, 8, and 13 are the only enzymes able to initiate the degradation of interstitial collagens, types-I, -II, and -III (Dasu, Hawkins, Barrow, Xue, & Herndon, 2004). Among these enzymes 55 MMP-1 is the only collagenase abundantly expressed by human fibroblast (Parks, 1999) during wound healing to remodel the ECM. In this study we showed that the tested kynurenines, with the exception of D-Kyn, not only suppress type-I collagen and fibronectin expression but also increase the rate of ECM degradation via increasing the expression and activity of MMP-1 (collagenase). In addition to MMP-1, scar formation is highly dependent upon lack of MMP-3 expression. Unlike MMP-1, MMP-3 has a wide array of ECM substrates such as fibronectin, proteoglycan, laminin, and types-IV, -IX, and -X collagen (Armstrong & Jude, 2002; Dasu et al., 2004). MMP-3 was also reported to activate other pro-metalloprotease including pro-MMP-1 (G. Murphy, Cockett, Stephens, Smith, & Docherty, 1987; Pupa, Menard, Forti, & Tagliabue, 2002). Li et al. (M. Li et al., 2009) demonstrated that variations in MMP-3 protein expression are mainly detectable in fibroblast-conditioned medium but not the cell lysate. Therefore, in this study fibroblast conditioned medium was used to evaluate the effect of kynurenines on MMP-3 expression. Although the level of MMP-3 protein was significantly increased in conditioned medium in response to kynurenines, the MMP-3 mRNA expression was not significantly affected by kynurenines. This finding suggests that kynurenines modulate MMP-3 expression at the post-translational level. Stereospecificity is fundamental to many biological reactions. A mounting body of evidence shows that chiral enantiomers differ significantly in biological activity, pharmacodynamics, pharmacokinetics, and toxicity. Comparison of the 56 effect of different Kyn isomers on ECM expression revealed that L-Kyn is the active enantiomer of the drug. Contrary to our expectation to observe higher ECM modulation in response to L-Kyn, fibroblasts treated with the same dose of L-Kyn and Kyn, which is a racemic 1:1 mixture of two enantiomers, demonstrated comparable ECM expression. It has previously been reported that in some cases the replacement of the racemic drug with the active enantiomer does not lead to the expected increase of drug potency (Borden, Chong, McLean, Slessor, & Mori, 1976; Raffa et al., 1993). This might be explained by direct pharmacodynamic or pharmacokinetic competition/interaction between two enantiomers. For instance, one of the isomers can provide specific protective effects for the potent enantiomer and facilitate its physiological effects. Further pharmacological studies are required to better understand the interaction between Kyn enantiomers. The presented data also revealed that KynA has comparable effects to Kyn in vitro regarding the modulation of ECM expression. Although Kyn and KynA have different molecular structures, they both bind to aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, which might explain their comparable effects on fibroblasts (DiNatale et al., 2010; Opitz et al., 2011). Several studies revealed the pivotal role of AHR signalling in ECM metabolism including stimulation of MMPs (MMP-1, MMP-9, and MMP-13) expression and suppression of collagen production (Andreasen et al., 2006; Ishida et al., 2010; Kung et al., 2009; K. A. Murphy, Villano, Dorn, & White, 2004). A study by Anguilera-Montilla et al. (Aguilera-Montilla et al., 2013) revealed the contribution of AHR in the MAPK/Erk kinase/extracellular-signal- 57 regulated kinase (MEK/ERK) signaling pathway. Also we have recently shown that Kyn induced MMPs expression is dependent on MEK-ERK1/2 mitogen-activated protein kinase (MAPK) signaling pathway activation (Y. Li et al., 2014). Direct comparison of the lasting effects of a single dose of either Kyn or KynA demonstrated that KynA administration outlasts Kyn in its antifibrogenic response. Therefore, less frequent application of KynA is required to get the same effect compared with Kyn, which can reduce the possible adverse effects of the medication. The added efficacious duration of KynA suggests a longer therapeutic half-life for KynA, which may translate to a more feasible dosing regime in the clinical setting. In vitro wound healing scratch assay showed enhanced keratinocyte migration in response to Kyn and KynA treatment. The enhanced keratinocyte migration may lead to accelerated wound re-epithelialization and reduced wound contraction and scar formation (Huang et al., 2002; Singer et al., 2009). In the current study we found that KynA and Kyn with concentrations over 25 µg/ml significantly increase the expression of MMPs and suppress the type-I collagen and fibronectin synthesis in vitro. Therefore, in order to test the antifibrotic effects of KynA and Kyn in a rabbit ear fibrotic model, wounds were daily treated with 50 µg of either Kyn or KynA/100 µl of cream per wound. Since kynurenines increase the expression of MMPs and suppress the expression of type-I collagen, application of the cream right after wounding may have compromised the formation of granulation tissue and impeded the early phases of wound healing. On the other hand, application of kynurenines at the latter 58 stages of wound healing might not be highly effective in prevention of scar formation. Therefore, in this study topical Kyn and KynA application started at day 3 post wounding in order to allocate enough time for granulation tissue formation and at the same time target the inflammation and proliferation phase of wound healing. Results from clinical and histological studies demonstrated that treated wounds are fully epithelialized and healed by day 35 post wounding. Kyn or KynA treated wounds did not show any significant delay in wound closure, indicating that the application of kynurenines at the mid-stages of wound healing does not delay the healing process. Topical Kyn and KynA application on the rabbit ear significantly improved the wound healing outcome through increasing the expression of MMP-1 and suppressing the expression of type-I collagen and fibronectin. Reduced collagen deposition was confirmed by down-regulation of type-I collagen and up-regulation of MMP-1 expression in KynA or Kyn treated wounds. Results from our in vivo experiments were further supported by our previous studies (Chavez-Munoz et al., 2012; Forouzandeh et al., 2010; Y. Li et al., 2006) showing that wound treatment with IDO-expressing skin substitute not only accelerates the wound healing process, as a skin substitute, but also decreases the inflammation and scar formation. In addition to evaluating the effect of KynA treatment on collagenase and collagen expression, it was found that KynA and Kyn suppress the expression of fibronectin in vitro and in vivo and, further, that KynA is more effective in reducing the fibronectin mRNA. Fibronectin is an abundant component of the provisional matrix that regulates different aspects of wound healing including cell attachment, migration, and differentiation, 59 matrix organization, and wound contraction (Welch, Odland, & Clark, 1990). Excessive fibronectin expression and accumulation are pathological features of fibrotic disorders (Cooper et al., 1979; Ghahary, Shen, Scott, & Tredget, 1993; Kischer & Hendrix, 1983; Kischer et al., 1989). By acting as a potent chemoattractant (Rennard, Hunninghake, Bitterman, & Crystal, 1981), fibronectin recruits fibroblasts into the wound bed and induces their differentiation into myofibroblasts (Kohan, Muro, White, & Berkman, 2010). Also, it has been shown that fibronectin promotes epithelial mesenchymal transition (EMT), a very important mechanism in fibrosis (Ding, Gelfenbeyn, Freire-de-Lima, Handa, & Hakomori, 2012; Freire-de-Lima et al., 2011). Inhibition of fibronectin accumulation has been used as an effective strategy to prevent renal fibrosis in vivo (McDonald et al., 2003). Another hallmark of fibrosis is high cellularity. Excessive fibroblast proliferation and abnormalities in myofibroblasts apoptosis can lead to excessive cellularity at the wound bed and scar formation (Tuan & Nichter, 1998). Several studies revealed that application of antifibrogenic agents (Shah, Foreman, & Ferguson, 1992; Younai et al., 1994) reduces fibroblast proliferation and reduces overall wound cellularity, thereby improving wound healing outcome. Our in vitro studies revealed Kyn and KynA effectively reduce the proliferation rate of the primary fibroblasts, without compromising cellular viability. The inhibitory effect of kynurenines on fibroblast proliferation was further confirmed by a significant reduction in tissue cellularity in response to KynA or Kyn treatment. In vitro wound healing assay also showed a significant reduction in fibroblast migration in 60 response to KynA treatment which may play a key role in tissue cellularity reduction in KynA treated wounds. In conclusion, the results of this study demonstrate the antifibrogenic efficacy of KynA, Kyn, and L-Kyn both in vitro and in vivo. For the first time, the stereospecific antifibrogenic effect of Kyn was also observed through independent assays of the L/D-enantiomers demonstrating that D-Kyn has no therapeutic effect. KynA and Kyn treatments enhance ECM remodeling via increasing the expression of ECM degrading enzymes (MMP-1 and MMP-3) and suppression of type-I collagen and fibronectin production. It was further concluded that Kyn and KynA administration in a topical cream at the mid-stage of wound healing offers a therapeutic strategy to reduce scarring in vivo, reducing SEI and ETI of healed wounds. Considering the possible adverse effects of Kyn, together with the prolonged therapeutic effects of a single KynA dose and the enhanced suppression of fibronectin, KynA has demonstrated, in this study, to be a suitable antifibrogenic candidate drug to improve healing outcome in patients that suffer from hypertrophic scarring and keloids. 61 3 Kynurenine modulates MMP-1 and type-I collagen expression via aryl hydrocarbon receptor activation in dermal fibroblasts 3.1 Introduction Fibrosis is a pathological condition characterized by high tissue cellularity, Excessive extracellular matrix (ECM) accumulation, ECM contracture, and distortion of normal tissue structure (Armour, Scott, & Tredget, 2007b; Kischer & Hendrix, 1983; Tredget, 1999). Skin fibrosis, such as post-burn and post-surgical hypertrophic scars (HSCs), has long lasting cosmetic, psychological, and functional consequences for the patients and immense economic burden on medical systems (Bock et al., 2006; Brown et al., 2008). The current treatment modalities for these devastating conditions are unsatisfactory, which raised a great need for innovation within the wound care industry. Recently, the antiscarring properties of kynurenine (Kyn), a tryptophan metabolite, were identified using the fibrotic rabbit ear model (Y. Li et al., 2014; Poormasjedi-Meibod, Hartwell, Kilani, & Ghahary, 2014). It was shown that the antifibrotic effect of Kyn is mediated through suppression of type-I collagen and fibronectin expression, enhancement of matrix metalloproteinases (MMP) production, and reduction of fibroblast proliferation and tissue cellularity. The aryl hydrocarbon receptor (AHR) is a cytoplasmic ligand-activated transcription factor, which is a member of the basic helix-loop-helix Per/AHR-ARNT/Sim family. Primary studies on the AHR signalling pathway focused on its activation by exogenous environmental contaminants, such as polyaromatic hydrocarbons (Mimura & Fujii-Kuriyama, 2003). However, the role of 62 endogenous AHR ligands, including tryptophan metabolites (Mezrich et al., 2010; Nguyen et al., 2010), is becoming more evident in regulating different physiological processes. Recent studies pointed to the role of AHR in regulation of ECM synthesis and degradation in different skin cells (Meng et al., 2009; K. A. Murphy et al., 2004; Villano, Murphy, Akintobi, & White, 2006). Therefore, the question examined here is whether AHR activation is involved in modulation of key ECM components by Kyn treatment in primary dermal fibroblasts. Daily topical administration of any anti-fibrogenic agent, including Kyn, is challenging in some patients, such as burn patients and those with split-skin donor sites, where the dressings are changed every 3 to 5 days and there will be no chance for daily application of the medication. To address this difficulty, the fabrication and functionality of a nanofibrous anti-fibrogenic dressing as a slow-releasing drug delivery system in an animal model were studied. To achieve this, Kyn was incorporated in nanofibers prepared by an electrospinning process. Following optimization of the drug release profile, the anti-fibrotic activity of the Kyn-loaded nanofibers was tested in vitro and in vivo. Findings showed that application of these medicated mats, extending the drug release up to 5 days, improved the wound healing outcome in a rat model by increasing the expression of MMP-1 and suppressing the production of type-I collagen and alpha smooth muscle actin (α-SMA). Furthermore, it was demonstrated that Kyn-dependent modulation of MMP-1 and type-I collagen is mediated through the AHR signalling pathway. 63 3.2 Materials and methods 3.2.1 Ethics statement Both Human and Animal Ethics Committees of the University of British Columbia approved all methods and procedures, as well as the use of animals and tissue specimens obtained from animals and humans. 3.2.2 Cell culture Primary human keratinocytes and fibroblasts were isolated and cultured from foreskin samples collected from healthy donors undergoing elective circumcision, using the protocol described elsewhere (M. Li et al., 2009). Keratinocytes and fibroblasts at passages 4â7 were used in all the experiments. 3.2.3 AHR immunocytochemistry and blockade of AHR activity Fibroblasts were plated on glass slides and allowed to adhere overnight. Cells were cultured in DMEM+2% fetal bovine serum (FBS) and treated with Kyn (50 µg/ml) for 30 min or 1 or 2 h. Cells maintained in DMEM+2% FBS were used as control. Subsequently, cells were washed with phosphate buffered saline (PBS), fixed using ice-cold methanol, and blocked with 5% normal goat serum. Slides were incubated overnight at 4°C with mouse anti-AHR antibody (1:50, Ab2769, Abcam, Cambridge, MA), washed and incubated with rhodamine goat anti-mouse IgG (1:2000, Chemicon International, Temecula, CA). Photographs were taken using a Zeiss LSM 510 confocal microscope. Fibroblasts were treated with Kyn (50 µg/ml) with or without increasing concentrations (1, 5, or 10 µM) of 6,2â²,4â²-trimethoxyflavone (TMF), AHR inhibitor, for either 6 h (messenger ribonucleic acid (mRNA) analysis) or 48 h (protein 64 analysis). Cytochrome-P450 (CYP1A-1) mRNA and MMP-1 and type-I collagen protein expression were evaluated in the cell pellet using quantitative polymerase chain reaction (Q-PCR) and Western blotting, respectively. Table 3.1 contains the list of the primers used in this study. Polyclonal rabbit-anti-MMP-1 Ab (1:2000, Abcam) and mouse-anti-Collagen-I (1:500, Developmental Studies Hybridoma Bank) were used in Western blotting. 3.2.4 Preparation of Kyn-containing electro-spun PVA fiber mats Polyvinyl alcohol (PVA, MW = 130,000 g/mol, Sigma-Aldrich, Ontario, Canada) was dissolved in distilled water at 10% w/w concentration at 80 °C for 8 h on a magnetic stirrer. Kyn (20 mg/ml, Sigma-Aldrich) was dissolved in sodium hydroxide (1 M, Fisher Scientific, Ottawa, ON), and pH was adjusted to 7 using concentrated hydrochloric acid (Fisher Scientific). The Kyn and PVA solutions were then mixed at a 1:7 ratio, yielding a final drug concentration of 2.5 mg/ml and PVA concentration of 8.75% w/w. The Kyn/PVA solution was electrospun under a voltage of 20 kV from an ES-30R power supply (Gamma High Voltage Research Inc.) and a distance of 15 cm. The PVA/Kyn nanofibers were collected on an aluminum-foil-covered copper plate with a surface area of 80 cm2, which enables deposition of medicated nanofibers with a Kyn concentration of 250 µg/cm2. To prevent immediate dissolution of Kyn-loaded PVA nanofibers in an aqueous environment, PVA nanofibers were heat treated either at 125 °C for 2 h or 80 °C for 30 min. Subsequently, a protective poly(lactic-co-glycolic acid) (PLGA 85:15, Sigma-Aldrich) shell was added to the PVA nanofibers, which were heat treated at 80 °C for 30 min, by dip-coating. For dip-coating, the medicated 65 PVA nanofibers, pre-cut to 1 cm Ã 1 cm squares, were submersed in a solution containing PLGA (20% w/w) in a 3:1 mix of tetrahydrofuran (THF) and dimethylformamide (DMF). After removal from the coating solution, the PVA fibers were dried under pressure applied by placing them between two steel plates. Upon drying, the coated mats were coated again in the PLGA solution for a second layer, followed by a second drying process without applied pressure. The PLGA-PVA composite was frozen overnight and lyophilized to remove the remaining organic solvent. The nanofiber morphologies were characterized using a Hitachi S-3000N scanning electron microscope with the software ImageJ for analysis. 3.2.5 In vitro drug release The medicated fibrous mats (1 cm2) were immersed in phosphate buffered saline solution (PBS, pH 7.4) in a 6-well plate at 37 °C. The solution was collected and replaced after 1, 2, 4, 8, 24, 48, 72, and 96 h of incubation. Kyn level was evaluated using the Ehrlichâs assay as described previously (Jalili, Rayat, Rajotte, & Ghahary, 2007). In brief, 0.5 mL of nanofiber incubated PBS was mixed with the Ehrlich reagent. Following 10 minutes of incubation at room temperature the absorption was measured at 490 nm by spectrophotometer. Using the results from the spectrometric analysis, cumulative release is determined and plotted against release time. 3.2.6 In vitro cytocompatibility assay The cytocompatibility of PVA/PLGA nanofiber mats (NF) and Kyn-incorporated PVA/PLGA nanofiber mats (Kyn+NF) was evaluated using MTT (3- 66 (4,5-dimethylthiazol-2-yl)_2,5-diphenyl tetrazolium bromide, Sigma-Aldrich) assay and live/dead® viability/cytotoxicity assay. Cell cultures were set up using a two-chamber cell culture system (6-well plates, Corning Incorporated, Corning, NY; cell culture inserts, Millicell, Millipore Corporation, MA) in which fibroblasts or keratinocytes (250 Ã 103) were cultured in the lower chambers while NF or NF+Kyn were placed in the upper chamber. Following 3 days of incubation, cells were subjected to MTT assay and flow cytometry using a live/dead® viability/cytotoxicity assay kit (Life Technologies-Invitrogen, Eugene, OR) as described elsewhere (Poormasjedi-Meibod et al., 2014). 3.2.7 Effects of Kyn on expression of MMP-1 and type-I collagen in fibroblasts Fibroblasts were cultured in a two-chamber cell culture system and treated as described above. Fibroblasts were treated with Kyn (50 µg/ml) as the positive control. After 48 h of treatment, cells were harvested and the expression levels of type-I collagen, MMP-1, and α-SMA were evaluated using western blotting. Mouse-anti-Collagen-I (1:500, Developmental Studies Hybridoma Bank), rabbit-anti-MMP-1 Ab (1:2000, Abcam, Cambridge, MA) and rabbit-anti-α-SMA (1:1000, #Ab32575, Abcam) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ab (1:3000 dilution, Bio-Rad) and HRP-conjugated goat anti-mouse Ab (1:3000 dilution, Bio-Rad) were used as the secondary antibodies. Beta-actin (β-actin) was used as the protein loading control. 67 3.2.8 Wound creation and treatment scheme Four full thickness wounds (8 mm in diameter) were created on the dorsal surface of male Long Evans rats (The Jackson Laboratories, Bar Harbor, ME) following hair removal. A donut-shaped silicone splint (Grace Bio-Labs, Bend, OR, #476684) was placed on top of the wound using an immediate bonding adhesive (Krazy Glue; Elmerâs Inc., Columbus, OH) to inhibit the wound contraction. The splint was matched with the edge of the wounds with the wounds centered within the splint. In each set of experiments, the order of treatment was changed to reduce the chance of bias in wound healing based on the position of wounds on the animalâs back. Tegadermâ¢ dressings (3M, St. Paul, MN) were used to cover the wounds for the first 2 days following the surgery. On post surgical day 3, wounds were treated with Kyn-containing cream (500 µg/ml) or covered by NF or NF+Kyn. Control wounds were left untreated to heal by secondary intention. Dressings were soaked in PBS, placed over the wounds, and covered with Tegadermâ¢. Dressings were changed every 4 days while cream was applied daily. 3.2.9 Histological analyses and immuno-staining Animals were killed on day 15 post-wounding and scars were harvested with a 0.5 cm margin of the surrounding unwounded tissue. Half of the samples were fixed in 10% neutral buffered formaldehyde, dehydrated, and embedded in paraffin. Sections (5 µm) were rehydrated and subjected to haematoxylin-eosin (H&E), CD3, and α-SMA staining using rabbit anti-CD3 antibody (1:100; Abcam, Cambridge, MA) and rabbit-anti-α-SMA (1:500, Abcam). For each wound five 68 randomly selected high-power fields (HPF, 400Ã magnification) were photographed using a Zeiss fluorescent microscope. Photographs were subsequently coded, and the number of CD3+ T-cells and α-SMA+ myofibroblasts were counted manually. The average number of these five HPFs was reported as the cell number/HPF. 3.2.10 MMP-1, type-I collagen, and α-SMA expression in the wounds Half of the scar was used for total RNA extraction using Trizol reagent according to the manufacturerâs instructions (Invitrogen). Following DNase treatment, total RNA (5 µg) was reverse transcribed into cDNA using a Superscript II First Strand cDNA Synthesis kit (Invitrogen). Q-PCR was performed to evaluate the expression of MMP-1, type-I collagen, and α-SMA in tissue samples. Table 3.1 contains the list of the primers used in this study. Table 3.1: List of primers used in this study. Rat-MMP-1 forward primer 5â²-TTGTTGCTGCCCATGAGCTT-3â² Rat-MMP-1 reverse primer 5â²-ACTTTGTCGCCAATTCCAGG-3â² Rat Col-1α1 forward primer 5â²-CAAGAATGGCGACCGTGGT-3â² Rat Col-1α1 reverse primer 5â²-GGTGTGACTCGTGGAGCCA-3â² Rat α-SMA forward primer 5â²-ACTGGGACGACATGGAAAAG-3â² Rat α-SMA reverse primer 5â²-CATCTCCAGAGTCCAGCAGA-3â² Rat GAPDH forward primer 5â²-CTTCCACGATGCCAAAGTTG-3â² Rat GAPDH reverse primer 5â²-GATGGTGAAGGTCGGTGTG-3â² Human CYP1A-1 forward primer 5â²-CAAGAGGAGCTAGACACAGTGATT-3â² Human CYP1A-1 reverse primer 5â²-AGCCTTTCAAACTTGTGTCTCTTGT-3â² Human GAPDH forward primer 5â²-GACAAGCTTCCCGTTCTCAG-3â² Human GAPDH reverse primer 5â²-CAATGACCCCTTCATTGACC-3â² Note: GAPDH = glyceraldehyde-3-phosphate dehydrogenase. 3.2.11 Statistics Data were expressed as Mean ± SEM of three or more independent observations. Statistical significance was calculated using a two-tailed unpaired 69 Studentâs t-test or a one-way analysis of variance with post hoc test in case of multiple comparisons. P-values < 0.05 were considered statistically significant in this study. 3.3 Results 3.3.1 Kyn modulates the expression of MMP-1 and type-I collagen via AHR activation in dermal fibroblasts To determine whether Kyn treatment activates AHR in dermal fibroblasts, immunocytochemistry was used to evaluate the changes in cellular compartmentalization of the AHR after Kyn treatment. Control, untreated fibroblasts, showed intense AHR staining predominantly in cell cytoplasm. Fibroblasts treated with Kyn showed a dramatic translocation of AHR to the nucleus within 30 min of treatment, which coincided with a parallel reduction in the cytoplasmic AHR staining. The AHR nuclear translocation was reversed after 1 h of treatment and the receptor was mainly detected in the fibroblast cytoplasm (Figure 3.1. A). CYP1A-1 expression was then examined to confirm the effect of Kyn on AHR activation. CYP1A-1 expression increased 3-fold in response to Kyn treatment (Figure 3.1. B). In order to confirm that Kyn-dependent induction of CYP1A-1 required the AHR activation, fibroblasts were co-treated with Kyn and a specific AHR antagonist, 6,2â²,4â²-trimethoxyflavone (TMF) (I. A. Murray et al., 2010). Co-treatment with TMF attenuated the Kyn-induced CYP1A-1 expression in a dose-dependent manner (Figure 3.1. C). To assess the role of AHR signaling in the Kyn-induced MMP-1 expression and suppression of type-I collagen expression, Kyn-treated 70 fibroblasts were incubated with increasing concentrations of TMF. Kyn-induced MMP-1 up-regulation and type-I collagen suppression were attenuated in response to increasing concentrations of TMF in a dose-dependent manner (Figure 3.2. A). These results suggest that AHR is required for Kyn-induced modulation of MMP-1 and type-I collagen expression in dermal fibroblasts. Figures 3.2. B and C depict the quantitative analysis of the data shown in Figure 3.2. A for MMP-1 and type-I collagen protein expression, respectively. 71 Figure 3.1. Kyn activates AHR. (A) Immunocytochemistry (ICC) for AHR in dermal fibroblasts treated with 50 µg/ml Kyn. (B) Q-PCR analysis of relative CYP1A-1 mRNA expression in Kyn-treated fibroblasts. (C) TMF antagonizes Kyn-dependent AHR-mediated CYP1A-1 expression. Dermal fibroblasts were treated with vehicle dimethyl sulfoxide (DMSO), Kyn (50 µg/ml), or Kyn in conjunction with increasing concentrations of TMF for 6 h. CYP1A-1 mRNA expression was evaluated using Q-PCR (*P-value < 0.05 and **P-value < 0.01, n = 4). 72 3.3.2 Characterization of nanofibers and in vitro drug release To generate a novel slow-releasing anti-fibrogenic dressing, a series of experiments on fabrication of a suitable nanofibers dressing were conducted. The electrospinning process yielded uniform polyvinyl alcohol (PVA) nanofibers with an average diameter of 309 ± 19 nm. The scanning electron microscope images of the Kyn-loaded as-electrospun PVA nanofibers and heat cross-linked nanofibers are shown in Figure 3.3. A and B, respectively. Scanning electron microscope images revealed weld formation among nanofibers at their junction points following heat treatment and increased fiber diameter (366 ± 57 nm) due to increased fiber fusion. Heat-treated PVA nanofibrous mats were dip-coated into polylactic-glycolic acid (PLGA) solution to protect the PVA nanofibers from immediate dissolution. After addition of two PLGA coats, scanning electron microscope imaging at 10,000Ã magnification showed a film-like structure with no identifiable pores on the surface (Figure 3.3. C). Drug release profiles for PVA/Kyn nanofibers before and after post-spinning modifications are shown in Figure 3.3. D. As expected from its high water solubility, Kyn-loaded PVA nanofibers without modifications disintegrated within 15 min after submersion in PBS solution, with all Kyn released. Nanofibers heat-treatment significantly reduced the solubility of the PVA mats in water, which is complimented by the lack of the immediate and complete Kyn release. However, there was no significant drug release after 4 h, and the cumulative release remained below 40% of the total amount loaded. 73 Figure 3.2. TMF antagonizes Kyn-dependent AHR-mediated MMP-1 up-regulation and type-I collagen down-regulation. Dermal fibroblasts were treated with vehicle (DMSO), Kyn (50 µg/ml), or Kyn in conjunction with increasing concentrations of TMF for 48 h. (A) MMP-1 and type-I collagen expression were evaluated using Western blotting. The Mean ± SEM ratio of (B) MMP-1 and (C) type-I collagen density to β-actin at the protein level, respectively. β-actin was used as protein loading control (*P-value < 0.05 and **P-value < 0.01, n = 4). 74 Addition of a PLGA shell around the PVA/Kyn nanofibers effectively reduced the burst release, with a 4-h release of 14.8% of the loaded drug, compared with the as-electrospun PVA nanofibers and generated the most linear release profile over the first 96 h. In addition, the cumulative release at 96 h was 85.7% for the dip-coated sample, indicating that release could continue beyond the 96 h study period. 3.3.3 The Kyn-loaded mats are cytocompatible To examine the effect of Kyn-impregnated nanofibers on cell proliferation and viability, fibroblasts and keratinocytes, treated with NF or NF+Kyn, were subjected to MTT and live/dead® viability/cytotoxicity assay. NF+Kyn significantly reduced the fibroblast proliferation as compared with the untreated cells or NF-treated fibroblasts (Figure 3.4. A). Interestingly, under similar conditions, NF and NF+Kyn significantly increased keratinocyte proliferation (Figure 3.4. B). As shown in Figure 3.4. C, fibroblasts and keratinocyte exposure to either NF or NF+Kyn did not significantly increase the percentage of dead cells in addition to cells with damaged membrane undergoing apoptosis as compared with the control. Figure 3.4. D and E depict the quantitative analysis of the data shown in Figure 3.4. C for fibroblast and keratinocyte viability, respectively. 3.3.4 Nanofiber-released Kyn modulates the expression of different ECM components in vitro To evaluate the functionality of the Kyn-impregnated mats, fibroblasts were incubated with NF or NF+Kyn. As shown in Figure 3.5. A, NF+Kyn-incubated medium, for 48 h, contained 54.2 ± 3.28 µg/ml of Kyn, which according 75 to previous studies [14, 15] was a proper dose to trigger a significant cell response. NF+Kyn-treated fibroblasts demonstrated a marked reduction in type-I collagen and α-SMA expression and a significant increase in MMP-1 expression in comparison with NF-treated fibroblasts or the control (Figure 3.5. B). NF-treated fibroblasts demonstrated comparable levels of type-I collagen, α-SMA, and MMP-1 expression to the controls, implying that the PVA/PLGA nanofibers did not modulate the level of ECM expression (Figure 3.5. B). Figures 3.5. C, D, and E depict the quantitative analysis of the data shown in Figure 3.5. B for type-I collagen, α-SMA, and MMP-1 protein expression, respectively. 3.3.5 Kyn-loaded dressings improved wound healing outcome To evaluate the anti-fibrotic effect of Kyn-impregnated mats in vivo, four full thickness wounds were generated on the backs of rats. On day 3 post surgery, wounds were covered with either NF or NF+Kyn dressings, changed every 4 days, or received daily Kyn cream. Wounds in the control group remained untreated and healed by secondary intention. To evaluate the effect of NF+Kyn on tissue cellularity and infiltrated immune cells, skin sections were subjected to H&E (Figure 3.6. A) and cluster of differentiation 3 (CD3) staining (Figure 3.6. B). The result showed a markedly lower number of CD3+ immune cells in NF+Kyn and Kyn cream treated wounds relative to those of untreated control wounds or wounds treated with NF only (Figure 3.6. B). 76 Figure 3.3. Scanning electron microscope photographs of electrospun nanofibers and Kyn release profiles before and after post-spinning modifications. (A) As-electrospun Kyn/PVA nanofibers prior to modifications. (B) Physically cross-linked Kyn/PVA nanofibers after heat treatment at 125 °C. (C) PLGA film shell obtained by dip-coating the Kyn-loaded PVA nanofibers in the PLGA solution. (D) Drug release profiles of PVA/Kyn nanofibers before and after post-spinning modifications. 77 Figure 3.4. Effect of nanofiber released Kyn on fibroblast and keratinocyte proliferation and viability. To determine the effect of nanofiber-released Kyn on dermal cell proliferation rate and viability, fibroblast and keratinocytes were cultured in two chamber plates with NF or NF+Kyn were placed in the upper chamber. Following 3 days of incubation cells were subjected to (A and B) MTT assay and (C) flow cytometry using live/dead® viability/cytotoxicity assay kit (*P-value < 0.05, n = 4). 78 Figure 3.5. Nanofiber released Kyn modulates the expression of different ECM components. (A) Nanofiber released Kyn level in medium was measured using Ehrlich assay. (B) Evaluation of type-I collagen, α-SMA, and MMP-1 expression at the protein level using Western blotting. The Mean ± SEM ratio of (C) type-I collagen, (D) α-SMA, and (E) MMP-1 density to β-actin at the protein level, respectively. β-actin was used as protein loading control (*P-value < 0.05 and **P-value < 0.01, n = 4). 79 Statistical analysis of infiltrating T-cells in the dermal layer demonstrated that NF+Kyn and topical Kyn cream significantly reduced the number of CD3+ T cells (15.72 ± 6.4 and 19.96 ± 4.08 cells/HPF, respectively, Figure 3.6. C) in comparison to the control (60±5.14 cells/HPF) and NF-treated wounds (60 ± 9.06 cells/HPF). Furthermore, dermal cellularity was significantly decreased in wounds treated with NF+Kyn (278.86 ± 22.49 cell/HPF) or Kyn cream (314.9 ± 23.86 cell/HPF) as compared with the control (485 ± 20.55 cells/HPF) or NF-treated wounds (466.63 ± 37.13 cells/HPF, Figure 3.6. D). 3.3.6 Kyn suppresses α-SMA expression and myofibroblasts To evaluate the effect of the NF+Kyn application on the number of myofibroblasts in vivo, skin sections were subjected to α-SMA staining. NF+Kyn treated wounds demonstrated significantly fewer α-SMA+ expressing cells, which is comparable with the wounds treated with Kyn cream (Figure 3.7. A). Untreated control wounds and NF-treated wounds showed higher numbers of α-SMA+ myofibroblasts that were randomly distributed in the dermis, while myofibroblasts were mainly localized at the skin appendages in normal skin (Figure 3.7. A). Statistical analysis of the number of the myofibroblasts in the dermal layer (Figure 3.7. B) revealed that NF+Kyn and Kyn cream treated wounds demonstrated a significant reduction in the number of α-SMA+ myofibroblasts (64.12 ± 25.48 and 60.58 ± 13.83 cells/HPF, respectively) in comparison with the NF-treated or control untreated wounds (207.5 ± 9.47 and 193.13 ± 12.94 cell/HPFs, respectively). 80 Figure 3.6. Effect of Kyn-incorporated dressings on tissue cellularity and CD3+ inflammatory cells. Wounds were either left untreated to heal by secondary intension or treated with NF, NF+Kyn, or Kyn cream. At day 15 post surgery animals were killed and skin samples were subjected to (A) H&E staining and (B) CD3 immuno-fluorescence staining. The arrows indicate the wound margin. The number of nuclei and CD3+ T-cells per high-power field was counted for 25 fields from each group of wounds. Panels C and D demonstrate the quantification and statistical analysis of CD3+ T-cells and tissue cellularity, respectively. Graphs show manual counting scores ± SEM per high-power field (HPF, 400x, *P-value < 0.05 and **P-value < 0.01, n = 5). 81 Q-PCR analysis confirmed the reduction of α-SMA expression in response to Kyn, delivered by either nanofiber mats or cream, compared with the control wounds (Figure 3.7. C). Also, as expected, wound treatment with either NF+Kyn or Kyn cream significantly increased MMP-1 expression and suppressed type-I collagen expression in comparison with NF-treated or control wounds (Figure 3.7. D and E, respectively). In general, the NF+Kyn and topical Kyn application showed comparable effects on reduction of α-SMA+ myofibroblasts population and modulation of ECM components, MMP-1, type-I collagen, and α-SMA, expression in vivo. 3.4 Discussion Previous studies on the AHR signalling pathway focused on its role in the induction of phase-I and -II drug metabolizing enzymes, such as the cytochrome P450 enzymes (Nebert et al., 2004). However, the creation of AHR null mice indicated the pivotal role of this receptor in multiple aspects of growth, development, and differentiation via modulating the expression of cytokines, transcription factors, ECM components, and regulators of ECM metabolism and cell adhesion (Gonzalez & Fernandez-Salguero, 1998; Hillegass et al., 2006; Puga et al., 2002). These studies expanded the AHR ligand inventory from exogenous environmental contaminants (Mimura & Fujii-Kuriyama, 2003) to a diverse variety of naturally occurring dietary molecules (Ashida et al., 2008; Ciolino et al., 1998) and endogenous AHR ligands including tryptophan metabolites such as Kyn (Mezrich et al., 2010; Nguyen et al., 2010). 82 Figure 3.7. Effect of Kyn-PVA/PLGA dressings on the α-SMA+ myofibroblasts and different ECM components expression at day 15 post-wounding. (A) Skin samples were subjected to immuno-fluorescence staining for α-SMA. (B) Quantification and statistical analysis of α-SMA+ myofibroblasts. Graphs show manual counting scores ± SEM per high-power field (HPF, 400Ã). Q-PCR analysis of relative (C) α-SMA, (D) MMP-1, (E) type-I collagen mRNA expression in tissue samples (**P-value < 0.01, n = 5). 83 AHR-dependent down-regulation of collagen expression and up-regulation of MMPs in response to 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and benzo(a)pyrene (BaP) (Meng et al., 2009; Villano et al., 2006) have been reported in different skin cells, including melanocytes, keratinocytes, and fibroblasts (K. A. Murphy et al., 2004; Ono et al., 2013; Villano et al., 2006). Kyn, TCDD, and BaP are all AHR ligands; therefore, here the question asked was whether AHR is involved in Kyn-induced MMP-1 up-regulation and type-I collagen suppression. Kyn-dependent AHR nuclear translocation and subsequent CYP1A-1 induction in dermal fibroblasts indicated the presence of a functional AHR pathway in these cells (Figure 3.1.). Our results also showed that Kyn-dependent modulation of MMP-1 and type-I collagen expression is abolished in response to TMF, confirming the involvement of AHR signalling pathway. AHR regulates the expression of its target genes via different mechanisms including (1) direct binding of ligand-activated AHR to the dioxin-responsive element in the gene promoter and (2) interaction with a variety of other signalling pathways (Beischlag et al., 2008; Haarmann-Stemmann et al., 2009; Puga et al., 2009). Earlier studies pointed to the AHR-dependent extracellular-signal-regulated kinase (ERK) ERK1/2 signalling pathway activation in response to TCDD (H. Yu et al., 2014). Moreover, the role of the ERK1/2 signalling pathway in the MMP-1 up-regulation (Brauchle, Gluck, Di Padova, Han, & Gram, 2000; Chaudhary & Avioli, 2000; Cortez et al., 2007) and type-I collagen down-regulation has been studied extensively. In fact, in our previous study (Y. Li et al., 2014), the role of the ERK1/2 signalling pathway in Kyn-dependent MMP-1 expression was 84 reported. Taking these pieces of evidence together, it is possible that Kyn interaction with AHR causes ERK1/2 activation, resulting in subsequent MMP-1 up-regulation and type-I collagen suppression. The scar-reducing effect of a topical Kyn cream in a rabbit ear model has been demonstrated previously (Y. Li et al., 2014; Poormasjedi-Meibod et al., 2014). Although daily application of this cream improved the outcome of wound healing, this method of application is not clinically feasible for large wounds where dressings need to be kept in place for 3â5 days. Frequent dressing changes not only disturb the healing process, extend the healing time, and increase the risk of wound infection (Church, Elsayed, Reid, Winston, & Lindsay, 2006) but also are an economic burden on patients and health care systems. Furthermore, dressing removal is reported to be the time of the greatest perceived pain in burn patients (Atchison, Osgood, Carr, & Szyfelbein, 1991; Geisser, Bingham, & Robinson, 1995; Weinberg et al., 2000). This undertreated pain can reduce the patient compliance and enhance the risk of post-traumatic stress disorders. Therefore, in this study nanofibrous dressings were developed as a slow-releasing drug delivery system for Kyn, which would reduce the need for frequent dressing changes. The electrospinning process was used to incorporate Kyn within PVA nanofibers with post-spinning modifications to control and prolong the Kyn release. High surface area to volume ratio of the nanofibers made it possible to deliver uniform, high, controlled doses of Kyn to the wound bed over the course of 4 days. Electrospinning PVA nanofibers as well as incorporating therapeutics into PVA matrices have been previously demonstrated 85 (Hadipour-Goudarzi, Montazer, Latifi, & Aghaji, 2014; Santos et al., 2014). Unlike in previous research, the primary challenge in this study was to control the release of a small hydrophilic drug over a minimum of 4 days. Reduction of the burst release is desirable for anti-fibrotic drugs in order to prevent the deleterious effects on the newly regenerated ECM and also warrant a prolonged efficacy during the remodeling phase. Without the flexibility of the material choice, post-spinning modifications become more important to tailor the release profile. While as-spun PVA nanofibers dissolved in the PBS and released 100% of the loaded Kyn in the first 15 min, PVA fibers heat-treatment resulted in a less mobile fiber network, lower rate of PVA dissolution in PBS, and prolonged Kyn release (up to 4 h). Addition of the PLGA shell to the PVA nanofiber hindered the PBS penetration into the drug-loaded layer and suppressed the Kyn burst release. Comparing the two post-spinning modification techniques showed that dip-coating yielded the most desirable release profile with the greatest long-term release; therefore, this formulation was selected as the lead formulation for further studies. Excessive ECM accumulation, including exaggerated collagen deposition and impairment of ECM breakdown, is a hallmark of fibrotic conditions (Arakawa et al., 1996; Ghahary, Shen, Scott, Gong, et al., 1993; L. Q. Zhang et al., 1994). It was previously shown that Kyn improves scar formation by decreasing type-I collagen and fibronectin expression and increasing the production of MMPs (Y. Li et al., 2014; Poormasjedi-Meibod et al., 2014). The presented in vitro and in vivo experiment in this study further validated that Kyn incorporated into nanofibers 86 has the same efficacy as free-Kyn (added directly to the cell culture medium or delivered by daily Kyn cream application). This finding confirms the preservation of the drugâs biological activity during the electrospinning process. In addition to confirming previous findings (Y. Li et al., 2014; Poormasjedi-Meibod et al., 2014) regarding the Kyn effect on key ECM components, the present study found that Kyn also suppresses the expression of α-SMA in vitro and in vivo and reduces the number of α-SMA+ myofibroblasts in the wound. Myofibroblasts, which possess the morphological and biochemical characteristics of both fibroblasts and smooth muscle cells, play a pivotal role in the wound healing process via deposition, remodeling, and contraction of the ECM (Gabbiani, 1981, 2003). These cells that express α-SMA in their stress fibers contract the deposited ECM, which is integral to normal wound closure. In the normal wound healing process, myofibroblasts disappear via apoptosis following the completion of re-epithelialization (Desmouliere, Badid, Bochaton-Piallat, & Gabbiani, 1997; Greenhalgh, 1998). However, the persistent presence of myofibroblasts in fibrosis leads to excessive ECM deposition, scar contracture, high tissue cellularity, and subsequent poor cosmetic outcome and loss of organ function (Desmouliere, Darby, & Gabbiani, 2003; Gabbiani, 1981; Moulin et al., 2004). In addition to being the key source of ECM expression, myofibroblasts are a significant source of other inflammatory and fibrogenic cytokines such as TGF-β1, CC chemokines, and monocyte chemotactic mediator-1 (Andoh et al., 2000; Phan, Zhang, Zhang, & Gharaee-Kermani, 1999). Through the expression of these cytokines, myofibroblasts potentially enhance the recruitment of 87 inflammatory cells and prolong inflammation. In addition, in this study lower number of CD3+ T-cells was found in Kyn-treated wounds, which may be caused by either direct anti-inflammatory effects of Kyn (Elizei, Poormasjedi-Meibod, Li, Jalili, & Ghahary, 2014; Fallarino et al., 2003) or indirectly through reducing the number of myofibroblasts. Myofibroblasts are reported to be present in most fibrotic pathological condition and in wounds often contribute further to contracture (Desmouliere et al., 2003; Gabbiani, 1981, 2003; Phan, 1996; H. Y. Zhang & Phan, 1999). The absence of myofibroblasts in the fetal scarless wound healing and the presence of different therapeutics that improve wound healing outcomes via inducing the myofibroblastsâ disappearance (Burgess et al., 2005; Cass, Sylvester, Yang, Crombleholme, & Adzick, 1997) indicate the role of these cells in scar formation and fibrosis. Considering the inhibitory effects of Kyn on α-SMA expression and myofibroblasts differentiation in wounds, I anticipate to see improvement in other fibrotic conditions upon Kyn application. In conclusion, the presented results showed that Kyn-dependent modulation of ECM expression is mediated through the AHR signalling pathway. Also, a slow-releasing drug delivery system was fabricated that can perform as an antifibrotic wound dressing in vivo. Moving toward a clinical application, these dressings could be used as a sustained drug delivery system to improve the outcome of wound healing in burn patients. 88 4 Development and application of anti-scarring nano-fibrous wound dressings to prevent the emergence of skin fibrosis 4.1 Introduction An ideal wound dressing should absorb the excess wound exudate, maintain a moist wound environment, minimize infection, and allow gas exchange. In addition, wound dressings must be easy to apply and remove to produce pain-free dressing changes and improve patient compliance (Benbow, 2010; Meaume, Teot, Lazareth, Martini, & Bohbot, 2004; Selig et al., 2012). In recent years, the role of wound dressings have evolved from plainly protecting the wound from infections and balancing the wound moisture to also acting as drug delivery systems to modulate the wound healing outcome (Boateng et al., 2008; G. H. Kim et al., 2011). Recently, electrospun polymeric nanofibers have become popular in wound healing applications (Choi et al., 2015; Ignatova et al., 2013; Thakur et al., 2008). When spun as overlaid mats, nano-fibrous wound dressings potentially offer several advantages over conventional dressings because of their inherent properties. Such substrates have a high void volume to accommodate a significant amount of exudate. While increasing the breathability of the dressing, the small pore size of the electrospun mats protects the wound from infection. Electrospun nanofibers have large surface area to volume ratio, which improves the drug loading capacity and mass transfer properties of the medicated mats. In addition electrospun dressings can provide a controlled drug release profile ranging from minutes to months depending on the application, 89 design, and material system (Ignatova et al., 2013; Meinel, Germershaus, Luhmann, Merkle, & Meinel, 2012; Thakur et al., 2008). For wound healing applications, drug loading studies have mainly targeted the early stages of the healing process in order to prevent/control infection, ease the pain, suppress excessive inflammation, and promote the wound healing process (Kong & Jang, 2008; Price et al., 2007; Thakur et al., 2008). Although the promotion of healing in patients is clearly desirable, its timely cessation is equally important. Prolonged âearly-stageâ healing events can lead to excess extracellular matrix (ECM) accumulation and emergence of deleterious fibrotic conditions such as post-burn hypertrophic scars (HSCs). High tissue cellularity and excessive ECM accumulation are among the main characteristics of these pathological conditions (Armour et al., 2007a; Ghahary et al., 1996; Ghahary, Shen, Scott, Gong, et al., 1993; Ghahary, Shen, Scott, & Tredget, 1993; Tuan & Nichter, 1998). Local application of therapeutic modalities that suppress the ECM accumulation, either by reduction of ECM deposition or stimulation of ECM degradation, showed promising results in the treatment of fibrotic diseases (Mead et al., 2003; Rahmani-Neishaboor et al., 2010). In our previous study using a fibrotic rabbit ear model, the anti-scarring properties of L-tryptophan metabolites, kynurenine (Kyn) and kynurenic acid (KynA), were demonstrated (Poormasjedi-Meibod et al., 2014). It was also shown that the anti-scarring effect of these small molecules is mediated through (1) suppression of type-I collagen and fibronectin expression, (2) increases in matrix metalloproteinase (MMP) expression, and (3) reduction of fibroblast proliferation 90 and migration. When applied as a cream, these molecules significantly improve the wound healing outcome in vivo. In spite of their anti-fibrotic efficacy, daily administration of Kyn or KynA cream on skin injuries is challenging in burn patients or at skin donor sites, because dressings are usually changed every 3 to 5 days. Subsequent studies examined the encapsulation of Kyn, a water-soluble agent, in polyvinyl alcohol (PVA) nanofiber mats as an improved formulation for wound dressing application. However, because both Kyn and PVA are water soluble, it was necessary to further process the Kyn/PVA mats and coat them with a hydrophobic polymer such as poly(lactic-co-glycolic acid) (PLGA) to obtain a durable controlled release profile upon application at the wound site (chapter 3). Here, the incorporation of KynA into nanofibers in order to develop anti-fibrogenic dressings is investigated. KynA solubility in organic solvents allowed the use of an organic-solvent-based electrospinning process with biocompatible hydrophobic polymers such as poly(methylmethacrylate) (PMMA). PMMA mats containing KynA were prepared by electrospinning. Increasing concentrations of poly(ethylene glycol) (PEG) were then added to the polymerâdrug solution to obtain the appropriate release profile. The findings showed that PMMA+10%PEG nano-fibrous mats could be developed and used as a wound dressing through which KynA is slowly released at the wound site. There it improves the healing outcome by modulating the expression of type-I collagen and fibronectin and enhances the MMP-1 expression in cell culture and in animal models. 91 4.2 Method and materials 4.2.1 Materials PMMA (Mw â¼ 350,000, Aldrich, where Mw is molecular weight), PEG (Mw â¼ 1,000, Aldrich), dimethylformamide (DMF) (Fluka, 98%), and KynA (Aldrich) were used without any purification. 4.2.2 Electrospinning Electrospinning solutions were prepared by dissolving PMMA and PEG in DMF at 7.5% w/v concentrations. The PEG concentration was varied from 1% to 20% (w/w to PMMA), and the weight % of KynA was 6% (w/w) with respect to the total polymer. The KynA/PMMA-PEG solution was then electrospun with the parameters listed in Table 4.1. All electrospinning experiments were carried out at room temperature in a horizontal orientation. Table 4.1. The electrospinning conditions used in this study. Polymer PEG 1KD (w/w%) Voltage (kV) Syringe pump (mm/min) KynA (w/w%) Solvent PMMA (350 KD) 0 24 0.1 6 DMF PMMA (350 KD) 1 24 0.1 6 DMF PMMA (350 KD) 2.5 24 0.07 6 DMF PMMA (350 KD) 5 24 0.07 6 DMF PMMA (350 KD) 10 24 0.05 6 DMF PMMA (350 KD) 20 24 0.05 6 DMF 92 4.2.3 Characterization of electrospun fibers The morphology of the electrospun nanofibers was investigated by high-resolution scanning electron microscopy (Hitachi S-3000N scanning electron microscope). The average diameter of the nanofibers was determined directly from scanning electron microscopy images using ImageJ software, and 25 fibers were analyzed per image. 4.2.4 Water contact angle measurements Water contact angles of electrospun nanofiber mats were measured using a contact angle measuring system (KSV Instruments). Nanofiber mats were cut into square specimens, 1 cm Ã 1 cm, and fixed on a testing plate. Subsequently, 10 µl of distilled water was dropped onto the prepared sample. The contact angle between water droplet and dressing was measured from the resulting photographs. Five measurements at different positions were performed for each sample. 4.2.5 Determination of the water absorbency of the electrospun mats The dry mats were weighed, incubated in water for 1, 2, 5, 10, 20, and 30 min, excess water was removed, and the mats were weighed again. The percentage of weight gain was calculated using the expression Wg = 100*(Wt â Wd)/Wd, where Wg is the weight gain and Wt and Wd are the weight of nanofiber mats after incubation and the dry weight of the mat, respectively. 4.2.6 KynA release assay KynA release was evaluated by the total immersion method at 37 °C in phosphate buffered saline (PBS) solution (pH 7.4), as a releasing medium. The 93 drug-loaded electrospun mats (30 mg) were immersed in PBS, and the solution was collected and replaced after 1, 2, 4, 8, 24, 48, 72, 96, and 120 h of incubation. KynA concentrations were determined by high-performance liquid chromatography (HPLC) using a Waters Millennium system that utilized a mobile phase of 10 mM sodium dihydrogen phosphate:methanol (73:27, by volume) at pH 2.8, flowing at 1 ml/min, a C18 reverse phase Novapak column (Waters), and a 20 µl injection volume, with absorbance detection at 330 nm. Two-chamber 6-well plates (Corning Incorporated, Corning, NY) were used to measure the drug release in the non-immersed setting. Known weights of the medicated electrospun mats and hydrogel (500 µl, Intrasite Gel Applipak Hydrogel Wound Dressing, Smith & Nephew) were placed in the upper chamber, and 500 µl of PBS was added to the lower chamber. PBS was collected and replaced after 24, 48, 72, 96, and 120 h of incubation. KynA concentration was determined using HPLC as described above. 4.2.7 Ethics statement Both Human and Animal Ethics Committee of the University of British Columbia approved all methods and procedures, as well as the use of animals and tissue specimens obtained from animals and humans. Written informed consent was received prior to conducting any human sampling of tissue specimens. 4.2.8 Cell culture Dermal fibroblasts and keratinocytes were isolated from foreskin samples collected from healthy patients undergoing elective circumcision, using the 94 protocol described elsewhere (M. Li et al., 2009). Fibroblasts and keratinocytes were cultured in Dulbeccoâs Modified Eagleâs Medium (DMEM, Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS) and keratinocyte serum-free medium (KSFM, Invitrogen Life Technologies, Carlsbad, CA) supplemented with bovine pituitary extract (BPE) (25 ng/ml) and epidermal growth factor (EGF) (0.2 ng/ml, Gibco), respectively. Keratinocytes and fibroblasts at passages 4-7 were used in all the experiments. 4.2.9 In vitro cytocompatibility assay The live/dead® toxicity assay was used to determine the cytocompatibility of PMMA+10%PEG nanofiber (NF) mats and KynA-incorporated PMMA+10%PEG nanofiber mats (Kyn+NF). Cell cultures were set up using a two-chamber cell culture system in which fibroblasts (2.50 Ã 105) were cultured in the lower chambers while NF or NF+KynA were placed in the upper chamber. Following 3 days of incubation, cells were subjected to flow cytometry using live/dead® viability/cytotoxicity assay kit (Life Technologies, Invitrogen, Eugene, OR). Some cells were treated with KynA (100 µg/ml) as controls. Cells with compromised membranes were stained red with the ethidium homodimer-1 (EthD-1), a live-cell impermeable nucleic acid stain. Cells with intact cell membranes convert nonfluorescent calcein-AM into bright green fluorescent calcein. 4.2.10 Cell proliferation assay To evaluate the effect of NF and NF+KynA on the fibroblast proliferation rate, fibroblasts were cultured and treated as described above. Cells were 95 harvested, and the total cell number was counted after 36, 72, and 108 h of treatment. Cells were treated with KynA (100 µg/ml) as the positive control. 4.2.11 In vitro anti-scarring activity determination The anti-scarring activity of the KynA-incorporated mats was determined in vitro using Western blotting to determine the expression levels of type-I collagen, fibronectin, and MMP-1. Fibroblasts, plated in two-chamber 6 well plate, were treated with NF, NF+KynA, or KynA (100 µg/ml) as described above. Following 48 h of incubation, cells were harvested and cell pellets were resuspended in 100 µl of cell lysis buffer (50 mM Tris-HCl, pH 7.4, 10 mM ethylenediaminetetraacetic acid (EDTA), 5 mM EGTA, 0.5% NP40, 1% Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich)). After the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockfield, IL), equal amounts of protein from the whole-cell lysates (20 µg) were separated by running the sample on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked and probed for MMP-1, fibronectin, and type-I collagen using rabbit anti-MMP-1 Ab (1:2000, Abcam, Cambridge, MA, USA), rabbit anti-fibronectin (1:1000, Santa Cruz Biotechnology, CA), and mouse anti-collagen-I (1:2000, Developmental Studies Hybridoma Bank), respectively. Horseradish peroxidase (HRP) conjugated goat anti-rabbit Ab (1:3000 dilution, Bio-Rad) and HRP-conjugated goat anti-mouse Ab (1:3000 dilution, Bio-Rad) were used as the secondary antibodies. An enhanced chemiluminescence (ECL) detection system (ECL; 96 Amersham Biosciences, UK) was used in all blots to detect the secondary antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the protein loading control. 4.2.12 Wound creation and treatment Male Long Evans rats (The Jackson Laboratories, Bar Harbor, ME) were used to evaluate the anti-fibrotic effect of the medicated mats. Briefly, eight rats were anesthetized and prepared for wounding. Four full thickness wounds (8 mm) were created on the back of each animal. Wounds were splinted using a donut-shaped silicone splint to inhibit wound contraction. The splints were fixed in their position using sutures, and the wound was centered within the splint. In each set of experiments, the order of treatment was changed to reduce the chance of bias in wound healing based on the position of wounds on the animalâs back. Tegadermâ¢ dressing (3M, St. Paul, MN) was used to cover the wounds for 3 days. On day 3 post-wounding, wounds were either left untreated to heal by secondary intention or treated with KynA-containing cream (500 µg/ml), NF, or NF+KynA. KynA cream was applied daily while dressings were changed every 4 days. Dressings were covered with a layer of Tegadermâ¢ and hydrated by hydrogel. Splints were removed on day 7 post-wounding, animals were sacrificed on day 15 post-wounding, and tissue was harvested. 4.2.13 MMP-1, fibronectin, and type-I collagen expression in the wounds Total ribonucleic acid (RNA) was extracted from the scar tissue using Trizol reagent according to the manufacturerâs instructions (Invitrogen). Following deoxyribonuclease (DNase) treatment and complementary DNA (cDNA) 97 synthesis expression, Q-PCR was used to evaluate the MMP-1, fibronectin, and type-I collagen expression. Q-PCR was carried out on the Applied Biosystems® 7500 Fast Real-Time PCR System, using the SYBR® Green PCR Master-Mix kit (Applied Biosystems, Warrington, UK). The following cycling conditions were used for Q-PCR: 95 °C/15 min with 40 cycles of 95 °C/1 min, 55 °C/30 s, and 72 °C/30 s. Table 4.2. contains the list of the primers used in this study. β-actin was used as the reference gene. 4.2.14 Statistics Data were expressed as mean Â± standard error of the mean (SEM) of three or more independent observations. Statistical significance was calculated using a two-tailed unpaired Studentâs t-test or a one-way analysis of variance with post hoc test in case of multiple comparisons. P-values < 0.05 were considered statistically significant in this study. Table 4.2. List of primers used in this study. Rat-MMP-1 forward primer 5â²-TTGTTGCTGCCCATGAGCTT-3â² Rat-MMP-1 reverse primer 5â²-ACTTTGTCGCCAATTCCAGG-3â² Rat Col-1α1 forward primer 5â²-CAAGAATGGCGACCGTGGT-3â² Rat Col-1α1 reverse primer 5â²-GGTGTGACTCGTGGAGCCA-3â² Rat fibronectin forward primer 5â²-GGGATCAAAGGGAAACACAG-3â² Rat fibronectin reverse primer 5â²-AGACGGCAAAAGAAAGCAG-3â² Rat β-actin forward primer 5â²-TATCGGCAATGAGCGGTTCC-3â² Rat β-actin reverse primer 5â²-GTGTTGGCATAGAGGTCTTTACG-3â² 4.3 Results and discussion 4.3.1 Fiber formation and characterization In order to fabricate the anti-fibrotic wound dressings, in this study a lead formulation was developed and characterized for incorporation of the drug of interest, KynA, into nano-fibrous mats. Here PMMA was used as the 98 electrospinning carrier with an efficient control of nanofiber formation and drug encapsulation. PMMA is particularly interesting for this application, as it is used extensively in orthopedic settings as a drug delivery vehicle for antibiotics because of its high biocompatibility and ease of manipulation (Evans & Nelson, 1993; Majid et al., 1985). PMMA is a hydrophobic polymer; therefore, only a small percentage of the encapsulated drug is released from monolithic forms with low surface area to volume ratios (Anagnostakos, Wilmes, Schmitt, & Kelm, 2009; Andollina et al., 2008). The release profiles may be modulated by the inclusion of water-soluble excipients (J. Jackson, Leung, Duncan, Mugabe, & Burt, 2011) or by increasing the surface area using electrospinning (D. G. Yu, Branford-White, White, Li, & Zhu, 2010). Various electrospinning conditions, including variable flow rate or the inclusion of different PEG concentrations, were used in this study. The ideal electrospinning parameters to create defect-free, continuous nanofibers were set at PMMA/PEG mass ratio = 9/1, voltage = 24 kV, flow rate = 0.05 mm/min, and a needle to collector gap = 20 cm. Although the PMMA polymer is usually very brittle and hard, when electrospun it forms a strong yet flexible mat that contour-fits uneven surfaces such as large wounds (macroscopic observation). Figure 4.1. shows the scanning electron microscopy images of the electrospun mats prepared using increased concentrations of PEG (1-20% w/w). The PMMA-PEG combinations were electrospun uniformly to provide homogenous mats. As shown in Figure 4.1, only cross-sectionally round bead-free PMMA nanofibers were formed. The measured fiber diameter range and average fiber diameter for the PMMA fibers with increasing concentrations of 99 PEG are listed in Table 4.3. The inclusion of PEG with low molecular weight decreased the spinning solution viscosity considerably, requiring slower flow rates in order to obtain bead-free fibers. Reduced spinning solution viscosity and flow rate resulted in formation of fibers with significantly smaller diameters. Polymer solutions with PEG concentrations over 20% w/w were not investigated because their viscosity was too low for optimal electrospinning. Table 4.3. Average diameter of electrospun PMMA fibers with increasing concentrations of PEG. Polymer Nanofiber diameter (nm) PMMA 522.44 ± 16.19 PMMA+1% PEG 677.43 ± 46.24 PMMA+2.5% PEG 559.80 ± 17.02 PMMA+5% PEG 628.46 ± 16.3 PMMA+10% PEG 605.09 ± 52.04 PMMA+20% PEG 500.36 ± 26.77 100 Figure 4.1. Scanning electron microscopy images of electrospun PMMA fibers as a function of the increasing concentrations of PEG. The solution concentration, voltage, and tip-to-collector distance were 7.5 w/v%, 24 kV, and 20 cm, respectively. 101 4.3.2 Contact angle and water uptake measurements PMMA has a particular advantage in the wound setting because it does not swell in an aqueous environment. Therefore, a dressing may stay localized to the initial placement position but may still absorb water into voids by capillary action. The hydrophobicity of the electrospun mats was evaluated by the values of the water contact angle. Data in Figure 4.2. A indicate a decrease in the hydrophobicity of the PMMA/PEG mats compared with the PMMA mats. As expected, inclusion of 10% PEG, which is a hydrophilic polymer, significantly decreases the values of the water contact angle compared with the PMMA mats (110.87 ± 9.35° and 154.32 ± 1.71°, respectively, Figure 4.2. B). From the application point of view, the wettability and water uptake capacity of a dressing is important for its ability to remove the excess exudate and balance the wound moisture. For this purpose, PMMA and PMMA/PEG mats with the weight ratio 9/1 were soaked in water for 1, 2, 5, 10, 20, and 30 min. The excess water was removed, and the percentage of the weight gain in relation to the primary dry weight of the electrospun mat was plotted (Figure 4.2. C). While the PMMA mats did not absorb noticeable amounts of water, the PMMA mats containing 10% PEG retained significant amounts of water and gained up to 300% of their dry weight after 30 min of incubation in water (Figure 4.2. C). This increased wettability and water uptake is well suited for balancing the wound moisture as well as allowing drug release in the wound environment. 102 4.3.3 Release characteristics of KynA In this study, KynA has been incorporated into nano-fibrous dressings because it has several advantages over Kyn. While having comparable anti-scarring activity to Kyn in vitro, previous in vivo studies indicated that KynA is more potent than Kyn in suppressing the expression of fibronectin (Poormasjedi-Meibod et al., 2014), which is essential for collagen deposition and assembly (Sottile & Hocking, 2002; Sottile et al., 2007). In addition, the use of KynA would be safer even if it diffuses out into circulation because (1) it is an end product in the Kyn pathway and, therefore, there is no chance that it gets metabolized to another product with unknown effects and (2) its diffusibility through the blood brain barrier is very poor, which limits its accessibility to the central nervous system (Fukui et al., 1991; Schwarcz et al., 2012). Finally, KynA is far less water-soluble than Kyn and easily dissolvable in organic solvents, such as DMF, which enable the use of an organic-based electrospinning process. In this study the release characteristics of KynA from the electrospun mats were investigated by the total immersion method using PBS as the releasing media. The percentage of the cumulative amount of released KynA from electrospun mats is reported in Figure 4.3. Owing to the high hydrophobicity and poor wettability, KynA-loaded PMMA nanofibers demonstrated the lowest level of drug release with no detectable burst release. 103 Figure 4.2. Contact angle and water uptake measurements of the nano-fibrous mats. The effect of 10% PEG addition on surface hydrophilicity/hydrophobicity was investigated through contact angle measurements. (A) Photomicrograph images of a single water drop on the electrospun mats. (B) The contact angle degree measurements. (C) The effect of 10% PEG addition on dressing wettability was measured as weight gain following incubation with water. 104 Many researchers have investigated inclusion of hydrophilic excipients into hydrophobic polymers in order to increase the drug release rate. PEG is a water-soluble polymeric excipient that blends well with hydrophobic polymers such as PMMA or PCL. Previous studies revealed a concentration-dependent increase in the release rate of loaded drugs in response to inclusion of PEG in hydrophobic polymers (J. Jackson et al., 2011; J. K. Jackson et al., 1997; Winternitz, Jackson, Oktaba, & Burt, 1996). Therefore, in the next step, increasing concentration of PEG was added to the polymeric solution to tailor the release profile. Addition of 1% PEG increased the burst release so that 18% ± 3.4% of the drug was released in the first 24 h. After the first 24 h, KynA released in a linear fashion with approximately 2% of loaded drug being released every 24 h. The burst phase of KynA release increased with increasing PEG concentration in the spinning solution (Figure 4.3.). While PMMA+2.5% and 5% PEG demonstrated moderate levels of burst release in the first 24 h, 42% ± 7.3% and 49.37% ± 1.5%, respectively, more than 90% of the loaded drug was released from the electrospun mats containing 10% or 20% PEG in the first 8 h (Figure 4.3.). Addition of a hydrophilic excipient such as PEG increases the porosity of the PMMA matrix, which subsequently enhances the KynA diffusion rate out of the nanofibers. Therefore, an increase in the concentration of the PEG leads to a marked increase in KynA release. In order to better mimic the drug release environment at the wound site, a two-chamber release study was designed (Figure 4.4. A). KynA-incorporated mats were placed in the upper chamber and covered with hydrogel to keep the 105 dressings moist. The amount of the released drug was measured in the PBS (500 µl) placed in the lower chamber and replaced every 24 h. As shown in Figure 4.3. Cumulative release profiles of KynA from electrospun PMMA fibers containing increasing concentrations of PEG. The total immersion method at 37°C in phosphate buffered saline solution was used. Figure 4.4. B, the amount of released drug in this setting is much less when compared with that in the total immersion method. This is not surprising, since the drug release rate from PMMA, which is a non-degradable, water repellant polymer, is mainly governed by diffusion (Fu & Kao, 2010). Volume of the available solvent highly affects the drug diffusion; therefore, reduced levels of drug release in the two-chamber setting compared with the total immersion technique are a direct consequence of the reduced moisture. Compared with the total immersion setting, where the PMMA+5% PEG dressings released 50% of 106 the loaded drug in the first 24 h, these dressings demonstrated a linear release profile with 1% drug release every 24 h in a two-chamber plate setting. PMMA mats that contain 10% and 20% of PEG release 28.04% ± 2.8% and 32.43% ± 0.6% of the loaded drug in the first 24 h, respectively (Figure 4.4. B). These mats demonstrated an almost linear release profile after the primary burst release. Comparison of the KynA release profiles of different nano-fibrous dressings showed that KynA-incorporated mats containing 10% PEG would be considered to be the lead formulation for further studies. 4.3.4 Cytocomptatibility PMMA, a non-toxic, non-biodegradable polymer, has high levels of biocompatibility, which expanded its application in the biomedical field from orthopedics to wound dressing manufacturing (Andersson et al., 2014; Evans & Nelson, 1993; Majid et al., 1985). PEG, a FDA approved water-soluble polymer, has frequently been used in tissue engineering, drug delivery, and surface modification of medical devices because of its biocompatibility and nonimmunogenicity (Bjugstad et al., 2008; Yin et al., 2010). Low molecular weight PEG is rapidly cleared from the plasma by kidneys, which makes the application of this polymer quite safe (Berglund, 1965; Berglund, Engberg, Persson, & Ulfendahl, 1969). Cytocomptatibility is one of the main features of a wound dressing. Therefore, primary human dermal fibroblasts were used to test the cytotoxicity of the electrospun nanofibers. Figure 4.5. A is a schematic representation of the two-chamber cell culture setting used for fibroblast treatment. 107 Figure 4.4. Cumulative release profiles of KynA from electrospun PMMA-PEG fibers. (A) Schematic diagram of the two-chamber setting used for the release studies. (B) KynA cumulative release profiles from PMMA nanofibers containing 5%, 10%, and 20% PEG. Figure 4.5. B displays the ethidium homodimer/calcein dual-parameter histogram of the fibroblasts treated with NF or NF+KynA for 48 h. The percentages of the live and dead cells were determined by using flow cytometry. As shown in Figure 4.5. B, exposure of fibroblasts to NF or NF+KynA did not significantly increase the percentage of dead cells in addition to cells with damaged membrane undergoing apoptosis, cells having high levels of ethidium homodimer staining, compared with the control. A quantitative analysis of these 108 data (Figure 4.5. C) revealed that more than 95% of harvested cells were positive for calcein staining, indicating that most of the detected cells are viable. 4.3.5 Nanofiber releasabled KynA modulates the fibroblast proliferation and the expression of type-I collagen, fibronectin, and MMP-1 in vitro Prolonged fibroblast activation and excessive cell proliferation, leading to high tissue cellularity, are key features of fibrosis (Armour et al., 2007a; Tredget, 1999; Tuan & Nichter, 1998). In a previous study (Poormasjedi-Meibod et al., 2014), it was shown that KynA was able to suppress the proliferation of fibroblasts in vitro. To confirm the preservation of KynA biological activity during the electrospinning process, fibroblasts were incubated with NF+KynA or empty NF, as shown in Figure 4.5. A, and the total cell number was counted after 36, 72, and 108 h. Fibroblasts treatment with NF+KynA or KynA significantly reduced the proliferation of dermal fibroblasts whereas NF alone does not affect the proliferation rate of these cells (Figure 4.6. A). Excessive ECM deposition by activated fibroblasts and deficiencies in matrix degradation, leading to ECM accumulation, is another hallmark of fibrosis (Arakawa et al., 1996; Armour et al., 2007a; Craig, 1975; Zeisberg & Kalluri, 2013). Comparison of hypertrophic scars with normal skin revealed a significant increase in levels of type-I collagen and fibronectin expression and marked reduction in the expression of ECM degrading enzymes such as MMP-1 (Ghahary et al., 1996; Ghahary, Shen, Scott, Gong, et al., 1993). KynA, as an antifibrotic agent, has been shown to effectively reduce the production of 109 collagen and fibronectin and increase the expression of different MMPs (MMP-1 and -3) (Poormasjedi-Meibod et al., 2014). Figure 4.5. Live/dead® viability/cytotoxicity assay. (A) Schematic diagram of the two-chamber setting used for the cell treatment. (B) Evaluation of the cytocompatibility of electrospun nanofibers. Dermal fibroblasts were cultured in the lower chamber while NF or NF+KynA were placed in the upper chamber. Following 3 days of incubation, cells 110 were subjected to flow cytometry using a live/dead® viability/cytotoxicity assay kit. (C) Quantification and statistical analysis of treated fibroblast viability. Therefore, in order to further evaluate the anti-fibrogenic effects of the medicated mats in vitro, the expression of type-I collagen, fibronectin, and MMP-1 was evaluated following 48 h of fibroblast treatment. Media containing KynA (100 µg/ml) was used as the positive control for cell response. As expected, type-I collagen and fibronectin expression was significantly reduced in fibroblasts treated with NF+KynA compared with untreated cells or cells treated with empty nanofibers (NF). Also, these cells demonstrated a significant increase in the expression of MMP-1 (Figure 4.6. B). NF treated and untreated cells have comparable levels of type-I collagen, fibronectin, and MMP-1 expression, implying that the PMMA/10% PEG nanofibers did not modulate ECM expression. Figures 4.6. C, D, and E depict the quantitative analysis of the data shown in Figure 4.6. B for type-I collagen, fibronectin, and MMP-1 protein expression, respectively (*P-value < 0.05, **P-value < 0.001 n = 4). As shown in this figure, KynA delivered by nanofibers has comparable biological activity, inhibition of fibroblast proliferation and modulation of ECM expression, with direct KynA administration using the same concentration, indicating the preservation of the drugâs biological activity during the electrospinning process. Application of therapeutic agents that suppress ECM accumulation has been studied extensively in order to prevent skin fibrosis. Previous animal studies and data obtained from clinical trials demonstrated the anti-scarring effect of interlukin-10, which is mediated by induction of MMPs and suppression of collagen expression (Kieran et al., 2013; Liechty, Kim, Adzick, & Crombleholme, 111 2000; Shi et al., 2014; J. H. Shi et al., 2013). In addition, application of neutralizing antibodies against transforming growth factor beta (TGF-β) isomers showed promising results in suppression of skin scarring in animal models (Shah, Foreman, & Ferguson, 1994, 1995). KynA as an anti-fibrotic therapy has significant advantages over these modalities. In comparison with any other anti-fibrotic proteins, KynA is quite stable and inexpensive. Moreover, as stated by Bos et al. (Bos & Meinardi, 2000) compounds with molecular weight lower than 500 Da easily penetrate through the epidermis and reach the dermis. Therefore, in contrast to anti-fibrotic macromolecules, KynA (Mw = 189.168) application eliminates the need for intradermal injection in order for the drug to reach the dermal fibroblasts. These advantages set the stage for application of KynA in animal models as an effective anti-fibrotic agent. 4.3.6 Nanofiber releasabled KynA modulates the expression of type-I collagen, fibronectin, and MMP-1 in vivo In spite of all advances in the development of novel anti-fibrotic therapeutic modalities for skin, application of topical creams/gels or intralesional injections remained the main methods for delivery of these modalities to the wound bed (Kontochristopoulos et al., 2005; Payapvipapong, Niumpradit, Piriyanand, Buranaphalin, & Nakakes, 2015). Development of a slow-releasing drug delivery method, which eliminates the need for frequent application of the medication and the pain associated with intradermal injections, will increase patient compliance. Following the fabrication of KynA slow-releasing dressings their efficacy in 112 reducing skin fibrosis was assessed in vivo using a rat model. Full thickness wounds were either treated with KynA-containing cream (500 µg/ml, applied daily) or dressed with NF or NF+KynA mats (changed every 4 days). Control wounds were left untreated to heal by secondary intension. Wounds in all treatment groups were closed at day 15 post-surgery. Q-PCR analysis of the harvested tissue showed a significant reduction in the expression of type-I collagen (Figure 4.7. A) and fibronectin (Figure 4.7. B) in KynA-treated wounds, delivered either through dermal cream or medicated nanofibers, compared with control or NF dressed wounds. Also, KynA-treated wounds were associated with increased levels of MMP-1 expression (Figure 4.7. C, *P-value < 0.05, **P-value < 0.01, n = 8). In general, both the PMMA/PEG-KynA nanofiber mats and topical KynA-cream application showed comparable effects on reduction of type-I collagen and fibronectin expression and induction of MMP-1 expression in vivo. These in vivo data clearly demonstrate both the formulation potential of the nanofiber mats for controlled delivery of KynA and the therapeutic efficacy of the dressings for the treatment for fibrotic conditions. 113 Figure 4.6. Evaluation of KynA-incorporated electrospun dressing with anti-scarring activity in vitro. (A) Inhibition of fibroblast proliferation by KynA-incorporated mats (NF+KynA). (B) Suppression of type-I collagen and fibronectin expression and induction of MMP-1 production by KynA-incorporated mats (NF+KynA). The Mean ± SEM ratio of (C) type-I collagen, (D) fibronectin, and (E) MMP-1 density to GAPDH at the protein level, respectively. GAPDH was used as protein loading control. 114 Figure 4.7. Effect of Kyn/PMMA-PEG dressings on ECM components expression at day 15 post-wounding. Four full thickness wounds were generated on the backs of the rats, and at day 3 post-wounding wounds were either treated with daily application of KynA cream or dressed with NF or NF+KynA. Dressings were changed every 4 days, and control wounds were left untreated to heal with secondary intension. At day 15 post- 115 surgery animals were euthanized. Total RNA was extracted from harvested tissue samples, and cDNA was synthetized. Q-PCR was used to evaluate the expression of (A) type-I collagen, (B) fibronectin, and (C) MMP-1 in tissue samples. 4.4 Summary and conclusion Nano-fibrous PMMA-PEG membranes containing the anti-fibrotic agent KynA were efficiently prepared using an electrospinning process, providing strong, contour fitting wettable mats suitable for application at the wound site. The performance of PMMA+10%PEG as a slow-releasing drug delivery system for KynA and anti-fibrotic wound dressing was evaluated in vitro and in vivo. These medicated mats effectively suppress fibroblast proliferation and modulate ECM expression by these cells in vitro. Finally, the data obtained from animal studies indicated that KynA-containing electrospun dressings could be used as an effective replacement for daily KynA cream application for prevention of dermal fibrosis. 116 5 Conclusions and suggestions for future work Each year in developed countries almost 15 million patients, 70% of whom are children, develop pathological scarring with considerable morbidity (Ardehali et al., 2007; Bayat et al., 2003). Scar formation is an expected consequence of the wound healing process where the normal skin (or other tissues) is replaced by fibrous tissue mainly composed of collagen. Scarring is considered abnormal when the amount of the deposited extracellular matrix (ECM) is excessive, when it adversely affects the normal tissue function, or when it is cosmetically distressing or disfiguring for the patients. Therapeutic modalities for managing post-burn or post-surgical hypertrophic scars have transitioned from invasive methods, such as surgical revisions and radiation, to topical and intralesional therapies that improve the wound healing outcome by targeting skin cells. In spite of these advances in the wound care industry, unfortunately the current therapeutic modalities for prevention of scarring are still unsatisfactory. Indeed, no single treatment has been universally adopted in clinical practice as the optimal management approach. Application of small molecules, proteins, and neutralizing antibodies has been investigated in order to develop novel anti-fibrotic therapies. For instance, the role of basic fibroblast growth factor (bFGF) in improvement of wound healing has been studied extensively in hard-to-heal wounds and burns. bFGF acts as a potent chemo-attractant and mitogen for keratinocyte, fibroblasts, and endothelial cells in addition to enhancing the ECM production and deposition (Akita, Akino, Imaizumi, & Hirano, 2008; McGee et al., 1988; Wang et al., 2008). In recent 117 years several groups investigated the role of bFGF in prevention of scar formation. These studies demonstrated that bFGF, applied topically on open wounds or delivered via a slow-releasing system, improved the wound healing outcome by increasing the expression of matrix metalloproteinase (MMP-1), suppressing the expression of α-SMA and blocking the TGF-β1 signaling pathway (Eto et al., 2012; H. X. Shi et al., 2013; Xie et al., 2008). Interleukin-10 (IL-10), an anti-inflammatory cytokine, is another molecule that has been extensively studied for its potential anti-fibrotic effects. Data obtained from a phase II randomized clinical trial and several animal studies demonstrated the anti-scarring properties of IL-10, which are mediated by suppressing α-SMA and collagen (type-I and -III) expression and stimulating MMP-1 and MMP-8 production (Kieran et al., 2013; J. H. Shi et al., 2013). In 2012 Armendariz-Borunda et al. (Armendariz-Borunda et al., 2012) reported the outcome of a controlled clinical trial regarding the anti-scarring effects of topical pirfenidone after burn injuries. Pirfenidone is a small anti-fibrotic molecule recognized mainly for its therapeutic effects in lung fibrosis. Pirfenidone gel was applied 3 times per day for 6 months, and its anti-scarring effects were compared with pressure therapy as a standard conservative treatment. The results showed that patients receiving pirfenidone demonstrated higher levels of improvement compared with the control group. Pirfenidone reduces fibrosis via inhibition of pro-inflammatory and pro-fibrotic cytokines, IL-6, tumor necrosis factor alpha (TNF-α), and TGF-β, suppressing the expression of collagen, and inducing the expression of MMPs (Di Sario et al., 2004; Hisatomi et al., 2012). 118 Another well-studied anti-fibrotic strategy is inhibition of the TGF-β signaling pathway, either by using ligand traps, such as neutralizing antibodies and soluble decoy receptors, anti-sense oligonucleotides targeting TGF-β, or small molecule receptor kinase inhibitors (Huang et al., 2002; Mead et al., 2003; Shah et al., 1995; Singer et al., 2009). The pivotal role of the TGF-β signaling pathway in tissue repair and fibrosis has been demonstrated in different organs (Pohlers et al., 2009; Verrecchia & Mauviel, 2007). TGF-β enhances matrix accumulation and contraction by induction of ECM production, inhibition of ECM degradation, and stimulation of myofibroblast differentiation (Desmouliere, Geinoz, Gabbiani, & Gabbiani, 1993; Pan, Chen, Huang, Yao, & Ma, 2013; Vaughan, Howard, & Tomasek, 2000). Application of exogenous TGF-β3, which is widely known for its anti-fibrotic effects, is another attractive anti-scarring modality. Multiple animal studies and phase I/II clinical trials demonstrated the efficacy of avotermin (human recombinant TGF-β3) in prevention of scar formation (McCollum et al., 2011; Occleston et al., 2011). In 2012, our group reported that local indoleamine 2,3-dioxygenase (IDO) expression in the transplanted xenogeneic skin substitute not only protects the skin graft from immune rejection but also prevents scar formation in a fibrotic rabbit ear model (Chavez-Munoz et al., 2012). IDO is the first and rate-limiting enzyme in the kynurenine (Kyn) pathway, which catabolizes tryptophan (Trp) to N-formylkynurenine. Local IDO expression results in Trp depletion and Kyn accumulation. Further studies by Li et al. (Y. Li et al., 2014) led to the 119 identification of Kyn as a potent anti-fibrotic agent the application of which in a dermal cream can significantly improve scar formation in a fibrotic animal model. The goal of this study was, therefore, to evaluate the anti-fibrotic potential of Kyn and its metabolites and also to develop slow-releasing drug delivery systems for these agents for their controlled and prolonged delivery to open wounds. To address the first objective of this study, which was to evaluate the anti-fibrotic potential of Kyn metabolites, a series of in vitro and in vivo studies were used to evaluate and compare the anti-scarring effects of kynurenic acid (KynA) and Kyn. In chapter 2 it was shown that KynA, which can effectively increase the expression of MMPs (MMP-1 and -3), decreased the expression of collagens (type-I and -III) and fibronectin and thus acted as a potent anti-fibrotic therapy when applied daily in a fibrotic rabbit ear model. KynA-treated wounds, which have reduced skin elevation index (SEI), epidermal thickness index (ETI), and tissue cellularity, demonstrate a significant decrease in type-I collagen and fibronectin expression and a marked increase in MMP-1 expression. While Kyn and KynA effectively reduce the ECM accumulation via enhancing MMP expression and suppressing the ECM deposition, like other anti-fibrotic strategies they have several advantages over alternative anti-scarring proteins (such as IL-10 and bFGF), as explained below: 1) Kyn and KynA are small molecules with molecular weights lower than 250 Da. Therefore, they can easily penetrate through the epidermis and reach the dermal layer where they target the fibroblasts to modulate the ECM expression by these cells. This property will eliminate the need for 120 intralesional injection of the agents, which is frequently required for macromolecular proteins. Moreover, topical application of these medications will minimize side effects and enhance patient compliance. 2) Kyn and KynA are non-toxic endogenous compounds that are naturally found in the body. As natural products, the regulatory process for introducing these agents to the clinic would be easier than for synthetic drugs. 3) Kyn and KynA are stable for a long period of time at 4 °C or at room temperature. The shelf life and stability of the drug are some of the main considerations in the manufacturing process and storage. 4) Compared with many other anti-fibrogenic factors (proteins), Kyn and KynA are inexpensive to either purchase or manufacture. That being said and in spite of their anti-fibrogenic efficacy, daily administration of Kyn or its metabolites directly on the wound is not hassle-free. Most patients with severe trauma such as large burn injuries require dressings that are usually changed every 3â5 days. This setting will limit frequent application of dermal creams containing Kyn/KynA. Therefore, a slow-releasing drug delivery system would be an appropriate method for controlled and prolonged delivery of these therapeutic modalities to the wound site. Application of nanotechnology in drug delivery is a promising methodology for increasing the therapeutic efficacy of the administered drugs. Nanotechnology-based models have several advantages over the conventional routs of drug delivery including (1) controlled and prolonged drug delivery would eliminate the need for frequent application of the medication, (2) improved and targeted delivery of large 121 macromolecules and poorly water-soluble drugs across the biological barriers in a tissue or cell specific manner, (3) improved drug stability and bioavailability and reduced side effects of the drug, and (4) co-delivery of multiple drugs for combination therapy (Farokhzad & Langer, 2009; Hughes, 2005; McNeil, 2011; Ruggiero, Pastorino, & Herrera, 2010). Different nano-formulations, such as nanofibers (NFs), liposomes, and microspheres, have been developed for drug delivery. As reported in chapters 3 and 4, electrospinning was used to incorporate Kyn or KynA into polymeric nanofibers in order to fabricate slow-releasing drug delivery systems that will precisely deliver a calculated dose of Kyn/KynA to the wound site over time. Electrospinning is a versatile and simple technique for fiber generation from polymeric solutions. Application of electrospun fibers for drug delivery has been extensively investigated because of their structural stability, improved drug encapsulation, and easily tunable drug release profile. Application of multi-nozzle, centrifuge-based, and free surface instruments facilitated the scaling up of the manufacturing process and its translation to clinic use (Brettmann et al., 2012; Krogstad & Woodrow, 2014; Luo, Stoyanov, Stride, Pelan, & Edirisinghe, 2012). A wide variety of natural and synthetic materials have been electrospun to manufacture topical bioactive dressings or tissue scaffolds in order to promote and improve the wound healing process (Choi et al., 2015; Dubsky et al., 2012; Rho et al., 2006; Zhou et al., 2013). When the water solubility of the Kyn was considered, polyvinyl alcohol (PVA), a hydrophilic polymer, was used as the main carrier for the electrospinning process. PVA, 122 which is a non-toxic, biocompatible, biodegradable, and non-carcinogenic polymer, has been used extensively for drug delivery in the forms of hydrogel, microspheres, or nanofibers (C. J. Kim & Lee, 1992; Zeng et al., 2005). The amorphous domains of PVA and high surface to volume ratio of nanofibers promote the fast dissolution of the electrospun mats and burst release of the incorporated drug. Different cross-linking methodologies, such as glutaraldehyde, borax, or heat treatment, are developed in order to reduce the solubility of the PVA nanofiber mat in water and control the drug release (Cencetti et al., 2012; Ramires & Milella, 2002). In chapter 3 it was shown that heat treatment leads to weld formation between PVA fibers and hinders the dissolution of the PVA mat in a moist environment such as a wound. As shown in chapter 3, while PVA nanofibers immediately dissolved in phosphate buffered saline (PBS) and released the incorporated drug, heat treatment prolonged the release up to 4 h, which might be caused by reduced solubility of the matrix. The effect of cross-linking on the PVA dissolution and drug release has been investigated previously. In 2014 Peresin et al. (Maria Soledad Peresin, 2014) reported a significant enhancement in the physical integrity of the electrospun PVA fibers following cross-linking using maleic anhydride and heat. As reported by Khan et al. (Samiullah Khan, 2014) cross-linking not only decreases the degree of PVA hydrogel swelling but also reduces the drug release from these hydrogels, which is due to tighter hydrogel structure and polymers entanglements following cross-linking. 123 As described in chapter 3, PLGA coating was added on top of the electrospun PVA nanofibers in order to further prolong the release profile. Presence of two layers of PLGA effectively reduced the burst release and extended the drug release up to 4 days tested. Application of dip-coating technique for drug loading or modulation of drug release profile has been studied previously (Acharya & Park, 2006; Farb et al., 2001). As reported by Gulati et al. (Gulati et al., 2012), formation of a thin PLGA film over the drug-loaded titania nanotube significantly improved the drug release characteristics and prolonged the drug release from 4 days to more than 30 days. Another study by Kim et al. (Hyeseon Kim, 2014) also confirmed the efficacy of PLGA coating on reduction of early burst release and prolongation of the release profile in drug-loaded vascular grafts. In chapter 4, poly(methylmethacrylate) (PMMA) was used for fabrication of KynA-containing nanofibrous dressings. KynA is partially soluble in water, but it has good solubility in organic solvents such as dimethylformamide (DMF). Application of PMMA polymer and inclusion of low molecular weight poly(ethylene glycol) (PEG) eliminated the need for post-spinning modification in order to control the release profile. Application of PMMA in drug delivery and its electrospinning have been described by different groups (Evans & Nelson, 1993; Kong & Jang, 2008; Majid et al., 1985). Although PMMA has high levels of biocompatibility (Amon & Menapace, 1991), fabrication of PMMA nanofibrous mats as an effective drug delivery system has not been investigated extensively. Andersson et al. (Andersson et al., 2014) reported the fabrication of an anti-microbial nanofibrous mat by using PMMA/poly(ethylene oxide) (PEO) as the 124 polymeric carrier. Their study proposed that these mats, which show effective anti-bacterial performance and high levels of tensile strength, can be used as an anti-infective wound dressing. As shown in chapter 4, KynA incorporation into PMMA/PEG mats leads to the fabrication of flexible, beadless nanofibrous mats, which can prolong the drug release up to 4 days tested. In order to address the last objective of this thesis, which was to evaluate the anti-fibrotic potential of the fabricated dressings, a rat excisional wound model was established and used to evaluate the anti-fibrotic effect of the manufactured Kyn or KynA slow-releasing drug delivery systems in open wounds. Hydrogel was added to the medicated nanofibers in order to provide the required moisture for drug release. Wounds covered with these medicated mats demonstrated a significant reduction in fibrosis and inflammation compared with the control untreated wounds. The local and controlled release of Kyn or KynA markedly improved the wound healing outcome, indicated by reduced tissue cellularity, suppressed collagen and fibronectin expression, and enhanced MMP-1 expression. In addition, Kyn-treated wounds showed a lower number of α-SMA+ myofibroblasts, which are responsible for ECM deposition and contraction. As reported in chapters 3 and 4, application of these medicated dressings, which are changed every 4 days, have an anti-fibrotic effect equal to the daily application of the Kyn or KynA-cream; therefore, these dressings can be implemented as an effective drug delivery method for these anti-scarring agents. In summary, in spite of all advances in the area of bioactive dressings there is no anti-fibrotic dressing on the market. Current delivery of anti-scarring 125 agents to the wound relies on intralesional injection or topical gels that must be applied two times per day to provide a significant improvement in the wound healing outcome. Therefore, application of the manufactured anti-fibrotic dressings not only improves the wound healing outcome but also increases the patient compliance by eliminating the need for frequent application of the medication and subsequent dressing changes. 5.1 Suggestions for future work This thesis has described the mechanisms underlying the anti-fibrotic effects of different Trp metabolites, Kyn and KynA. In addition, the electrospinning process has been used to incorporate these agents into nanofibers. These medicated nanofibrous mats have been used as slow-releasing drug delivery systems for controlled delivery of Kyn or KynA to the wound bed in order to improve the wound healing outcome. Although the fabricated dressings significantly reduced scar formation in animal models, this research only demonstrates proof-of-principle, and these systems require further improvement. My suggestions for future research to improve these anti-fibrotic dressings are as follows: 1) In this study, the rat has been used as the selected surgical wound model to evaluate the anti-fibrotic effect of these dressings in vivo. The rat model is not the ideal fibrotic model; therefore, use of the Duroc pig, which better mimics scarring in human, is suggested. Ultimately, clinical trials using burn patients are required to further confirm the efficacy of the dressings developed. 126 2) Although the fabricated wound dressings can be used as an effective slow-releasing drug delivery system for open wounds, their topical application is not feasible in closed wounds, such as surgical incisions. As such, fabrication of slow-releasing implants with nanofiber or microsphere structures will provide a better drug delivery system for Kyn or its metabolites in closed wounds. Biodegradable polymers should be used for fabrication of these wound care products in order to eliminate the need for the drug delivery device retrieval through a second surgery. In addition, in this setting the release profile should be prolonged in order to support a single dosing approach at the time of surgery. This approach will facilitate the clinical translation of this technology in closed wounds. 3) Fabrication of anti-fibrotic sutures is another possible advancement to this thesis. Besides having the common mechanical requirements of a suture, these wound care products slowly release Kyn or its metabolites at the wound site and prevent/reduce the scar formation following suturing. In the next step other therapeutic modalities such as anti-microbial agents can also be incorporated into the design to reduce the rate of post-surgical infections. 4) In this study the focus was on incorporation of anti-fibrotic agents into dressings. In the next step other medications such as pain killers, anti-inflammatory drugs, anti-infective agents (antibiotics or silver nanoparticles), growth factors, and cytokines can be added to the formulation in order to develop a multifunctional dressing. These dressings can be used to address 127 multiple complications, including infection and excessive inflammation, which might emerge during the course of wound healing. 5) Keeping the dressings moist in order to obtain the desired release profile in vivo was a challenge in this study. Here, hydrogels were applied on top of the dressing to provide enough moisture for the nanofibrous dressings. In the next step water-absorbing polymers can be incorporated in the nanofiber mat fabrication process. In this case the fabricated mat will have two types of nanofibers, one that carries and delivers the anti-fibrotic agents and another that acts as a hydrogel. The need for application of external hydrogel will be eliminated though this novel design. 6) In addition to addressing the post-burn or post-surgery skin fibrosis, this technology can be used in other organs or clinical settings where the patient is facing fibrosis. For example, anti-fibrotic drug-eluting stents or catheters can be manufactured where a biodegradable polymer containing Kyn or its metabolites covers the outer layer of the stent. The outer layer can be added to the stent through electrospinning, electrospraying, or dip-coating. 128 References Acharya, G., & Park, K. (2006). Mechanisms of controlled drug release from drug-eluting stents. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivs 2.5 Canada
Graduate
Development and application of anti-fibrogenic dressings
Text