Oxidative Stress and Biomaterials

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OXIDATIVE STRESS AND BIOMATERIALS

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OXIDATIVE STRESS AND BIOMATERIALS

Edited by

THOMAS DZIUBLA Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, United States

D. ALLAN BUTTERFIELD Department of Chemistry, Markey Cancer Center, Spinal Cord and Brain Injury Research Center, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, United States

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © 2016 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 978-0-12-803269-5 For Information on all Academic Press publications visit our website at http://www.elsevier.com/

Publisher: Joe Hayton Acquisition Editor: Cari Owen Editorial Project Manager: Lucy Beg Production Project Manager: Sruthi Satheesh Designer: Mathew Limbert Typeset by MPS Limited, Chennai, India

CONTENTS List of Contributors ix Preface xiii

1. A Free Radical Primer

1

Prachi Gupta, Andrew Lakes and Thomas Dziubla 1.1  Free Radical Biology—Importance 1 1.2  RED/Ox Chemistry 3 1.3  Biological Oxidation Events 17 1.4  Conclusion and Final Thoughts 27 References 27

2. Oxidative Stress, Inflammation, and Disease

35

Shampa Chatterjee 2.1 Introduction 35 2.2  ROS and Oxidative Stress: A Major Activator of Inflammatory Pathways 37 2.3  Inflammation: A Major Cause of Oxidative Stress 40 2.4  Oxidant Stress and Inflammation in Cellular Transformation, Apoptosis, and Necrosis 42 2.5  Exploring the Link Between Oxidative Stress and Inflammation and the Onset of Various Diseases 43 2.6  Antioxidants and Anti-Inflammatory Agents: Perspectives in Therapeutics 47 2.7  Conclusions and Perspectives 51 Abbreviations 51 References 52

3. Oxidative Stress, Inflammation, and the Corrosion of Metallic Biomaterials: Corrosion Causes Biology and Biology Causes Corrosion 59 Jeremy L. Gilbert and Gregory W. Kubacki 3.1 Introduction 59 3.2  Oxidation, Reduction, and Tribocorrosion at Metallic Biomaterial Surfaces 61 3.3  Immune Cells, Inflammation, and ROS 67 3.4  Metal Ions and Wear Debris Effects on Local Tissues 73 3.5  Reduction Reactions and Cellular Viability 76 3.6  ICIC of CoCrMo and Ti Alloys: ROS Effects on Corrosion and Wear 79 3.7  Summary and Conclusions 83 Acknowledgments 84 References 84 v

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Contents

4. Oxidative Stress and Biomaterials: The Inflammatory Link

89

Isaac M. Adjei, Glendon Plumton and Blanka Sharma 4.1 Introduction 89 4.2  FBR to Biomaterials 90 4.3  Effect of Physicochemical Properties of Biomaterial on Inflammation 95 4.4  Relationship Between Inflammation and Oxidative Stress 98 4.5  Oxidative Stress as By-Product of Inflammatory Response to Biomaterial 102 4.6  Impact of Oxidative Stress on Implanted Cells and Induction of Inflammation 107 4.7 Conclusion 109 References 109

5. Nanoparticle Toxicity and Environmental Impact

117

Yiqun Mo, Aihua Gu, David J. Tollerud and Qunwei Zhang 5.1  Introduction 117 5.2  Nanotoxicology 118 5.3   Free Radicals, Reactive Oxygen Species, and Oxidative Stress 119 5.4   Nanoparticle-Induced ROS Generation and Oxidative Stress 120 5.5   Inflammation and Nanoparticles 123 5.6  Systemic Toxicity 125 5.7   Mechanisms of Nanoparticle Toxicity 125 5.8   Genotoxic Effects of Nanoparticles 128 5.9   Ecotoxicity of Nanoparticles and Its Environmental Impact 129 5.10 Conclusion 133 Acknowledgments 134 References 134

6. In Vitro Cellular Assays for Oxidative Stress and Biomaterial Response

145

Mihail I. Mitov, Vinod S. Patil, Michael C. Alstott, Thomas Dziubla and D. Allan Butterfield 6.1  Introduction to the In Vitro Cellular Assays 145 6.2  Choice of Cell Lines and Animal Models 146 6.3  “Real-Time” Cellular Assays for Detection of Oxidative Stress 149 6.4  Fluorescent Probes and Dyes Based Detections 150 6.5  Seahorse FX Technology Based Assays 157 6.6  EPR Methods 166 6.7  “Static” Assays for Detection of Oxidative Stress 167 6.8 Conclusion 175 Abbreviations 176 References 179

Contents

vii

7. Redox Interactions Between Nanomaterials and Biological Systems 187 Devrah Arndt and Jason Unrine 7.1 Introduction 187 7.2  Oxidative Stress by Inorganic Nanomaterials 189 7.3  Oxidative Stress by Organic Nanomaterials 192 7.4  Oxidative Stress and Nanomaterial Surface Chemistry 194 7.5  Techniques for Evaluating Oxidative Stress Due to Nanomaterial Exposure 196 Acknowledgments 200 References 200

8. Hydrocyanines: A Versatile Family of Probes for Imaging Radical Oxidants In Vitro and In Vivo 207 Kousik Kundu and Niren Murthy 8.1 Introduction 207 8.2  The Hydrocyanines: A New Family of Fluorescent ROS Probes 208 References 221

9. Oxidation State as a Bioresponsive Trigger

225

John R. Martin and Craig L. Duvall 9.1 Introduction 225 9.2  Oxidation-Responsive Polymer Systems: Phase Transition Versus Polymer Degradation 225 9.3  Utilizing Oxidation-Responsive Polymers in Drug Delivery 227 9.4  Utilizing Oxidation-Responsive Polymers in Biodegradable Tissue Engineering Scaffolds 240 9.5 Conclusion 244 References 246

10. Antioxidant Polymers as Biomaterial

251

Robert van Lith and Guillermo A. Ameer 10.1 Introduction 251 10.2  Passive Delivery of Antioxidant Molecules by Polymers 255 10.3  Intrinsically Antioxidant Polymers: Nonenzymatic Antioxidants 263 10.4  Intrinsically Antioxidant Polymers: Enzymatic Antioxidants 274 10.5  Intrinsically Antioxidant Polymers: Metal-Chelating Polymers 278 10.6  In Vivo Oxidative Stress Modulation With Antioxidant Polymers 281 10.7  Conclusions and Perspectives 286 References 289

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Contents

11. Oxidation of Total Joint Implants and Antioxidant Strategies: Designing Implants for Oxidative Stress Resistance

297

Ebru Oral 11.1  A Brief History of Total Joint Implants 297 11.2  Oxidation Mechanisms of Total Joint Implants 299 11.3  In Vitro Simulation of Oxidation and Characterization 303 11.4  The Introduction of Antioxidants into Medical Grade UHMWPE 309 11.5 Conclusion 315 References 315

12.  Targeted Antioxidant Interventions for Vascular Pathologies

323

Elizabeth D. Hood, Vladimir V. Shuvaev and Vladimir R. Muzykantov 12.1 Introduction 323 12.2  Vascular Oxidative Stress and Inflammation in Dangerous Acute Conditions 324 12.3  Markers of Oxidative Stress and Inflammation 326 12.4  Antioxidant Interventions and Untargeted Delivery Systems 329 12.5  Targeted Delivery of AOEs 334 12.6  Conclusion: Challenges and Perspectives 341 References 342

13.  Oral Mucositis as a Target for Antioxidant Biomaterial Therapy

351

Nihar M. Shah 13.1 Introduction 351 13.2  Pathophysiology of OM 352 13.3  Management and Treatment of OM 353 13.4  Oxidative Stress Management and Antioxidant Therapy for OM 354 13.5  A Case for Curcumin as an OM Therapeutic 356 13.6  Challenges With Curcumin Delivery 357 13.7  Advances in Curcumin Delivery Technologies 357 13.8  Curcumin Delivery From Poly(Beta-Amino Ester) Polymers 361 13.9 Conclusion 367 References 368 Index 373

LIST OF CONTRIBUTORS Isaac M. Adjei J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States Michael C. Alstott Markey Cancer Center, University of Kentucky, Lexington, KY, United States Guillermo A. Ameer Biomedical Engineering Department, Northwestern University, Evanston, IL, United States; Department of Surgery, Feinberg School of Medicine, Chicago, IL, United States; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, United States; Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, IL, United States Devrah Arndt Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States D. Allan Butterfield Department of Chemistry, Markey Cancer Center, Spinal Cord and Brain Injury Research Center, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, United States Shampa Chatterjee Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA, United States Craig L. Duvall Department of Biomedical Engineering,Vanderbilt University, Nashville, TN, United States Thomas Dziubla Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, United States Jeremy L. Gilbert Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, United States; Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY, United States; Institute of Medical and Biological Engineering, University of Leeds, Leeds, United Kingdom Aihua Gu State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China Prachi Gupta Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY, United States

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x

List of Contributors

Elizabeth D. Hood Department of Systems Pharmacology and Translational Therapeutics, Center for Translational Targeted Therapeutics and Nanomedicine of the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Gregory W. Kubacki Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, United States; Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY, United States Kousik Kundu Nalco-Champion, An ECOLAB Company, Houston, TX, United States Andrew Lakes Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY, United States John R. Martin Department of Biomedical Engineering,Vanderbilt University, Nashville, TN, United States Mihail I. Mitov Markey Cancer Center, University of Kentucky, Lexington, KY, United States Yiqun Mo Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, KY, United States Niren Murthy Department of Bioengineering, University of California, Berkeley, CA, United States Vladimir R. Muzykantov Department of Systems Pharmacology and Translational Therapeutics, Center for Translational Targeted Therapeutics and Nanomedicine of the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Ebru Oral Orthopaedic Surgery, Harvard Medical School; Harris Orthopaedic Laboratory, Massachusetts General Hospital, Boston, MA, United States Vinod S. Patil Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY, United States Glendon Plumton J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States Nihar M. Shah Bluegrass Advanced Materials, LLC, A268 ASTeCC, Lexington, KY, United States Blanka Sharma J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States

List of Contributors

xi

Vladimir V. Shuvaev Department of Systems Pharmacology and Translational Therapeutics, Center for Translational Targeted Therapeutics and Nanomedicine of the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States David J. Tollerud Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, KY, United States Jason Unrine Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States Robert van Lith Biomedical Engineering Department, Northwestern University, Evanston, IL, United States Qunwei Zhang Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, KY, United States

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PREFACE After decades of free radical biology research and development, the conclusions are clear; there are no simple solutions to oxidative stress relateddisorders. Dietary antioxidants are not enough to stave off the countless diseases that involve excess production of reactive oxygen species. The failed clinical trials have shown that there is no panacea to use for all conditions. Much like the combustive processes involved in oxidation, such disappointments result in scorched earth that turns researchers off from continued development in the area. However, out of the wreckage of these shattered therapeutic dreams emerges a deeper understanding of the way the body deals with oxidation events, inflammation and attempts at reverting to homeostasis. As the entire field of biomaterials explores more deeply the ability to control the inflammatory response, improve material biocompatibility and even delve into the field of regenerative medicine, this understanding provides fertile ground for exploring new ways to improve the materials and devices we create for biomedical applications. With the above paragraph in mind, this book, we believe, serves as a starting point to introduce investigators to the importance of oxidative stress to biomaterials and broaden the work being done in the area. To facilitate this objective, we separated the chapters into three main sections: (1) a background summary of oxidative biology and its links to inflammation and biomaterial biocompatibility; (2) an overview of analytical approaches currently available to characterize oxidative stress responses; and (3) a series of examples of in which researchers have taken advantage of oxidative stress biology to improve biomaterial function. We hope as you read this book you will agree with us that the future of biomaterials must include on some level, a mechanism by which such substances interact with cellular redox systems. We look forward to seeing what new, phoenix-like devices are created out these antioxidant ashes. Sincerely, Thomas Dziubla, D. Allan Butterfield

xiii

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CHAPTER ONE

A Free Radical Primer Prachi Gupta1, Andrew Lakes1 and Thomas Dziubla2 1

Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY, United States Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, United States

2

1.1  FREE RADICAL BIOLOGY—IMPORTANCE Free radicals, represented with a superscript “dot” (A • ), are defined as any atom or molecule containing an unpaired electron, which has a strong tendency to gain another electron to achieve a nonradical state [1]. These molecules are considered to be highly reactive and are capable of reacting with a nonradical molecule in their quest for self-stabilization. Radical species can be formed through a variety of mechanisms, one of which is abstraction of an electron from an atom or a molecule. Radicals can also be generated by the splitting of a molecule at a very high-energy state. A classic example would be radiation-induced homolysis of a water molecule into a hydroxyl radical and a hydrogen atom [2] (Eq. (1.i)) H 2O + eV → H• + OH• (1.i) As these radical ions exist in a high and unstable potential energy state, they can react in variety of ways. For example, two radical species can react with each other to form a nonradical molecule or one radical can donate an electron to another yielding two stable compounds [3]. As oxygen serves as the primary player in most biological free radical reactions, free radicals are also commonly known as reactive oxygen species (ROS). In a similar way, nitrogen base free radicals are called reactive nitrogen species (RNS). Table 1.1 lists some of the most common free radicals/oxidants/ROS important in biology. Most of the essential cellular matrix components (protein, cellular membrane, DNA, lipids, PUFAs (polyunsaturated fatty acids), etc.) in physiological systems are stable nonradical entities and perform their regular function of energy production in the form of ATP and maintain the cellular redox balance. But, the presence of more than the basal level of free radical molecules/ROS can lead to reactions of ROS with alternative cellular components in a quest to stabilizing themselves, damaging the chemical integrity of cellular Oxidative Stress and Biomaterials. Doi: http://dx.doi.org/10.1016/B978-0-12-803269-5.00001-2

© 2015 2016 Elsevier Inc. All rights reserved.

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

Table 1.1  Potential Free Radical Species ROS/RNS Symbol

ROS/RNS

Symbol

Superoxide Hydroperoxyl Nitric oxide

O2− • OOH NO•

Hydroxyl Peroxyl Nitrogen oxide



Peroxynitrite Hypochlorous acid

ONOO− HOCl

Singlet oxygen

1

LH+OH•

OH ROO• NO•2 O2

L•+H2O O2 LOO•

LOOH LH

Figure 1.1 Lipid peroxidation cycle in presence of excess free radicals, specifically hydroxyl radical ( OH • ).

biomolecules [4,5]. One well-known example is lipid membrane damage via lipid peroxidation, where the hydroxyls radical react with PUFAs of the cellular membrane, extracting an electron and yielding a lipid free radical. If this reaction phenomena is not controlled in a timely manner, it can initiate a chain reaction of free radical molecules with intact lipids resulting in overall cellular membrane damage [6] (Fig. 1.1). Although most of the research concerning the role of ROS/RNS has been done towards the potential cellular damage and subsequent pathological events, they do serve beneficial role to the body under certain conditions. When present at optimum concentrations, they help maintain the redox homeostasis in the cellular environment that regulates cell functioning and cell signaling and responds to endogenous and exogenous stimuli [7]. Under normal physiological conditions, a balance between generation and elimination of ROS/RNS via endogenous antioxidant enzymes/ molecules helps the redox-sensitive signaling proteins to function properly [8]. Endogenous ROS generating enzymes like myeloperoxidase and NADPH oxidase (NOX), also known as phagocyte oxidase, actually help the neutrophils perform their phagocytic function against microbial intrusion. In a process known as the respiratory burst, NOX-catalyzed superoxide production further forms hydrogen peroxide with the help of superoxide

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A Free Radical Primer

dismutase (SOD), which in turn converts to hypochlorus acid (HOCl), which has strong bactericidal properties [9,10]. oxidase SOD • NADPH + O2  NADPH  → O− → 2  

Myloperoxidase → HOCl H 2O 2    (1.ii)

Indeed, the dual nature of biological oxidation and reduction processes is the very reason for its importance in biomaterial/tissue interaction. Through a proper understanding of the underlying chemistry, it is possible to design next generation materials which can harness this intrinsic biological signaling mechanism. This chapter will cover the key points in free radical chemistry, the various pathways and vocabulary and finally, the way these processes occur in a biological setting.

1.2  RED/OX CHEMISTRY 1.2.1  Oxidation/Reduction Reactions and Voltage Potentials Originally, the term “oxidation” was described as a process of any element, primarily metals, to combine with oxygen to form metal oxides and “reduction” defined as a process that will convert the metal oxide back to pure metal. For example, conversion of magnesium (Mg) to magnesium oxide is oxidation, while smelting of magnesium oxide to magnesium at high temperature in presence of carbon is reduction. Later, the discovery of electrons changed the definition of oxidation–reduction to the transfer of electrons from one species to another. As per the law of conservation of mass applied to electrons, oxidation and reduction are always linked to one another. Meaning, if one species is oxidized, the counter reactant species will be reduced [11,12]. 2Mg + O2 → 2MgO Oxidation (1.iii) MgO + C → Mg + CO Reduction (1.iv)

By definition, oxidation of any given element or molecule will involve a loss of electron and the element/molecule will be known as a reducing

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

agent while the reduction of any molecule will be a gain of electron and that molecule will be known as an oxidizing agent [13]. In reference to free radicals or ROS they are commonly known to be strong oxidizing agents and have a tendency to get reduced [14,15]. This is because most radicals have one unpaired electron, and the addition of another electron (a.k.a. getting reduced) with the opposite electron spin would help in stabilizing the electron pair, taking them to a more inert state. However, this is not always true. Oxidizing tendency of any free radical will depend on its affinity to gain an electron or its own reduction potential against the affinity or potential of the coreactant. It is possible that free radical “A” gets reduced in presence of “B” but might get oxidized itself by losing another electron in presence of “C” because the reduction potential or electron gaining affinity of “C” is greater than “A.” 1.2.1.1  Types of Redox Reactions 1.2.1.1.1  Corrosion and Rusting

Iron has served as a common example of corrosion, which is the electrochemical oxidation of metals in presence of oxygen to form respective oxides. In reference to iron, it is specifically termed as formation of “rust” (Eq. (1.v)) [16]. 4Fe + 3O2 → 2Fe2O3 Oxidation of Fe to Fe3 + (1.v) In presence of an acid, iron (II) is oxidized to iron (III) by reaction with hydrogen peroxide, which acts as an oxidizing agent [17], although iron (III) can be reduced back to iron (II) in the presence of stronger reducing agents or free radicals, such as superoxide anion [18]. This oxidation–reduction chemistry of iron with hydrogen peroxide is popularly known as the Fenton and Haber–Weiss reaction mechanisms (see Section 2.4) and are critical in maintaining the redox state of the cell. 2Fe2 + + H 2O2 + 2H+ → 2Fe3 + (oxidized ) + 2H 2O(reduced)Oxidation of Fe 2 + toFe3 + (1.vi) 1.2.1.1.2  Nitrification and Denitrification

Nitrification often occurs naturally and is a biologically oxidative process where ammonia is oxidized to nitrite followed by formation of nitrate by nitrifying bacteria. On the other hand, reduction of nitrate to nitrogen in the presence of an acid is termed as denitrification and is often used as water purification process [19]. These nitrates have the ability to diffuse

A Free Radical Primer

5

through the cellular membrane and play a significant role in the production of RNS in a cellular environment. + Nitrification : NH3 + O2 → NO− 2 ( oxidized ) + 3H (1.vii) + 2e−( ammonia to nitrite) − + − (1.viii) NO− 2 + H 2O → NO3 ( oxidized ) + 2H + 2e ( nitrite to nitrate )

− + Denitrification : NO− 3 + 10e + 12H → N 2 ( reduced ) (1.ix) + 6H 2O (oxidized)

1.2.1.1.3  Dismutation Reaction

Dismutation or disproportionation is a specific kind of redox reaction, where both oxidized and reduced forms of a chemical species are produced. For example, superoxide free radicals produced in mitochondria dismutate to hydrogen peroxide and oxygen (Eq. (1.x)) [20] or ascorbyl radical to ascorbate (vitamin C) and dehydroascorbate (DHA) (Eq. (1.xi)) in order to maintain intracellular nutrient requirements for the cells [21,22]. + 2O− → H 2O2 ( reduced ) + O2 (oxidized ) (1.x) 2 + 2H

(1.xi) 2 ascorbyl • + H+ → ascorbate(oxidized) + DHA(reduced) 1.2.1.1.4  Cellular Respiration

Oxidation of glucose to carbon dioxide with simultaneous reduction of oxygen to water is another example of natural oxidation–reduction reaction, and is required for energy production in living organisms [23]. C6H12O6 + 6O2 → 6CO2 + 6H 2O (1.xii) All the reactions shown (Eqs. (1.v)–(1.xii)) and their tendency to either undergo oxidation or reduction are controlled by their reduction potential. Reduction potential (Eo) is defined as a tendency of a chemical species to be reduced by gaining an electron and is defined with electrochemical reference of hydrogen, which is globally given the reduction potential of zero [24]. As this is an electric potential, it is measured in volts and each chemical species has its own intrinsic reduction potential. Numerically, the more positive the potential, the stronger the affinity of the species is to acquire an electron and get reduced.

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1.2.1.2  Redox Potential A normal redox reaction example could be as given below (Eq. (1.xiii)): (1.xiii) Fe3 + + Cu+ → Fe2 + + Cu2 + and this can be broken down into two parts: (1.xiv) Fe3 + + e− → Fe2 + Reduction (1.xv) Cu+ → Cu2 + + e− Oxidation For a combined redox reaction, overall redox potential is estimated by o o ∆E = E acceptor − Edonor (1.xvi)

Electrochemical potential or reduction potential stated above is directly related to the Gibbs free energy (∆G ) of the reaction: (1.xvii) (∆G ) = −nF ∆E n, number of electrons associated with the reaction F, Faraday’s constant. For a reaction to proceed, total Gibb’s free energy (∆G ) must be negative or ∆E should be positive. In Eq. (1.xv), Cu is the electron donor with redox potential of Cu2+ to Cu+ is + 0.16 V and that of Fe+ to Fe2+ is 0.77 V. Therefore, overall redox potential becomes + 0.61 V. To state this simply, for a system to undergo a redox reaction, the redox potential of a species to be reduced should be higher than the species to be oxidized. Table 1.2 lists some of the common redox potential of certain half-cell redox couples and of some important biomolecules important to physiological redox environment.

1.2.2  Thermodynamic Treatment (Ellingham Diagram) Voltage potential is not the only way to determine the direction of reaction. As stated earlier, for a reaction to proceed, ∆G should be negative which requires ∆E to be positive, but ∆G is also a function of temperature (Eq. (1.xviii)). An alternative method to determine the direction of a reaction can be obtained by a simplified thermodynamic analysis of the reaction equilibrium. (1.xviii) ∆G = ∆H − T ∆S where T, reaction temperature

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A Free Radical Primer

Table 1.2  Half-Cell Reduction Potentials of Biologically Significant Molecules Redox Couple Eo (V)

+0.816

O2 + 4H+ + 4e− → 2H 2O −

NO2 + e



NO− 3

+

+1.04

NO− 2 −

+ 4H + 3e +

→ NO + H 2O

+0.96

→ 2H 2O

+0.82



H2O2 + 2H + 2e

Fe3 + + e− → Fe2 +

+0.77

+

+0.68



O2 + 2H + 2e

→ H2O2

2I2 + 2e− → 2I−

+0.54

2H2O + O2 + 4e− → 4OH−

+0.40

2Cu

2+



+ 2e

2 cytochromec

+0.34

→ Cu 3+

2 cytochrome b

3+



+ 2e

2H + 2e +

+0.070

→ 2 cytochrome b



→ succinate

Fumarate + 2H + 2e −

+0.254

2+



+ 2e

+

+

→ 2 cytochromec

2+

+0.031 0.00

→ H2 +



FAD + 2H + 2e

→ FADH 2 ( free coenzyme )

−0.22

FAD+ + 2H+ + 2e− → FADH 2 ( in flavoprotein ) −0.166

Oxaloacetate + 2H+ + 2e− → malate +



Pyruvate + 2H + 2e +

+



NAD + 2H + 2e +

+

→ NADH + H −

NADP + 2H + 2e −

O2 + e



−0.185

→ lactate +

−0.320

+

→ NADPH + H

+

−0.324 −0.33

O− 2 +



Succinate + CO2 + 2H + 2e Na + + e− → Na

→ α − ketoglutarate + H2O

−0.324 −2.71

ΔS, entropy change ΔH, enthalpy change. The temperature dependence of a reaction using Eq. (1.xviii) is also utilized in redox chemistry to drive the direction of reactions of metal oxides and sulfides to pure metal. This phenomenon is usually illustrated in the form of Ellingham’s diagram, which represents the stability of a metal oxide as a function of temperature in reference to ΔG (Figure 1.2). Free energy versus temperature plot for a particular metal shows the free energy values

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

for its metal oxide formation where slope depicts the ΔS values while the y-intercept gives ΔH. The position of the line of a metal with reference to another helps in depicting the metal oxide reduction ability in presence of another metal. To illustrate, the lower the position of metal ( Mg + O → MgO) is on Elligham’s diagram (Fig. 1.2), the more stable its oxide is at a particular temperature than the one that lies above it ((4 / 3)Al + O2 → ( 2 / 3)Al2 O3 ). Therefore, magnesium can reduce aluminum oxide to metallic aluminum. Looking at the diagram, all the metal H2/H2O ratio

–8

10–4

10–2

–2

10

2 +O O4 e3 M 4F

100

200 4Cu

+ O2

U O = 2C 2

1

=6

1

M

–2

10

O

M

NiO

O2

i+

2N

300

2Co

+ O2

o = 2C

1

M

=2

–4

10

M

2Fe +

O2

eO = 2F

2

10

–6

10 2

10

400 O

Zn

500

O2

+ Zn

=2

2

M

C

OK

10 10–6

O3 Fe 2

2

Standard free energies of formation of oxides (–∆Gº = RTIn pO ) kJmol–1 O2

H

pO2

10–4

–6

10 10–8

CO/CO2 ratio

600

O n+ 2

Si +

/3

–8

10

M

4

10

M M

nO

= 2M

2M

700

O3 Cr 2

=2

O2

r+

C 4/3

M M MnO

B

10–10

B

iO 2

O2

=S

–12

iO 2

O2 Ti +

10

6

=T

10

800

l 2O 3 /3 A

l 3A

4/

900

=2

O

Mg

M + Mg

1000

+ O2

O2

2

M

=2

O2 a+

6

10

B

8

B

O

Ca

=2

10

10–14

–16

10

2C

Change of state

M

Element

Melting Point Boiling Point

1100

Oxide

8

10

M B

M B

–18

10

10

10 1200

200

0

400

600

800

1000 1200 1400 1600 Temperature (ºC)

1800

–200

14

–100

10

–70

10

–60

10

–50

10

–42

10

–38

10

12

10

–34

10

–20

10

2200 2400

10

CO/CO2 ratio H2/H2O ratio 10

2000

–30

10

–28

10

10

10

–26

10

–22

10

–24

10

Figure 1.2 Ellingham’s diagram. The figure is reproduced with permission from the University of Cambridge Dissemination of IT for the Promotion of Materials Science (DoITPoMS) website. (www.doitpoms.ac.uk).

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9

oxide formations have a positive slope while carbon oxidation to carbon monoxide has a negative slope and it cuts across many of the metals at particular temperatures. Therefore, carbon becomes a very useful reducing agent for metal oxides at higher temperatures. For example, carbon can reduce manganese oxide (MnO) to its metallic form Mn once the reaction temperature goes above 1400°C while it will be able to reduce TiO2 to Ti above 1600°C [25–27]. As highlighted here, while the reduction potential of two reacting molecules decides their affinity to get reduced or oxidized, it is important to remember that the change in other system properties (eg, temperature) can alter the fate of one species getting reduced in presence of another.

1.2.3  Combustion Sequences and/or Metal Oxides Combustion is a classic example of free radical reactions and generation. It is a high temperature exothermic process involving multiple redox reactions of a fuel (hydrocarbons) with an oxidant mostly oxygen resulting in oxidized products primarily carbon dioxide and water, to generate heat and light. The overall combustion process of oils is described by the reaction:  n + 1 Cn H 2( n +1) + 4n +  O2 → nCO2 + (n + 1)H 2O (1.xix)  2  While this equation is a useful simplification of the combustion process, the exact chemistry of combustion is highly complex, multifaceted, and not easy to describe. Molecular oxygen in its ground state is a very stable molecule and unreactive to hydrocarbons until a catalyst is introduced. However, at elevated temperatures as high as 2200°C, oxygen converts into highly reactive singlet oxygen (O21) [28] and is known to have high oxidizing power. Singlet oxygen can react with and break carbon–carbon or carbon–hydrogen bonds of large hydrocarbons into smaller molecules and, subsequently, hydrogen and water. In the process, it is also capable of initiating numerous radical chain reactions via reaction with molecular hydrogen (H2) resulting in hydroxyl radical (OH• ) and proton (H+). Combination of many such reactions results in the generation and simultaneous consumption of hydroperoxyl (HCOO• ), formyl (HCO• ) radicals, and carbon monoxide [29,30]. As for hydrocarbon pyrolysis, the process involves generation of various aliphatic and aromatic radicals.

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Below are some of the common series of radical reactions that can occur during combustion process: O2 + H+ → O•− + OH• (1.xx) O12 + H 2 → H+ + OH• (1.xxi) C3H8 + M → C2H5• + CH•3 + M (1.xxii) C2H•5 + M → C2H4 + H+ + M (1.xxiii) C3H8 + OH• → C3H•7 + H 2O (1.xxiv) C3H8 + H+ → C3H•7 + H 2 (1.xxv) C3H8 + O•− → C3H•7 + OH• (1.xxvi)

C3H•7 → C3H6 + H+ (1.xxvii)

Decay tounsaturated hydrocarbons These radicals react with oxygen radical to produce formyl radical and formaldehyde and CH•3, CH2, H2CO finally oxidize to CO2 and H2O. C3H6 + O•− → C2H5 + HCO (1.xxviii) C2H4 + O2 → 2CO + H 2 (1.xxix) CO + (1 / 2)O2 → CO2 (1.xxx) Looking at all the reactions that are involved in the process of producing heat energy via oxidation of long/short chain hydrocarbon fuels, it is clear that a milieu of free radicals are generated. Yet, a spatial relationship also exists in radical production, which is described by the “zone theory.” This concept divides the combustion process into four zones: zone 1-free flame zone (fuel zone), zone 2-high temperature flame zone (>1200°C), zone 3-postflame thermal zone (600–1200°C), and zone 4-gas quench cool and surface catalysis zone (1200°C Hydrocarbon + O2 = heat energy

Free radicals

Incomplete reaction Postflame zone 600° > T >1200°C partial combustion NO, semiquinone, phenoxyl radicals

Oxidative phosphorylation

Partial oxygen reduction/ proton leak

Carbohydrates+ O2 = ATP (cellular energy)

(O2+e– = O2–) Oxygen reduction to superoxide

Cool zone T < 600°C Fe2+/ Cu2+

EPFRs

ROS/RNS production 2+ H2O2 Fe / Cu2+

.

OH– + OH

Figure 1.3  Analogous comparison of various stages of combustion zone theory with cellular respiration during energy production along with free radical generation [27].

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

formation. In both cases, the role of radicals produced at this stage are boosted by the presence of transition metal ions. Transition metal ion combination with radicals makes more environmentally stable EPFRs and are reactive to other biological species. During cellular metabolism, transition metal ions act as a catalyst towards production of more reactive and damaging oxidants namely hydroxyl radical (OH• ).

1.2.4  Fenton/Haber–Weiss Chemistry Both the Fenton and Haber–Weiss reactions are associated with iron and the production of hydroxyl radicals. The Fenton reaction describes the • formation of hydroxide (OH−) and hydroxyl (OH ) radical by a reaction between Iron (II) (Fe2+) and hydrogen peroxide (H2O2) [33], Haber– Weiss reaction is where hydroxyl and hydroxide ions are generated from •− the reaction of H2O2 and superoxide ion (O2 ) catalyzed by iron [34]. Fe2 + + H 2O2 → Fe3 + + OH− + OH• O•2− + H 2O2 → OH− + OH• + O2

Fenton reaction (1.xxxi)

Haber - Weiss reaction (1.xxxii)

The Haber–Weiss cycle is actually a two-step reaction, where the ferric ion reduces to ferrous ion via reaction with superoxide, which, in turn, reacts with H2O2 to form OH− and OH• ions, converting ferrous back to ferric ion. 3+ O•− → Fe2 + + O2 (1.xxxiii) 2 + Fe

Fe2 + + H 2O2 → Fe3 + + OH− + OH• (1.xxxiv) Henry J.H. Fenton reported for the first time the oxidation power of H2O2 and Fe2+ towards tartaric acid in 1876 but never mentioned the existence of the hydroxyl radical intermediate in the oxidation process, • although the reaction was named after him [35]. The existence of OH was proposed by two German chemists, Fritz Haber and Joseph Joshua Weiss in 1934 via reaction of hydrogen peroxide and superoxide in presence of iron as a catalyst [36]. Formation of this hydroxyl radical via transition metal catalyzed reaction has gained plenty of attention over the years and is considered to be one of the key processes for the production of the highly reactive OH• in the cellular redox chemistry. Not only hydroxyl radicals but also Fenton/Haber–Weiss chemistry gives rise to several other intermediates including hydro peroxides (HOO• ), superoxide (O2−), etc. during various chain initiation and

A Free Radical Primer

13

propagation reactions shown in Eqs. (1.xxxv)–(1.xliii). OH• acts as the chain carrier with the ability to react with Fe2+, H2O2, or any organic species to propagate the reaction. But in some instances, chain termination also comes into effect via combination of two radicals (OH• / OH• or OOH• / OH•) producing just hydrogen peroxide, water, and oxygen as shown in Eqs. (1.xlii) and (1.xliii) [37]. The existence and propagation of any of these reactions highly depends on the density of iron in its required oxidation state as well as the rate constant. An important condition for iron-induced reduction of hydrogen peroxide is the low pH requirement between 3 and 6. Fe2 + + H 2O2 → Fe3 + + OH− + OH• (1.xxxv) K = 5.7 × 102 M−1 s−1 Fe3 + + H 2O2 → Fe2 + + H+ + OOH• / O− 2 −3 −1 − 1 (1.xxxvi) K = 2.6 × 10 M s OH• + H 2O2 → OOH• / O− 2 + H 2O 7 −1 − 1 (1.xxxvii) K = 3.3 × 10 M s 2+ Fe3 + + OOH• / O− + O2 + H + 2 → Fe (1.xxxviii) K = 3.1 × 105 M−1 s−1

Fe2 + + OH• → Fe3 + + OH− (1.xxxix) K = 3.2 × 108 M−1 s−1 3+ Fe2 + + OOH• / O− + H 2 O2 2 → Fe 6 −1 − 1 (1.xl) K = 6.6 × 10 M s − • OOH• / O− 2 + OOH / O2 → H 2O2 (1.xli) K = 2.3 × 106 M−1 s−1 • OOH• / O− 2 + OH → H 2O + O2 (1.xlii) K = 8.9 × 109 M−1 s−1

OH• + OH• → H 2O2 (1.xliii) K = 5.2 × 109 M−1 s−1

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

Chain propagation and termination reactions associated with iron and hydrogen peroxide with rate constant measured at pH = 5 [38–40]. Fenton chemistry is commercially utilized to treat water pollution, contaminated soils, sludge, etc. by oxidizing the pollutants such as benzene, formaldehyde, rubber chemicals, and pesticides. The rate constant for the initial reaction between Fe2+ and H2O2 is generally observed to be around 102 M−1 s−1 but in biological systems, this rate of reaction with free Fe2+ is not enough for the oxidation to occur. However, when bound to ADP, ATP, or citrate, the oxidation rate increases by at least 2 orders of magnitude, resulting in a reaction rate fast enough for the iron-catalyzed redox process to occur [41].

1.2.5  Thiol Chemistry (–SH;–SS–) Thiol–disulfide reactions are one of the most important biological redox reactions. In biology, oxidation/reduction governs the metabolic redox state of a cell. Thiol–disulfide interchange/exchange reactions are significant in itself where a thiol (RSH) reacts with another disulfide containing molecule (R′SSR″) to give a new oxidized disulfide (R′SSR) and the corresponding reduced thiol (R″SH) [42]. The reaction is base catalyzed and is proposed to proceed through SN2, 2 step reactions as follows [43,44]: RSH  RS− + H+ (1.xliv) RS− + R′SSR″  R′SSR + R″S− (1.xlv) H+ + R″S−  R″SH (1.xlvi) d[ R″RS− ] = kRS− [ RS− ][ R′SSR″] (1.xlvii) dt d[R″S− ] = kobs [ RSH ]total [ R′SSR′ ] (1.xlviii) dt kRS− = kobs (1 + 10 pK a − pH ) (1.xlix) where R, attacking group; R′, central group; R″, leaving group

A Free Radical Primer

15

kRS− , calculated rate constant dependent on thiolate concentration but independent of pH kobs, observed rate constant dependent on total thiol concentration and pH. In this reaction mechanism, the thiolate anion (RS−) acts as an active nucleophile or reactive species to propagate the reaction. In the cytosol, glutathione plays a key role in the thiol redox chemistry as it is the most abundant small molecule cellular antioxidant (see Section 1.3.2 for an in-depth discussion on glutathione redox reactions). One of the important factors that contribute towards the occurrence of this thiol exchange reaction is the pKa of the corresponding thiol and the pH of the reaction environment [45]. For example, a thiol with pKa value of 10, 0.1% of the total thiol will form thiolate at pH 7 while at pH 8, 10 times higher thiolate ions would be present in comparison [43]. In other words, we can say that thiol–disulfide interchange is most favorable at pH near to the pKa values of the thiol. Oxidation of glutathione (GSH) (pKa = 9.33) to its oxidized form (GSSG) is faster at higher pH of 9.43 with rate constant k = 45 L/mol s as compared to at pH of 8.46 with k = 9.1 L/mol s [46]. As the pKa is a function of the structure of thiol molecule, chemical properties of thiol molecules involved in the reaction will dictate the extent of reaction and their existence in reduced or oxidized form. For instance, the reaction of 2-mercaptoethanol (pKa = 10.14) with Ellman’s disulfide (5-(3-carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid) (pKa = 4.5) is faster than mercaptoethanol with oxidized glutathione (GSSG) (pKa = 9.33) by the order of 104 in water [47,48]. Also, the exchange reaction is faster when the pKa of a nucleophilic thiol (RSH) is as high as possible and that of the corresponding disulfide molecule (R′SH/R″SH) is as low as possible. Stearic interference is another factor that determines the rate constants of the thiol–disulfide exchange reactions. It is most pronounced when there is any carbon substitution at α-position to sulfur. For instance, reaction of bis (t-butyl) disulfide with 1-butylthiolate (k = 0.26 M−1 s−1) is 106 times faster than that with t-butylthiolate (k = 10−7 M−1 s−1). Similarly, charge on the thiolate anion also affects the rate constants [49]. The effect is higher when the charged entity is near to the sulfur group [50]. The rate of reaction is often correlated with the Brønsted plot, which displays the relationship between pKa of the reacting thiols/ disulfide and the rate constant. The slope of the plot is defined by the

16

Prachi Gupta, Andrew Lakes and Thomas Dziubla

13. Thiol-disulfide interchange 1.0

4

3

0.6 2 0.4

log KRS–

θ = [RS–]/([RS–]+[RSH])

0.8

1

0.2

0

0.0 5

6

7 K p a

8

9

Figure 1.4  Correlation between pKa of reacting thiol and rate constant (kRS−)/degree of dissociation (θ) where kRS− was calculated using Eq. (1.l), considering pKa of R′SH and R″SH groups as 8.5. This figure is reproduced with permission from [43].

Brønsted coefficient (β), which normally lies between 0.4 and 0.5 for different thiol–disulfide exchange reactions. Eq. (1.l) is generally recommended for calculating kRS− values for the thiolate content [51] and Fig. 1.4 shows the graphical correlation between pKa of thiol versus log(kRS− ) at pH 7 for a thiol–disulfide exchange reaction. Fig. 1.4 also shows the degree of dissociation (θ) of the thiol and observed rate constant for the thiol/disulfide exchange reaction are predicted by Eq. (1.li). Therefore, the highest observed rate constant for an exchange reaction is RSH observed at a pK a that is equivalent to the pH of the solution, pH = 7 in the figure.

log(kRS−) = 6.3 + 0.59pK a RSH − 0.40 pK a R′SH − 0.59pK a R″SH (1.l) kobs = θkRS− (1.li)

A Free Radical Primer

17

1.3  BIOLOGICAL OXIDATION EVENTS 1.3.1  Oxygen and Nitrogen Currency Oxygen and nitrogen are the most abundant diatomic gaseous molecules in the atmosphere and play a significant role in regulating both, human and plant metabolism. Nitrogen is known to be highly inert and even oxygen (O2) in its ground state is a stable molecule. But O2 at a higher energy state form such as singlet oxygen (O21) [52] or in the reduced form of superoxide (O•− 2 ), it can become an active source of free radical generation leading to disruption of the cellular redox state [53], while oxide forms of nitrogen (NO, NO2) are generally produced enzymatically and can readily diffuse through lipid membranes and lead to formation of intracellular free radicals or RNS. Indeed, these processes has led to the Superoxide Theory of Oxygen Toxicity which states that the partially reduced form of oxygen, ie, superoxide via enzyme, auto-oxidation, mitochondrial electron transport chain, heme proteins, etc. is a primary cause of cellular toxicity [54]. Oxygen, upon its one electron reduction to superoxide, form can further yield hydrogen peroxide via the dismutation process [55]. Hydrogen peroxide can reduce to hydroxyl radicals in the presence of transition metal ions like Fe2+/Cu2+, a highly reactive radical in cellular biology capable of reacting with DNA, lipid membranes, proteins, etc. [1]. Reactivity and reduction potential at various stages of oxygen radical production chain are different and are often enzyme catalyzed. For example, dismutation of superoxide to hydrogen peroxide mainly occurs via SOD-catalyzed reaction [56,57], but a part of hydrogen peroxide production also takes place via a two-step process of oxygen conversion to peroxyl radicals followed by further reduction to H2O2. In the oxygen-derived free radical production chain, H2O2 is not considered a highly reactive molecular species but serves as an intermediate to the production of hydroxyl radical (OH• ) [58] via Fenton reaction, which is highly reactive towards all the cellular components and can lead to complete cell damage. Similarly, superoxide ion by itself is not very reactive towards nonradical species but it can react very quickly with NO• or phenoxyl radicals [59]. This radical has a unique selectivity towards its oxidizing properties. For example, it does not readily react with free NADH/NAD+ but can easily oxidize enzyme-bound (lactate dehydrogenase) NADH to NAD+ [60]. Apart from being an oxidizing agent (oxidation of ascorbate to ascorbyl radical), superoxide can also serve as a reducing agent where it can reduce cytochrome c or Fe3+ to Fe2+ which is one of the steps in cellular redox cycling discussed later [61,62].

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

Singlet oxygen 1

O2

hv E°= –0.95V

Dioxygen O2

Superoxide radical e– E°= –0.33V

O2•–

Peroxide radical

e–

O22– K = 4.5×105 M–1S–1 E°= +0.94V

H+

E°= –0.46V

HO2•

O2•– +H+ K = 8×107 M–1S–1 E°= +1.06V

Peroxyl radical

2H+

H2O2

Fe2+ K ⇒ 2×109 M–1S–1 E°= +0.32

Hydrogen peroxide

OH•

Hydroxyl radical

Figure 1.5  Free radical production/reactive oxygen species with oxygen as a precursor [1].

In reference to biological free radical production, oxygen nucleated free radicals are produced from all the oxygen that is inhaled or consumed by organisms and mitochondria is considered to be the main source of superoxide radical production due to oxygen leakage in electron transport chain during ATP production and is reported to be produced by complexes I and III [63–65]. Reported in vitro studies show that about 4% of the oxygen consumed is converted into superoxide, though in vivo studies have reported about 10-fold lower production, which is still a significant amount of ROS and translates to about a 10-µM intracellular concentration of superoxide [66]. This charged radical readily diffuses through mitochondrial membranes and can react with biomolecules like proteins, PUFAs, or produce other reactive species (H2O2, OH•) (Fig. 1.5). Although gaseous nitrogen is an extremely stable and inert molecule, nitrogen in its oxide forms, such as nitric oxide (NO), acts as a precursor to a variety of free radicals collectively known as reactive nitrogen species, which are equally capable of carrying out cell damage if produced in excess. Nitric oxide ( NO• ) due to its lipophilic property readily diffuses through cell membranes from the atmosphere where it is a byproduct of various combustion processes [67]. More importantly, in the cellular environment, it is produced as a result of the reaction of l-arginine and oxygen with NADPH in the presence of nitric oxide synthase (NOS) to give nitric oxide, l-citrulline, and NADP+ [68]. Nitric oxide by itself is not very reactive towards nonradical species similar to superoxide anion but can generate highly reactive free radicals such as nitrogen dioxide (NO•2 ) via a slow reaction with oxygen (O2) [69]. NO•2 with the Eo value of 1.04 V is a potent oxidant in metabolic redox system and in RNS chain, it further reacts with NO• leading to production of N2O3, which eventually decomposes to give another reactive nitrite (NO2−) free radical [70]. In the presence of superoxide, NO• reacts to form peroxynitrite (ONOO−),

19

A Free Radical Primer

O2

NO

NO

2NO + O2 L-arginine

+ NADPH + O2

2NO 2

N2O3 RSH/RNH2

O2– NO + NO2– ONOO–

NO2–

OONOH

NO2

NO2

ONOO–

OH

Thiol nitrosation (RSNO, RSOH) Nitrosation of amine RSH/RNH2

Figure 1.6  Reactive nitrogen species production.

which in itself is also a powerful oxidizing molecule [71,72]. It is present in the form of acidified peroxynitrous acid (ONOOH) or its activated form ( • NO2 … OH• ), both forms are capable of oxidizing most biological molecules such as DNA, proteins, and lipids. NO• also takes part in the modification of some biological molecules with thiols or amine groups during the process called nitrosation via direct reaction with thiols or via a metal bound NO• reaction with thiols/amines, resulting in formation of nitrosothiols (RSNO) or nitrosamine (RN2O), which are also regarded as nonradical RNS [73,74] (Fig. 1.6).

1.3.2  Cellular Redox Chemistry Within the cellular environment, there exists an equilibrium between oxidants and antioxidants. Major players of these processes are defined in Fig. 1.7. Of particular note is the small molecule thiolated antioxidant, glutathione (GSH), which is the most abundant small molecule in eukaryotic cells, roughly found intracellulary from 1 to 10 mM and about 1 to 10 µM extracellular [75–78]. Synthesized within the cell from the three amino acids, glutamate, glycine, and cysteine, GSH production is rate limited by cysteine content, a semi-essential amino acid in biology. Cysteine transported from extracellular space in the disulfide form, cystine [79].

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

G6PD HNO3

NADP+

NADPH

AO/GSH Glu, Cys, Gly ONOO– NOS Arg O2

NO XO COX

NADPHOX

GSS

LPO, PCO

GSSG

2GSH

AO/ GSH O2– OH Fe2+ Cu2+ SOD

GST GSH GS-LPO GS-PCO

GR

GCL

H2O

L, P CAT

GPx ROOH, RH, H2O, O2 H2O2 Cl–

H2O + O2

MPO HOCl

GSH/AO

GSA, HCl, H2O

Abbreviations: AO, antioxidant CAT, catalase G6PD, glucose-6-phosphate dehydrogenase GCL, glutamate cysteine ligase GPX, glutathione peroxidase GR, glutathione reductase GSA, glutathione sulfonamide GSH, glutathione GSS, glutathione synthetase GSSG, glutathione disculfide GST, Glutathione S-transferase L, lipid LPO, lipid peroxide MPO, myeloperoxidase NOS, nitrous oxide synthase P, protein PCO, protein carbonyl SOD, superoxide dismutase

Figure 1.7 Significant cellular redox molecule interactions (green, glutathione processes; red, oxidant species reduced by glutathione processes). (For interpretation of the references to color in this figure legend, see the color plate.)

Reaction of cysteine with glutamic acid in presence of glutamate– cysteine–ligase (GCL) followed by the reaction with glycine via glutathione synthetase (GSS) results in GSH production [79]. GSH may act in several mechanisms as an antioxidant, whether directly through electron donation to ROS/RNS in the intracellular or extracellular space, or via enzymatic routes such as with glutathione S-transferase (GST) to irreversibly reduce toxic species like lipid peroxides and protein carbonyls [80]. In addition to direct chemical reaction with oxidative species, GSH participates in several mechanisms to regenerate other important antioxidant enzymes such as glutathione peroxidase (GPx), glutaredoxin (GRX), and peroxiredoxins nonspecifically. Oxidized glutathione (GSSG) may be reduced back to GSH via glutathione reductase (GR), which is driven by NADPH and the glucose-6-phosphate dehydrogenase (G6PD) cycle via oxidation of NADPH to NADP+. There are also other thiol-based oxidoreductases such as thioredoxin, which utilizes cysteine thiol–disulfide exchange (instead of GSH) and is regenerated with thioredoxin reductase, similarly utilizing NADPH for regeneration akin to GSSG with GR. Depending on the cellular compartment, redox equilibrium may vary. For instance, the cytosol (GSH:GSSG 100:1) and nucleus (GSH:GSSG > 100:1) maintain a reducing environment [76,81], whereas there are high concentrations of oxidants in the endoplasmic reticulum (GSH:GSSG of 1–3:1 [82], where disulfide bonds fortify the

A Free Radical Primer

21

protein structures), mitochondria (GSH:GSSG of 20–40:1 [81]), secretory pathways, and extracellular space (GSH:GSSG 20:1 [78]). It is important to note that extracellularly, while the GSH:GSSG ratio is around 10–20:1, cysteine and cystine are in greater concentration by about an order of magnitude, and maintain the oxidative environment around cysteine:cystine of 0.2:1 [78]. In a similar manner, NADPH is a reducing coenzyme involved in processes like fatty acid synthesis and is required to drive various redox reactions in the metabolic pathway. Therefore, NADPH:NADP+ (200:1) ratios are found to be in the same order of magnitude as GSH/GSSG redox equilibrium to drive the reaction forward. On the other hand, another coenzyme NADH plays an essential part in both reduction and oxidation in general, hence a significant concentration of both oxidized and reduced forms is maintained in the cell with NAD+ at higher concentrations. Cytoplasmic NADH, which is produced by oxidation of cytoplasmic NAD+, acts as an electron donor and is transported to the mitochondrion to reduce mitochondrial NAD+ to NADH, which in turn is oxidized again during oxidative phosphorylation process to generate ATP. It is postulated via several experimental studies that NAD+/NADH ratio in cytoplasm is around 700:1, though the overall cellular ratio varies between 0.5:1 and 4:1. This ratio is involved in regulation of several metabolic enzymes such as glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase used in conversion of pyruvate to acetyl-CoA.

1.3.3  Radical Generation in Metabolism and Role of Enzymes in Redox Cycle ROS/RNS production takes place in a cellular environment via multiple metabolic pathways with the aid of specific enzymes at various sites in a cellular matrix and its organelles. In that, mitochondria are one of the most important cellular components with the greatest potential to produce free radicals ranging from hydroxyl to peroxynitrite to hypochloride radicals. Figure 1.8 depicts the role of mitochondrion in the redox of cycle of a cell. Mitochondria are known to be the energy producer or ATP production center of the cell which takes place via tetravalent reduction of oxygen (O2) in the presence of mitochondrial cytochrome c oxidase (COX) also called complex IV [83]. About 2–4% of this oxygen taken by mitochondria undergoes univalent reduction to superoxide ion (O2.−) via complex I enzyme (NADH-ubiquinone oxidoreductase) to a concentration of about 10 µM [66,84]. Oxidation of NADPH to NADP+

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Prachi Gupta, Andrew Lakes and Thomas Dziubla

by O2 in the presence of NOX is another source of O•− 2 which occurs as an important inflammatory response in phagocytes against bacterial infection and this process is often termed as oxidative burst [85–87]. NOX also facilitates O•− 2 production in nonphagocytic systems like fibroblasts, endothelial cells, and smooth muscle cells, although rate of O•− 2 production here is one-third than that in neutrophils [7,88,89]. This superoxide anion is further converted to hydrogen peroxide by mitochondrial manganese superoxide dismutase (Mn-SOD) [90,91]. Apart from controlling the O•− 2 concentrations inside the mitochondrion, cytosolic CuZn-SOD also catalyzes the superoxide dismutation to H2O2, 104 times faster than an uncatalyzed reaction (k = 2 × 109 M−1 s−1) [92]. Hydrogen peroxide is also known to be formed by two electron reduction of O2 by cytochrome P-450 or acetyl coenzyme A oxidase [93–95]. As described earlier, hydrogen peroxide is relatively stable and membrane permeable nonradical ROS. In the cellular system, it can be converted to water and oxygen via the enzymes like catalase, glutathione peroxidase (GPx), etc. [96,97], or it can be further reduced to a highly reactive hydroxyl radical under transition metal ion catalytic conditions (oxidation of Fe2+ or Cu2+ to Fe2+ or Cu3+) (Fig. 1.9) [98] based upon Fenton/Haber– Weiss style reactions. In this pathway, oxidized iron or copper (Fe/Cu3+) is reduced back to ferrous or cuprous ions (Fe2+/Cu2+) by superoxide ion present in the vicinity, which in turn produces more hydroxyl radicals. The concentrations of superoxide anion, transition metal ions significantly define the free radical production rates [99,100]. Other active routes of OH• production are through disproportionation of peroxynitrous acid intermediate (HO…NO2) to hydroxyl and nitrous oxide (NO2.) radical [101]. Due to the extremely high reactivity of the hydroxyl radical, it is very difficult to monitor its real-time concentration in vivo (Fig. 1.8). Peroxynitrite is a product of reaction of superoxide with endogenously generated nitric oxide ( NO• ) radical [102,103]. NO is also generated in the cellular matrix via reduction of L-arginine in presence of nitric oxide synthase (NOS). NOS has three isoforms namely: (1) NOS1 (NOSI) found in neural tissues, (2) NOS II or iNOS, inducible NOS, which is found in various cell types stimulated upon inflammatory response, and (3) eNOS found in endothelium [104]. Mitochondrial NOS (mtNOS) is another isoform, which facilitates the formation of NO• inside the mitochondria. This NO formation plays an important role in regulating the mitochondrial respiration and has the potential to reversibly inhibit cytochrome oxidase activity, which is responsible for superoxide production [105]. Reaction of superoxide with nitric oxide under physiological

23

A Free Radical Primer

H2O2

GSSG

NADP+

H2O2 (10–8M)

HO

B BH2

ROOH (10–6M/s)

H2O + ROH

NADPH + H+

GSSG

ONOO–

DH2

NADP+

2GSH

GPer

NO

Peroxisomes Cat . H2O2

Cat RH

O2

DH2

D

H2O2

SOD

2GSH

NADPH + H+

A

O2 (10–11M)

SOD 2H2O

AH2

Cytosolic Endopiasmic enzymes reticulum

O2

Mitocondria

H2O2

O2

UQH

O.2–

ecSOD

H2O2

D

Fe

.

Lipid radicals

OH

EXTRACELLULAR ENDOTHELIUM

Xanthine oxidase

eNOS

PLASMA MEMBRANE

nNOS NO

O2. –

PM oxidoreductase/ NAD(P)H oxidase SR/T-Tubule NAD(P)H oxidases

ONOO–

CYTOSOL PLA2-dependent processes

O.2–

CuZnSOD

H2O2

GPx CAT

H2O

VDAC? Electron transport chain eNOS

MnSOD GPx H2O2 H2O O.2– NO

ONOO– Mitochondrion

Figure 1.8  Examples of various routes of metabolic free radical production and detoxification pathways.

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conditions to produce peroxynitrite is found to be three times faster than hydrogen peroxide production and again is also a highly reactive RNS just like OH.− [106]. Hydrogen peroxide also contributes to the production of hypochlorite radical upon reaction with chloride ion (Cl−) in activated leukocytes by the action of myeloperoxidase (neutrophils and monocytes) and eosinophil peroxidase (eosinophils) [107,108]. Their ideal function is to act as antimicrobial agents, but excess or unregulated production can result in fragmentation and aggregation of proteins [108,109]. As for the production of singlet oxygen, it is known to be generated at the catalytic sites of multiple enzymes in the cellular matrix or via dismutation of unstable oxidation products like superoxide both spontaneous and enzyme catalyzed. It is also known to be produced as a byproduct of Table 1.3  Role of Enzymes in Carrying Out Various Redox Reaction and Maintaining the Cell Homeostasis Enzyme Function Refs.

SOD (CuZn-SOD, Mn-SOD, Fe-SOD) NOS (eNOS, iNOS, NOSI) NADPH oxidase GPx Myeloperoxidase (abundant in neutrophils) Catalase (in peroxisomes) 5-Lipoxygenase Cyclooxygenase Xanthine oxidase

Monoamine oxidase (outer mitochondrial membrane)

Catalyzes dismutation of superoxide to hydrogen peroxide Oxidation of l-arginine with production of nitric oxide (NO) Superoxide production from dioxygen molecule Reduction of GSH to GSSG with simultaneous conversion of hydrogen peroxide to water Produces HOCl from H2O2 and Cl− ion, oxidation of tyrosine to tyrosyl radical in presence of H2O2 Decomposition of hydrogen peroxide to water and oxygen Inducible source of ROS production in lymphocytes ROS generation in TNF-α stimulated cells Source of ROS in diabetes mellitus catalyzes oxidation of hypoxanthine to xanthine with simultaneous production of H2O2, xanthine further catalyzes to uric acid and H2O2, sometimes can also produce superoxide radical Catalyzes oxidative deamination of biogenic amines, large source of H2O2 and OH• radical as well

[112,113] [68,114] [68,115] [116,117] [118] [119,120] [121,122] [123,124] [125]

[126]

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O2

O2



Fe3+

Fe2+

– OH + OH

H2O2

GR

GPx H2O + O2

NADP+ + H+

GSH

GSSG

NADPH

Figure 1.9  Concept of redox cycle in maintaining the healthy redox state of a cell.

peroxyl radical reaction between hydrogen peroxide and hypochlorite via the Russell mechanism, found to occur in stimulated neutrophils mostly during the respiratory burst phenomena (antimicrobial cell lysis process) [110,111] (Table 1.3; Fig. 1.9).

1.3.4  Known ROS Targets and Concerns Proteins, PUFAs, and carbohydrates are very common targets to ROS. These molecules constitute very important cell organelles including lipid membranes, mitochondria, DNA, and other nucleic acids. Reaction with these biological components starts with the excess production of ROS and insufficient endogenous antioxidants available to stabilize them, resulting in disruption of the cellular redox balance. Extent or affinity of ROS/ RNS to react with the cellular components varies with the type of ROS depending on its reduction potential. For instance, hydroxyl and peroxynitrite radicals discussed in Section 3.1 have relatively higher reduction potential amongst other oxidants (Table 1.2) and are considered to be the most reactive towards lipids, DNA, etc. It is almost difficult to detect or measure their quantity in vivo as their rate constants in the range of 107– 1010 M−1 s−1 almost match their diffusion rates [127,128]. In theory, oxidation of proteins, peptides, and amino acids by these free radicals can take place via several routes including hydrogen abstraction (favorable with aliphatic amino acids), electron transfer, dimerization, disproportionation, rearrangement, and many more [129,130]. Protein and amino acids have multiple sites of attack on their backbone and their reactivity depends on stability of the resulting radical and degree of carbon hydrogenation [131]. Aromatic amino acids have a tendency to undergo addition reaction with free radicals rather than hydrogen abstraction due to their stabilizing resonance property forming hydroxylated/quinone molecules after reaction with hydroxyl radical [132,133].

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One of the most sensitive target sites to free radicals are mitochondria and their DNA components (mtDNA) [134,135]. This is because, mitochondria is the key production site of ROS/RNS where superoxide anion concentration is found to be 5–10 times higher than in the cytosol, with mtDNA to the closest proximity in comparison to nuclear DNA to the cytosolic ROS [136,137]. There are numerous amount of studies demonstrating that the mitochondrial dysfunction due to excess ROS/RNS production is a kick-off event towards the final stage of cell apoptosis or necrosis [127]. The mitochondrial dysfunction is thought to start via mtDNA damage and protein deactivation. This leads to the incapability of the mitochondria to maintain its membrane potential, resulting in the net loss of ATP production, destructuring of the mitomembrane (inner and outer both), resulting in release of excess calcium ion (Ca2+), cytochrome c, and lysosomes. These apoptogenic proteins/ enzymes finally activate the apoptotic pathway leading to cell death [138]. Another potential target to ROS like singlet oxygen, peroxynitrite, or hydroxyl radical is the cellular lipid membrane, which is made of unsaturated fatty acids. It is thought that the central carbon of the unsaturated fatty acids (L) is attacked by ROS/RNS to produce lipid free radical ( L• ), which oxidizes further to its alkoxyl ( LO• ) and peroxyl ( LOO• ) radical forms [139]. These radicals, considered to be important intermediates in lipid oxidation process, again react with another fatty acid chain (L) to produce more L• radical and the chain reaction continues. In this propagation event, radicals like NO• can act as antioxidant towards termination of the chain by forming stable alkyl nitrites (LONO2) or as pro-oxidant to form more LO• and NO2 through the peroxynitrite pathway [140]. Due to this process, lipid fatty acid chains are often considered as secondary free radicals as they form reactive intermediates that propagates the lipid peroxidation chain reaction resulting in membrane degeneration and membrane protein dysfunction [106]. This degeneration leads to loss of membrane integrity making membrane bound protein even more susceptible to free radical attack. Alkyl peroxyl radicals (another form of secondary radicals) generated by decomposition of alkyl hydroperoxide in presence of transition metal ion also contribute in the propagation of lipid peroxidation [141–144]. (1.lii) LOO• + NO• → LOONO LOONO → LO• + NO2 Chain propagation(14% ) (1.liii) LOONO → LONO2 Chain termination( 86% ) (1.liv)

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1.4  CONCLUSION AND FINAL THOUGHTS As seen, the exact mechanisms by which free radical chemistry progresses are extremely complex, with multiple interacting pathways. Yet, even an incomplete understanding has led to major impacts into the way we approach fields as diverse as metallurgy, combustion, and most relevant here, biology. As we gain new knowledge in the field of free radical biology, the opportunities to improve our biomedical treatments and therapies also grow. Researchers have already started harnessing these opportunities to develop new and exciting biomaterials that no longer remain “inert,” but rather actively respond and control design a more nuanced level of biomaterial/host interaction.

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CHAPTER TWO

Oxidative Stress, Inflammation, and Disease Shampa Chatterjee Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA, United States

2.1 INTRODUCTION Oxidative stress is a term coined to denote the state of imbalance between the generation of oxidants (ie, free radicals and ions collectively called reactive oxygen species (ROS) and the availability of endogenous antioxidants to scavenge this ROS resulting in an excess production of oxidants [1–3]. As this ROS produced in biological systems can readily react with lipids, proteins, and DNA, an excessive production of ROS can be detrimental. Indeed studies have shown that ROS either through direct damage to biomolecules or modifications in proteins and genes are pivotal in triggering signaling cascades that lead to the onset of numerous pathologies and ultimately cell injury and death [4–7]. It is now well established that ROS and intermediate free radicals are the primary source of oxidative damage. Paradoxically, there is also extensive evidence to suggest that ROS serve as critical signaling molecules in maintenance of physiological function such as cell growth, proliferation, and survival [8–10]. ROS produced in vivo by cells, such as endothelial, inflammatory, and immune cells, has two faces; first is its participation in redox signaling and the second is its role in oxidative stress or injury. Redox signaling occurs when low levels of ROS produced induce activation of signaling pathways to initiate biological processes while oxidative stress defines high levels of ROS production that damages biomolecules. Although work over the last few decades implicates oxidative stress in the pathophysiology of many diseases, it seems increasingly evident that both redox signaling and oxidative stress underlie conditions ranging from cardiovascular to neurodegenerative diseases [11]. Both ROS-induced modifications of proteins and DNA as ROS-induced oxidative damage can lead to the onset of signaling that seems to be at the heart of inflammatory Oxidative Stress and Biomaterials. Doi: http://dx.doi.org/10.1016/B978-0-12-803269-5.00002-4

© 2015 2016 Elsevier Inc. All rights reserved.

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disorders. Inflammation is a “host defense” mechanism against pathogens which involves an enhanced or exaggerated ROS generation by activated inflammatory and immune cells. ROS that is produced as part of the inflammatory response facilitates clearance of tissue invasive bacteria, but when produced for prolonged periods can promote oxidative stress and chronic inflammation associated disorders. Additionally although inflammation induces oxidant injury, the reverse sequence of events is also true. Thus inflammation and oxidative stress are inextricably interrelated. While most of these modifications lead to irreparable damage, some modifications are more subtle and fully reversible. The reversible modifications can initiate signaling cascades known as “redox signaling.” This chapter reviews the role of oxidative stress and inflammation in the onset of signaling that underlies the pathology of several diseases. This link between oxidative stress and inflammation is highlighted in Figs. 2.1 and 2.2. The factors that cause inflammation and its amplification, leading to oxidative stress and the reverse sequence of events (oxidative stress induced inflammation), and the intersection of these are also reviewed. Finally, the antioxidant regulatory mechanisms that modulate the balance between host defense, inflammation, and oxidative stress are discussed.

Environmental agents

Cellular oxidants

Low ROS generation Signaling pathways

High ROS generation

Oxidative stress

Lipids Proteins DNA

Mutation

Pro-inflammatory mediators

Inflammation

Cell proliferation

Cell survival

Cellular transformation Carcinogenesis

Cell invasion/angiogenesis

Cell death Cell growth and tissue remodeling

Figure 2.1  Interrelationship between ROS, oxidative stress, inflammation, and cellular physiology and pathology.

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Transcription factors NF-κB, AP-1, HIF-1α, Nrf-2

ICAM/VCAM, MCP-1, TNF-α, TGF-β

Oxidative stress

Inflammation

MAP Kinase (ERK, JNK, p38Kinase) PI3-Kinase Growth and remodeling

Figure 2.2 ROS and redox-dependent signaling pathways in oxidative stress and inflammation. ROS is reported to activate transcription factors NF-κB, AP-1, and HIF-1α that drive expression of pro-inflammatory genes leading to induction of proteins such as CAMs, MCP-1, TNF-α, IL-1, and transforming growth factor (TGF-β). ROS can also modify tyrosine kinases, such as Src, Ras, PI3K, EGFR, MAPK, that is, p38MAPK, JNK, and ERK. Activation of these redox-sensitive pathways results in numerous cellular responses.

2.2  ROS AND OXIDATIVE STRESS: A MAJOR ACTIVATOR OF INFLAMMATORY PATHWAYS ROS is a term that encompasses a wide variety of free radicals, ions, and reactive species that are generated by enzymatic and nonenzymatic sources in various cell types. For a summary of these players see chapter “A Free Radical Primer.” Briefly, the major ROS players in biological systems are superoxide anion (O2−), hydrogen peroxide (H2O2), the highly reactive hydroxyl radical (HO·), singlet oxygen (1O2), and ozone (O3). Besides ROS, the other physiologically important reactive species are the reactive nitrogen species comprising of nitric oxide (NO·) and the reactive radical, peroxynitrite anion (ONOO−, produced via interaction of O2−· and NO·). Nitric oxide can be converted into peroxynitrous acid and ultimately into hydroxyl radical and nitrite anion (NO2−) [12]. ROS are produced by biological systems through normal metabolic pathways. Cells also possess endogenous enzymes such as superoxide dismutases (SOD) which protect against ROS-induced damage by scavenging these free radicals [13–15]. Enzymatic sources of ROS are xanthine oxidase (XO), cyclooxygenases (COX), lipooxygenases, myeloperoxidases

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(MPO), cytochrome P450 monooxygenase, uncoupled nitric oxide synthase (NOS), peroxidases, and NADPH oxidase (NOX) while nonenzymatic sources are leakage from electron transport chain, free iron, xenobiotic, etc. [16–18]. High levels of ROS from external sources (such as environmental pollutants) or produced endogenously (via activation of XO, NOX, or mitochondrial pathways) can result in direct or indirect damage or both. Direct damage to proteins, DNA, and lipids alters these moieties, affecting their normal cellular functions. Indirect damage can occur via modification of proteins (by oxidation of amino acid residue side chains) or DNA base pairs [19,20]. As several of these proteins act as regulatory and transcription factors for cellular processes, modification of their structure activates signaling cascades that lead to altered cellular function. The biological consequences include a loss of transforming activity as well as mutagenicity and genotoxicity. Redox-sensitive (or ROS-regulated) transcription factors such as nuclear factor kappa B (NF-κB) are, when in an inactive form, localized in the cytoplasm of cells and bound to cytosolic proteins like IκB. Oxidants or ROS drive modification of IκB proteins, such as phosphorylation by a serine kinase, inhibitor of NF-κB kinase (IKK), which leads to its degradation, thus freeing NF-κB that can then translocate to the nucleus where, either alone or in combination with other transcription factors, it induces expression of several genes that express inflammatory proteins. ROS from various sources, external or endogenous (XO or NOX derived or mitochondrial ROS), have been demonstrated to be needed for NF-κB activation [21–24]. Besides NF-κB, other transcription factors, such as activator protein-1 (AP-1), the hypoxia-inducible factor (HIF-1α), peroxisome proliferator activator receptor gamma (PPAR-γ), β-catenin/Wnt, and nuclear factor like 2 (Nrf-2), have also been observed to be ROS regulated [25–27]. The activation primarily occurs via their release from a complex and subsequent translocation into the nucleus where it can “turn on” the expression of specific genes that have DNA-binding sites for the transcription factor in its promoter region. Transcription factors, such as the ones mentioned above, can alter hundreds of genes that induce expression of cellular adhesion molecules (CAMs), growth factors (such as the vascular endothelial growth factor, VEGF), cytokines, and chemokines. Cytokines are mediators of inflammation signaling; they bind to their respective receptors and either initiate ROS production or activate kinases or transcription factors that lead

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Table 2.1  Redox Regulated Transcription Factors, Chemokines, and Cytokines

Transcription NF-κB factors AP-1 HIF-1α Nrf-2 p53 PPAR-γ Cytokines

TNF-α and -β (tumor necrosis factor) IL-1 to -15 (Interleukins)

Immune response, inflammation, proliferation Cell differentiation, survival, apoptosis, cytokine synthesis Vascularization, angiogenesis, cell migration, tumor invasion Antioxidant enzymes, inhibits inflammation Apoptosis, anti-proliferative, DNA repair Lipid handling, adipose regulation, inflammation Recruits neutrophils, macrophages Pro-inflammatory, mobilizes neutrophils, promotes angiogenesis Immune-regulatory; inhibits growth and activation Monocyte migration

TGF-α and -β (transforming growth factor) Chemokines MCP-1 and -3 (Monocyte Chemoattractant Protein) MIP-1α (Macrophage Macrophage activation, allergy Inflammatory Protein-1 RANTES (regulated on Cell migration activation, normal T-expressed and secreted, CCL5) IL-8 Neutrophil activation

to induction of other inflammatory signals. Chemokines are cytokines of low molecular weight (7–15 kDa) that bind to their respective receptors; some of these are released at the site of infection and drive inflammatory responses. Overall cytokines and chemokines induce recruitment of well-defined subsets of immune cells (leukocytes, polymorphonuclear neutrophils (PMN), macrophages, dendritic cells, etc.) [28–31]. Table 2.1 summarizes the redox or ROS-regulated players (transcription factors, cytokines, and chemokines) that are involved in various aspects of cellular and tissue inflammatory response. A pivotal event in the inflammatory response is the recruitment and adherence of phagocytes or immune cells (PMN, macrophages, monocytes) to the blood vessel wall. This is followed by extravasation of the adherent immune cells into the vascular wall. Once inside the tissue, these cells release ROS required for clearance of tissue invasive bacteria [32,33]. Excessive ROS produced during the inflammatory response can, in turn, cause further

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damage and exacerbate the oxidative stress-inflammation cycle. In general, the longer the inflammation persists, the higher the risk of pathologies such as atherosclerosis, cancer, and neurodegenerative diseases (Fig. 2.1). Adhesion molecules, cytokines, chemokines, and growth factors released by ROS-induced signals promote inflammation; conversely inflammatory processes by releasing ROS and other oxidants also promote oxidative stress and injury. Excessive ROS from external or other sources can, by generating lipid peroxidation products and other adducts, lead to oxidative damage. This also causes the onset of inflammation as peroxidized adducts trigger immune cell recruitment to “phagocytose” cell debris, etc. This is discussed in Section 2.4.

2.3  INFLAMMATION: A MAJOR CAUSE OF OXIDATIVE STRESS Inflammation is a pathological condition characterized by immune cell infiltration (monocytes, macrophages, lymphocytes, PMN, and plasma cells) into the vascular wall, extravasation of the immune cells into the tissue and release of ROS by these cells leading to tissue injury. PMN generate ROS predominantly via the enzyme NADPH oxidase 2, the activity of which is mediated through the assembly of catalytic subunit gp91phox (NOX2). The O2− produced spontaneously or enzymatically (SOD) dismutates to H2O2. H2O2 also gives rise to other highly reactive radicals, such as hydroxyl radical (·OH) or hypochlorous acid (HOCl) when catalyzed by the enzyme, MPO. Both these radicals can cause tissue injury besides oxidizing a variety of proteins, etc. ROS generated by inflammatory cells (besides causing direct oxidative stress to remove pathogens) also stimulates pathways that lead to amplification of inflammation. ROS-induced activation of kinases (or enzymes that facilitate phosphorylation), such as protein kinase C (PKC), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), leads to the activation of transcription factors, which in turn triggers the generation of pro-inflammatory cytokines and chemokines. Once triggered, these cytokines and chemokines bind to their respective receptors (such as plateletderived growth factor receptor,VEGF receptor, and epidermal growth factor receptor (EGFR) that are well established to generate ROS [34,35]. ROS is thus both upstream and downstream of the inflammation cycle (Fig. 2.2). Besides facilitating the production of cytokines such as tumor necrosis factor-alpha (TNF-α), interleukins (IL-1 and -6), and chemokines such

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as monocyte chemoattractant protein-1 and 2 (MCP-1/2), macrophage inflammatory protein (MIP-1/2) and RANTES (regulated on activation, normal T cell expressed and secreted), ROS induces (via transcription factor NF-κB) the expression of plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibition factor (MMIF), inducible NOS (iNOS), matrix metalloproteinases (MMP) 9, and MMP-2 [36]; all of which are important in recruitment and adherence of various subsets of immune cells. Recruitment and adherence are complex processes that involve cytokine-chemokine gradient to control influx and CAMs to regulate immune cell-vessel wall interaction. This is followed by PAI, MMIF, and MMPs induced disruption of adherens junctions that facilitate migration of immune cells into tissue. Postactivation, immune cells release oxidants and several proteases. Neutrophils produce neutrophil elastase, a serine protease which has direct antimicrobial activity. Besides ROS, other free radicals like HOCl are also produced when H2O2 is catalyzed by MPO, an enzyme which is highly abundant in inflammatory and immune cells. The highly reactive HOCl can oxidize a variety of molecules, including proteins, to cause tissue injury and dysfunction. ROS generation and oxidative stress can continue the cycle of inflammation, thus contributing to a chronic state which drives several inflammatory pathologies [37,38]. Recent advances in understanding the complexities of inflammatory signaling point to discrete pathways that are activated with recognition of molecular patterns derived from either pathogens or oxidative damage. Thus immune receptors can sense pathogen-associated molecular patterns (PAMPs), derived from invading pathogens or danger-associated molecular patterns (DAMPs) as a result of endogenous stress and trigger downstream signaling cascades. The multiprotein complex that assemble upon sensing PAMPs or DAMPs are termed as inflammasomes; these initiate a caspase signaling pathway that leads to cell injury and death [39–41]. Inflammasome induced cell injury and apoptosis contributes to and amplifies the pathology of inflammation diseases. Indeed, inflammasomes have been linked to a variety of inflammatory diseases from neurodegenerative (multiple sclerosis (MS), Alzheimer’s disease (AD), and Parkinson’s disease (PD)) to metabolic (atherosclerosis, type 2 diabetes, and obesity) [42]. Although the canonical pathway for inflammasome activation is via the PAMP-DAMP “sensing,” there are several reports that ROS and other oxidants activate the inflammasome complex. This would point to a “ROS induced inflammation induced oxidative stress” process [43,44]. As shown in Fig. 2.3, ROS are essential secondary messengers in

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Non pathogen mediated

Pathogen mediated

UV radiation

DAMPs

TLR2

PAMPs, DAMPs (bacterial toxins)

ATP Fatty acids Uric acid

LPS

ROS

NF-κB, AP-1

NLRP3 Inflammasome

ASC

Caspase-1 IFN-, TNF Pro-IL-1β, Pro-IL-18

IL-1β, IL-18

Inflammation Figure 2.3  Activation of the inflammasome. PAMPs and DAMPs arising from bacterial or viral ligands (such as LPS) or some environmental stress factors (asbestos, silica, alum) or endogenous agents of cellular stress (ATP, uric acid). In response to DAMPS and PAMPs, NLRP3 inflammasomes are activated (by among other agents, ROS) and recruit adaptor protein ASC and pro-caspase-1, which is cleaved to caspase-1. Caspase-1 facilitates the cleaving of Pro-IL-1β, Pro-IL-18 (which is expressed via transcription factors NF-κB and AP-1) to IL-18 and IL-1β, potent mediators of inflammation.

DAMP-PAMP–induced inflammasome activation and subsequent inflammatory signaling.

2.4  OXIDANT STRESS AND INFLAMMATION IN CELLULAR TRANSFORMATION, APOPTOSIS, AND NECROSIS Alterations or damage that arise from oxidative stress or from inflammation can cause cellular changes that may lead to cell death by necrosis, apoptosis, aging or to cell survival mechanisms such as autophagy where oxidized or defective proteins are degraded. ROS-induced cell death occurs either directly by damaging cell membrane/cellular structures or by activating caspases (enzymes that drive cell death) via lipid peroxidation products such as 4-hydroxy-2-nonenal

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(HNE). HNE, when accumulated in cells, can induce apoptosis by activation of the “death receptor” or the “mitochondrial” pathway. The death receptor pathway is activated when lipid peroxides induced cytokines activate “initiator” caspases (8 and 10) which cleave “effector or executioner” caspases that in turn cleave intracellular proteins to trigger apoptosis. The mitochondrial pathway is activated when ROS and products of lipid peroxidation and damage lead to mitochondrial outer membrane permeabilization, which results in both caspase-dependent and caspase-independent apoptosis. To make matters worse, ROS and HNE can also activate transcription factors NF-κB and AP-1, propagating the pro-inflammatory signaling cascades [45,46]. ROS and redox signals participate in cell protection mechanisms such as autophagy. Complexes and adducts of peroxidized proteins, such as HNE-protein Michael-adducts, are degraded by the autophagy mechanism. Autophagy, which involves the clearance of (defective or mutated) proteins and organelles, is characterized by the formation of an autophagosome or vacuole by the fusion of membrane edges. Once generated, this vacuole eliminates its contents through the canonical lysosomal degradation pathway. Reports show that ROS (superoxide anion and H2O2) are crucial in autophagy as treatment with antioxidants partially or completely reverses the process. ROS probably act via autophagosome activation that degrades defective and damaged proteins or autophagy execution, that is, activation of lysosomal cysteine proteases that are crucial for protein degradation [47–49]. Low ROS levels reportedly act as secondary messengers of signal transduction pathways involved in cell growth, transformation, and apoptosis [3,37,38,50]. These occur via activation of signaling pathways that result in the induction of either growth stimulatory genes like c-fos, c-jun, and c-myc or cell death stimulating enzymes (caspases, etc.) [51–53].

2.5  EXPLORING THE LINK BETWEEN OXIDATIVE STRESS AND INFLAMMATION AND THE ONSET OF VARIOUS DISEASES Both oxidative stress and inflammation can cause injury to cells [54]. Arguably, one cell type that is uniquely sensitive to this injury is the vascular endothelium that is the site for inflammation. Besides serving as a conduit to transport nutrients to organs and acting as a barrier between blood flow and tissue, the endothelium is also a site for adherence of immune

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and other cells. These immune cells are recruited to and adhere to the vessel wall in response to any pro-inflammatory stimuli. Following this adhesion, these cells can extravasate into the surrounding tissue, and release ROS and other oxidant radicals and metabolites that cause damage both to the endothelial cell lining and to tissues. Thus inflammation leads to endothelial dysfunction [55,56]. Forming a positive feedback loop, endothelial dysfunction in turn promotes a pro-inflammatory environment as evidenced by increased endothelial expression of adhesion molecules, chemokines, cytokines, and chemoattractants [54,57]. Endothelial dysfunction has been accepted as an early determinant in the development of atherosclerosis and diabetes [58]. Yet, besides the endothelium, other cell types, such as neuronal and epithelial cells, can undergo oxidative stress contributing to a diverse set of pathologies. Overall excessive ROS, aberrant inflammatory cytokine and chemokine expression, increased COX-2, and NF-κB activation are just some of the molecular factors that contribute to inflammation-induced atherosclerosis, carcinogenesis, and neurodegenerative diseases as discussed below.

2.5.1  Cardiovascular Disease Atherosclerosis and associated vascular pathologies, such as aneurysms (an abnormal bulge or ballooning in the vessel wall) and restenosis (blockage of stents inserted in patients with cardiovascular disease or CVD, to keep arteries open) have been attributed to inflammation-induced thickening of the vascular wall or plaque formation or thrombosis. The onset of inflammation in CVD occurs in response to oxidative stress induced oxidized low-density-lipoprotein cholesterol or infection. This then signals for immune cells such as monocytes to bind to this site which is also facilitated by several CAMs (which are induced by ROS-activated pro-inflammatory transcription factors and chemokines/cytokines). Monocytes ingest modified lipids and lipoproteins and get transformed into macrophages and eventually foam cells that initiate fatty streaks [59]. At the same time, PMN and other immune cells (macrophages, lymphocytes, etc.) are recruited into this region and release additional mediators (cytokines, chemokines, and growth factors) [33], leading to the activation of immune cells. This again leads to further ROS production and endothelial damage. Besides ROS released from activated immune cells, other free radicals like HOCl can oxidize and damage a variety of molecules, including proteins, to cause tissue injury and dysfunction. Byproducts of MPO reactions are abundantly present in atherosclerosis plaques.

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2.5.2 Cancer The three stages of carcinogenesis (1) initiation, (2) promotion, and (3) progression of cell proliferation leading to malignant tumors [60,61], involve ROS-induced mutations, chromosomal aberrations and sister chromatid changes. However, it seems that the major role of ROS occurs during the promotion and progression stage when ROS induces genes that regulate cell differentiation and growth. During the last two stages, inflammation via production of oxidants, cytokines and chemokines, increases cell proliferative and angiogenic genes, growth factors and blood vessel proliferation, all of which accelerate tumor growth. In the tumor microenvironment, chronic inflammation, in the form of immune cell infiltration (ie, tumor-associated macrophages, leukocytes, mast cells, dendritic cells, natural killer cells, neutrophils, eosinophils, and lymphocytes) causes further oxidant damage. In the final or progression stage, cancerous growth which is benign is stimulated toward more rapid cell proliferation and thus malignancy. A key characteristic of carcinogenesis is the increased ability of cancer cells to survive via activated survival pathways. These, too, are activated by ROS and oxidants. Indeed ROS-induced kinase pathway of ERKPI3K-Akt signaling and the downstream mammalian target of rapamycin (mTOR) activation is involved in cancer cell growth and survival and represents an important factor of resistance to chemotherapy [62–66]. Once activated, the mTOR pathway further produces ROS; thus ROS is both upstream and downstream of mTOR [67]. Besides these pathways, ROS also activates the transcription factor, Nrf-2 which upon translocation, binds to the antioxidant responsive element, initiating the induction of a number of antioxidant genes. This stimulates cellular antioxidant defenses and enables cancer cells to survive even with chemotherapy [68,69].

2.5.3  Degenerative Disease Neurodegeneration, the progressive dysfunction and loss of neurons in the central nervous system (CNS), is a major cause of cognitive and motor dysfunction. The major cells of the CNS are neurons and glial cells (astrocytes, oligodendrocytes, and microglia) [70]. The CNS is separated from the rest of the body by the blood-brain barrier (formed by tight junctions of the endothelial cells of the CNS blood vessels) that restricts entry of nutrients and cells, including immune cells, which are absent in the CNS. The microglia are the immune cells of the CNS and in response to injury

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or infection, they release pro-inflammatory cytokines and ROS that may actually contribute to neurodegenerative diseases [71]. Indeed a central feature of neurodegenerative diseases, such as PD and AD, is the activation of microglia and the resident CNS macrophages. AD is characterized by amyloid plaque deposition by amyloid-β peptide (Aβ) chelating transition metal ions (Cu2+, Zn2+, and Fe3+). These bound transition metals of Cu2+ and Fe3+ can promote chemical reactions resulting in the altered oxidation state of both the metals. In the presence of transition metals, H2O2 is catalyzed to the highly reactive toxic OH˙ free radical [72]. PD is characterized by deposition of aggregates (Lewy bodies) of the protein, α-synuclein. Mutations in α-synuclein and other related proteins have been implicated in PD. These mutations in α-synuclein have been reported to result in mitochondrial dysfunction, leading to excess ROS production, potentially indicating a role for oxidative stress in PD [73,74]. In diseases like MS, several cells of the immune system are recruited into the CNS by the high lipid content generated by myelin. Redox metals (iron, etc.) act as a catalytic center for lipid production and iron plaques deposited over myelin invoke a huge inflammatory response that triggers recruitment of tissue macrophage and T cells entering into CNS to cause substantive damage and demyelination to CNS [75]. In all these diseases, repeated immune attacks result in an amplification of the inflammatory cascade similar to that described in earlier sections. The resultant ROS and oxidative damage drives neuronal death and plaque formation in glia that lead to scars and consequently highly diverse neurological deficits [76,77].

2.5.4  Metabolic Syndrome This refers to a series of diseased states such as glucose intolerance, obesity, diabetes, and dyslipidemia. Recent studies show a positive association between inflammatory markers and obesity indices. Indeed weight loss of obese patients has been extensively reported to be associated with a decrease in inflammatory biomarkers and with an improvement of metabolic parameters such as glucose tolerance and insulin sensitivity [78–81]. The inflammation accompanying metabolic syndrome seems to differ from canonical inflammation in its response to oxidative stress or pathogens. Addition of fat to the adipose tissue requires anatomical space for new adipocytes. For this regulation of blood flow and thus nutrient supply has to be in concert with fat accommodation needs. Often this does

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not occur and there is both inadequate blood supply to the adipose tissue and exaggerated adipocyte growth in other organs, both of which trigger the onset of local tissue inflammation. A characteristic feature of metabolic syndrome is elevated amounts of fatty acids (nonesterified fatty acids) in circulation, which leads to high levels of lipids (very low density lipoprotein and high density lipoprotein) or lipotoxicity. Some of these lipids bind to receptors such as members of the Toll-like receptor (TLR) family, which are known to be activated by bacterial lipoproteins (TLR2 and 4). The engagement of either receptor leads to translocation of NF-κB and regulation of an inflammation response [82,83]. Additionally, exposure of adipocytes to ROS or oxidative stress due to other factors induces cellular kinases, such as MAPK (p38MAPK, JNK, and extracellular signal-regulated kinase), IKKβ, mTOR, and PKC. Some of these kinases are involved in impairment of insulin action through the stimulation of insulin receptor substrate serine phosphorylation. These kinases are also upstream of AP-1 [84,85]. It is generally thought that a controlled inflammatory response is protective and becomes detrimental if dysregulated. Thus, the inflammatory state is assumed to have both physiological and pathological components. But this rationale is not sufficient to explain why certain inflammatory responses occur only in pathological settings with no physiological counterpart (obesity, metabolic and neurodegenerative diseases) while for others (inflammation with certain infections) there is no pathological component. This just illustrates that our understanding of the complexities of the inflammatory processes in vivo is far from complete.

2.6  ANTIOXIDANTS AND ANTI-INFLAMMATORY AGENTS: PERSPECTIVES IN THERAPEUTICS Since there is a link between several diseased states and the production of ROS, oxidants and the inflammation trigger are gradually being understood, and the role of antioxidant (and anti-inflammation) therapy as a defense against several pathologies is being explored. In general the strategies in use are (1) the use of antioxidants, (2) prevention of inflammation, and (3) boosting of repair processes.

2.6.1  Use of Antioxidants Antioxidants are classified as exogenous (natural or synthetic) or endogenous compounds that are capable of removal of free radicals and ROS

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either by scavenging or by inhibiting their precursors, or by binding to metal ions that catalyze ROS generation [86,87]. The major endogenous antioxidant enzymes are the glutathione redox system (glutathione peroxidase and glutathione-S-transferase), SOD, and catalase. The nonenzymatic antioxidants are ascorbic acid (vitamin C), alpha-tocopherol (vitamin E), glutathione (GSH), carotenoids, and flavonoids. Antioxidants therapy in the form of vitamin C, vitamin E, or combinations has been found to be effective in preclinical experimental models of inflammatory disease. However, the results from clinical trials have been mixed [88,89]. While several large observational and placebo-controlled double-blind studies suggested an antioxidant-intake induced lowering of the risk of coronary artery disease [90–92], other studies were not conclusive [93,94]. Yet, some studies suggest potential benefits among certain subgroups. A recent trial of vitamin E in Israel, for example, showed a marked reduction in coronary heart disease among people with type 2 diabetes who have a common genetic predisposition for greater oxidative stress [95]. Besides vitamin-based antioxidants, ROS scavenging compounds (edaravone, N-acetylcysteine or NAC and alpha lipoic acid, idebenone) and food supplements (flavonoids) have also been studied. Studies on edavarone conducted on 252 ischemic stroke patients showed that edaravone significantly improved the functional outcome of patients [96]. NAC (Acetadote), a direct ROS scavenger and also provider of amino acid cysteine, which is the precursor for the rate-limiting step of the synthesis of GSH has also been used. In ROS-induced liver injury, where lipid peroxidation and DNA damage to hepatocytes may lead to cirrhosis or end stage liver failure, NAC is being increasingly utilized in a clinical setting [97]. Indeed liver damage from acetaminophen is routinely treated with intravenous NAC administration [98]. Some systematic reviews and meta-analyses have suggested that NAC prevents exacerbations and improves symptoms in chronic obstructive pulmonary disease patients [99] with no effect on lung function parameters [100]. Dietary antioxidants or naturally occurring compounds such as flavonoids have been extensively studied for their vast antioxidant, biological, and anti-inflammatory properties [101,102]. Epidemiological studies have shown that higher dietary intake of flavonoids is protective against CVDs [103,104], cancers [105], and some other chronic diseases [106]. Similarly consumption of chocolate, green tea, and soy has been reported

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to improve endothelial functional, lower blood pressure and reduce low density lipoprotein cholesterol to various degrees. However, the data on other flavonoid-rich food sources were not conclusive [93]. Overall studies that showed a protective effect concluded that antioxidants are much more effective in prevention of disease, rather than in the treatment of an already established active pathology. There are several causes for this. First, is the challenge of delivering the antioxidant to the target site, that is, the site of ROS production or oxidative damage. Dietary intake involves absorption through the gut which is not an efficient process; besides most of the absorbed amount is destroyed in the liver by process called “first pass metabolism.” Administration via the intravenous route though slightly better is ineffective in delivering antioxidants to cellular and subcellular regions. In recent years, nanotechnology-based antioxidant delivery systems are being developed for targeted delivery; however, their effectivity in a clinical setting has not been evaluated yet [107,108]. The second challenge is lack of an index or marker of baseline nutritional status or oxidative damage status. In the absence of this, the “antioxidant dose” and its effect on various “risk” populations are unclear. The third point is that antioxidants are often administered or delivered late in disease progress. All these observations have led to what is termed as the “antioxidant paradox” or the concept that giving large doses of dietary antioxidant supplements to human subjects has little or no preventative or therapeutic effect [86].

2.6.2  Prevention of Inflammation In following this strategy, two major directions emerge; one is the inhibition of ROS production either by blocking xanthine-XO pathways or by blocking especially NADPH oxidase, whose sole function is ROS production, which is also another seemingly promising strategy [109,110]. The vasculature is a source of NADPH oxidase which produces most of the ROS and plays an important role in vascular damage. In general, oxidative stress and vascular inflammation are closely interrelated to endothelial dysfunction and vascular damage. The second is blocking onset of inflammation signaling. A challenge to reducing inflammation through pharmacologic intervention is the multiple cellular pathways by which the inflammatory response can be mediated. Based on the role of COX enzyme systems in signaling (via production of prostaglandins and thromboxane, TxA2) that regulate

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gastrointestinal, renal, vascular functions, and inflammation, pain, and fever, COX blockers or nonsteroidal anti-inflammatory drugs have been used as a pharmacologic intervention. However, other pro-inflammatory pathways exist for the cell to recruit neutrophils to damaged or exercised muscle, including the alternative lipoxygenase pathway and the NF-κB–mediated induction of pro-inflammatory genes [111,112]. As exaggerated inflammasome activity is considered to drive the pathogenesis of neurodegenerative and metabolic diseases, reagents that target the inflammasome components and products (IL-1β, IL-18) have been used to treat inflammatory conditions. These include the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-1β antibody canakinumab, the soluble decoy IL-1 receptor rilonacept, IL-18-binding protein, soluble IL-18 receptors, and anti-IL-18 receptor monoclonal antibodies [113,114].

2.6.3  Boosting of Repair Processes Accelerating repair processes often involves boosting of the inflammatory signaling cascades for resolution of the injury. One of the major events is the clearance of apoptotic cells in the injured tissue [115] during which phagocytes ingest apoptotic cells and release large amounts of inhibitory mediators. The abundant neutrophils infiltrating the infarcted myocardium represent a large pool of short-lived inflammatory cells, programmed to undergo apoptosis. Though the fundamental biology of the inflammatory response suggests that macrophage-mediated clearance of apoptotic infiltrating neutrophils may be crucial in triggering anti-inflammatory and pro-resolving signals, the significance of these interactions in the infarcted heart has not been investigated. Regardless of the rationale, the practice of using ice, compression, and elevation in managing acute inflammation is well ingrained. Although a potential role for the use of physical agents, such as cryotherapy, in attenuating the neutrophilic response has been demonstrated in the laboratory [116,117], the actual clinical evidence supporting the efficacy of these practices is limited. Scavenging ROS by antioxidants or other agents as a mode of therapy for treatment of diseases has been a challenging field. This is because the link between ROS-oxidative stress-disease differs with each pathology; in some diseases oxidative damage is the major event in the pathology while in others it represents one of the events along with others and is a late consequence of the disease. Thus the timing of intervention is of paramount importance.

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2.7  CONCLUSIONS AND PERSPECTIVES In this chapter we presented the salient observations and studies related to the role of ROS and other oxidants in oxidative stress, inflammation, and in the initiation and progression of some disease pathologies. In doing so, we highlight the interrelation between ROS, oxidative stress, and inflammation and the fact that each of these leads to the other in a “feed forward” mechanism (Fig. 2.1). In general, inflammation has beneficial and detrimental aspects; the beneficial activity is critical for host response to microbial and other pathogens. With that in mind, the therapeutic inhibition of “ROS-inflammation” pathways has to be a well-engineered, balanced, and nuanced approach. To enable that, the identification and understanding of the oxidative stress-inflammation pathways with emphasis on the spatial and temporal aspects of molecular signaling and cellular events is necessary so that molecular targeted therapies can be approached to modulate disease-specific “redox signaling-inflammation” pathways.

ABBREVIATIONS AD Alzheimer’s disease AP-1 activator protein ARE antioxidant responsive element BBB blood–brain barrier CAM cellular adhesion molecules CNS central nervous system COX cyclooxygenases CVD cardiovascular disease DAMP damage associated molecular patterns ERK-PI3K-Akt  extracellular kinase- phosphoinositide 3-kinaseprotein kinase B GSH glutathione HIF-1α hypoxia inducible factor-1 HOCl hypochlorous acid ICAM intercellular adhesion molecule IKK I kappa B kinase IL interleukins JNK c-Jun-N-terminal kinase MAPK mitogen-activated protein kinase MCP-1/2 monocyte chemoattractant protein-1 and 2

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MIP-1/2 macrophage inflammatory protein MMIF macrophage migration inhibition factor MMP matrix metalloproteinases MOMP mitochondrial outer membrane permeabilization MPO myeloperoxidases mTOR mammalian target of rapamycin NAC N-acetyl cysteine NF-κB nuclear factor kappa B NOX2 NADPH oxidase 2 Nrf-2 Nuclear factor like 2 NSAID nonsteroidal anti-inflammatory drugs OH hydroxyl radical PAI-1 plasminogen activator inhibitor-1 PAMP pathogen associated molecular patterns PD Parkinson’s disease PGE prostaglandins PKC protein kinase C PMN polymorphonuclear neutrophils PPAR γ peroxisome proliferator activator receptor gamma RANTES  regulated on activation, normal T cell expressed and secreted ROS reactive oxygen species SOD superoxide dismutase TLR Toll like receptor TNF-α or β tumor necrosis factor-alpha/beta TxA2 thromboxane VCAM vascular cell adhesion molecule VEGF vascular endothelial growth factor XO xanthine oxidase

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CHAPTER THREE

Oxidative Stress, Inflammation, and the Corrosion of Metallic Biomaterials: Corrosion Causes Biology and Biology Causes Corrosion Jeremy L. Gilbert1,2,3 and Gregory W. Kubacki1,2 1

Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, United States Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY, United States Institute of Medical and Biological Engineering, University of Leeds, Leeds, United Kingdom

2 3

3.1 INTRODUCTION Metallic biomaterials (ie, alloys that comprise medical devices) have been in use for over 80 years, particularly in dental applications, and since the 1960s in a wide range of orthopedic, cardiovascular, spinal, and other applications [1]. Indeed, metallic biomaterials have been and will continue to be an essential part of medical devices for the foreseeable future, because of their unmatched strength, toughness, modulus, and fracture and fatigue resistances. The principal alloys used in medical devices consist of 316L stainless steel, cobalt-chromium-molybdenum (CoCrMo) alloys and titanium alloys. More recently alloys made from tantalum, zirconium, and others are being incorporated into medical devices, while alloys made from magnesium, iron, zinc, and others are under consideration for use as degradable metallic medical devices [2,3]. An overwhelming preponderance of the medical devices in use today are made from CoCrMo, Ti, and 316L stainless steel based alloy systems and hence, this chapter will focus primarily on these alloys. However, much of what will be discussed is related to other systems in use or under development as well. In terms of biocompatibility (ie, the ability of a biomaterial to perform its intended function with an appropriate host response [4]), metallic biomaterials have been thought to be essentially inert. That is, they have Oxidative Stress and Biomaterials. Doi: http://dx.doi.org/10.1016/B978-0-12-803269-5.00003-6

© 2015 2016 Elsevier Inc. All rights reserved.

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minimal interaction with the host mostly associated with protein adsorption and ultimately fibrous encapsulation, and do not elicit any active interactions directly with the host (ie, they are bioinert and not bioactive). Indeed, biocompatibility of metallic biomaterials has essentially been linked to the corrosion and wear resistance of these alloys. “The more corrosion resistant—the more biocompatible” has been the basic operating principle for these biomaterials [5]. Indeed, the prevailing paradigm for study of metallic biomaterials– biology interactions has been based on the concept that the biological system reacts to tribological and corrosion (ie, tribocorrosion) processes wherein generation of wear particles and release of metal ions (cations) stimulates adverse local tissue reactions (ALTRs) [6,7]. These tribological and corrosion damage processes are thought to occur first and then the biological system reacts to the particles and ions generated. This, conceptually, is a one-way arrow of interaction: wear and corrosion cause biology. Recent work, however, has shown that the electrochemical processes that can occur at metallic biomaterial surfaces (both oxidation and reduction) can have a strong effect on the local biological system [8–12] and that the inflammatory system and oxidative stress reactions can significantly alter metallic corrosion reactions [13]. That is, the interactions between metallic biomaterials and biology are two ways, where corrosion and wear cause biology and biology causes corrosion. Here, two important ideas have been developed: (1) reduction reactions on going at metallic biomaterial surfaces generate reactive oxygen intermediates that can adversely affect local cells adjacent to the metal surface and (2) reactive oxygen species (ROS) generated by mononuclear phagocytic cells can directly corrode these alloys. These two new insights into metallic biomaterial–biology interactions present a new paradigm where there is a positive feedback between biology and corrosion. Corrosion (oxidation and reduction) causes biology and biology causes corrosion. This chapter will focus on the consequences of corrosion on biology (both metal ion release and the role of reduction intermediates), and the consequences of biology on corrosion (inflammatory cell-induced corrosion, ICIC [13]). To do so, basic background on corrosion and electrochemistry at metallic biomaterials surfaces will be presented including both oxidation and reduction reactions. Then, the basic phagocytic cell types and the processes of cell-based ROS generation are described. Wear and metal ion effects on periprosthetic tissues will be discussed, followed by the effects of reduction reactions on cells. Finally, the recently

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observed effects of inflammatory cells and ROS on the direct corrosion of CoCrMo and Ti alloy implant surfaces will be described, including within modular taper junctions.

3.2  OXIDATION, REDUCTION, AND TRIBOCORROSION AT METALLIC BIOMATERIAL SURFACES Corrosion of metallic biomaterials, which most often occurs concurrently with wear processes (so-called mechanically assisted corrosion, MAC, or tribocorrosion [14]), consists of a set of electrochemical reactions that include both oxidation reactions (metal ion and metal oxide generation) and reduction reactions (see Fig. 3.1). It is critical to remember that corrosion consists of both oxidation and reduction [15,16]. Oxidation reactions take metal atoms (neutral charge) present at the surface of the metallic biomaterial and react with them into metal (positively charged) cations in solution and/or metal oxides. These cations and oxides can be released into the adjacent electrolyte (body fluids), or, in the case of oxides, reform the passive film on the metal surface after it is breached mechanically. In general, what chemical species can engage in an electrochemical half-cell reaction depends on the electrode potential of the interface. Net oxidation reactions for a single half-cell reaction will only occur if the electrode potential is above the equilibrium half-cell potential for that specific reaction, while net reduction reactions will only occur if the electrode potential is below the reduction half-cell equilibrium potential for a particular reaction [17]. During corrosion, where there are no external sources or sinks for electrons beyond the electrode surface, the electrode potential will be established by the condition where there is a balance between oxidation reactions and reduction reactions. That is, the rate of charge generation due to oxidation reactions will be balanced by the rate of charge consumption by a (set of) reduction reaction(s). This so-called “mixed potential theory” is a basic concept in corrosion science [17]. The major alloys of Ti, CoCrMo, and 316L stainless steel have passive metal oxide thin films that spontaneously form on the surface within milliseconds when in contact with oxygen, whether as molecular oxygen or water, or both (see Fig. 3.1). These passive metal-oxide thin films are the ultimate self-healing, smart, nanomaterials used in biomaterials. They are typically only a few nanometers thick (2–10 nm) and are typically generated by the reaction of metal with water as shown below for chromium.

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Reduction

Oxidation M→

Mn+

+

1

ne–

mM + nH2O → MmOn +

2nH+ + 2ne–

O2 + H2O + e– → 2OH–

2H+

Anodic currents

H2O +I

2

Mn+

H2O2 1/2O2 + H2O

2OH– OH

O2–



O2

Solution

HO2

+I

MmOn

Oxide Metal

Cathodic currents

M Anode

M

e– Cathode

Figure 3.1  Schematic of typical oxidation and reduction reactions on an oxide film covered metallic biomaterial exposed to the body environment (ie, aqueous electrolyte). Corrosion must consist of both oxidation, which generates metal ions (cations in solution) and/or metal oxides (which can form in solution or as a passive thin film on the metal surface). Electrons liberated by the oxidation reactions remain in the metal and move to sites where reduction reactions occur. Positive charge passes from metal to solution at the anodic sites and there are ionic currents (flow of charged species) in solution to the cathodic site, where the electronic circuit is completed by reduction reactions (which move negative charge from metal to solution). Reduction reactions take electrons from the metal and reduce oxygen and water to form hydroxide ion and other reactive oxygen intermediates like hydrogen peroxide, hydroxyl radical, hydroperoxyl radical, etc.



+ − 2Cr + 3H 2O → Cr (3.i) 2O3 + 6H + 6e

Ion release is also an electrochemical reaction where metal atoms oxidize to cations which are released into solution.

2+ Co → Co (3.ii) + 2e−

The driving force for both oxide formation and ion release is the free energy of their respective reactions. The free energy for oxidation of the metals in these alloy systems is highly exothermic (ie, spontaneous). This means that these alloys have a high driving force for oxidation and will

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Ionic dissolution H+

Sliding asperity

Mn+

Mn+

Debris

Repassivation

Oxide Metal e–

Plastic deformation

Oxide

Figure 3.2  Schematic of MAC of passive oxide film covered metallic biomaterials. Apposing hard surface asperities under load and in contact with the metal surface will induce abrasion and disruption of the oxide film exposing the underlying metal for a very short time. Within milliseconds the nanometer thick passive oxide film reforms electrochemically (oxidation reactions of Fig. 3.1). Metal cations and hydrogen ions get released by the bare metal surfaces (when metal cations are soluble). The underlying local surface metal is typically subjected to stresses sufficient to cause plasticity (ie, dislocation motion) and development of additional breaches in the oxide film. The oxide film immediately trailing the moving asperity is regrown by high-field oxide growth mechanisms in a matter of a few milliseconds.

oxidize rapidly if the conditions are right (eg, breach in the oxide). The primary factor keeping these alloys from oxidizing is the presence of the thin passive oxide film that spontaneously forms on their surface. Corrosion processes for many metallic biomaterials are driven by mechanical factors [14,18]. MAC or tribocorrosion (see Fig. 3.2) occurs when the surface oxide is breached (by abrasion, surface yielding, or other processes). This can occur, for example, by abrasion of a hard counterface asperity against the metal surface. The oxide film is removed and immediately behind the breach bare metal that will rapidly release ions and repassivate the oxide film is exposed. Debris (both oxide and metallic) is generated from the tribological process, and corrosion reactions are also present generating free electrons in the metal, releasing ions and oxide particles into solution (in some cases) and reforming the oxide film. It should be noted that not all alloys release ions and oxides in similar proportions. This will depend on the relative solubility of ions into the body environment and the potential across the interface. For example,

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titanium ions in neutral aqueous electrolytes have an extremely low solubility on the order of 10−10 M [19]. Therefore, when titanium experiences MAC, virtually all of the reaction is related to oxide film formation and only a small proportion goes to ion formation. The oxide film growth process occurs by the low temperature— high field growth mechanism described by Cabrera and Mott [20], and Guentherschulze and Betz [21,22]. This model says that an extremely high electric field (on the order of 107V   /cm or higher) is developed at the metal solution interface by electron tunneling from the metal to oxygen atoms in solution. The oxygen traps the electron and thereby creates a high electric field across the interface that further drives oxidation reactions and film formation. The rate of film growth, described by the film forming current (ie, the electrons from Eq. (3.i)) flowing across the interface, depends on the electric field (voltage/oxide thickness). This has been described by V



i =α

B dx = Ae x dt (3.1)

where x is the oxide film thickness, V is the voltage drop across the interface, and α, A, and B are constants. As the oxide thickens (x increases) the currents rapidly fall to a limiting value and a limiting thickness is reached beyond which the oxide does not grow significantly further. When the oxide is breached, ionic release (for CoCrMo and 316L SS) is significantly increased over that which occurs across an intact oxide. Once reformed, the oxide film acts as a kinetic barrier limiting the rate of further oxidation reactions (both ionic and oxide forming). Ions continue to passively dissolve through the oxide film into the solution, but at rates that are very small and result in CoCrMo and Ti alloys being some of the most corrosion resistant metals known. That is, corrosion rates for these alloys are extremely low because of the barrier effects of the oxide, not because there is no driving force for corrosion. When the oxides are breached, until they can repassivate (within milliseconds), the corrosion rates can increase over a million fold [23]. While an overwhelming preponderance of the studies of the role of corrosion on the local and systemic biological response, and the performance of metallic biomaterials in general, has focused on the products of the oxidation reactions (metal ions and oxide and metallic particles), there are a number of additional effects that have remained unstudied and

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unappreciated related to the reduction of half-cell [15]. These include the role of the electrons released from oxidation reactions and which are consumed in subsequent reduction reactions which generate reduction products that have significant biological effects. Some of these factors will be described below. The reduction half-cell in corrosion is present in all corrosion reactions. It is required to take up the accumulated free electrons released from the oxidation reactions and sweep them from the metal surface. This prevents significant charge accumulation from taking place. Very little focus has been given to the specific reduction reactions that are possible in the living system and for the most part researchers have simply assumed that the primary reduction reaction is oxygen and water reacting with electrons in the metal as (see Fig. 3.1)

1 O2 + H 2O + 2e− → 2OH− (3.iii) 2

However, this is highly simplified and does not reflect the wide range of redox-sensitive molecules that are generated or available in the biological system. In biological systems, reduction reactions also will depend on the electrode potential. While oxygen reduction is the presumed major reduction reaction, there are a number of other potential reactions, including reduction of disulfide bonds, reduction of water, and reduction of other redoxbased biological molecules and process that are possible. Indeed, biological systems utilize a wide range of oxidation and reduction reactions to maintain redox homeostasis, including glutathione levels [24] and to synthesize proteins and energy [25]. Even with the oxygen reduction reaction shown above, there are a number of reactive oxygen intermediates that can be generated at metallic biomaterials surfaces during reduction that have biological effects. These include [26–28]:



O2 + H 2O + e− → HO2• + OH− HO2• + H 2O → H 2O2 + OH• H 2O2 + 2e− → 2OH− O2 + 2H 2O + 2e− (3.iv) → H 2O2 + 2OH−

These reactions show that oxygen reduction, while ultimately resulting in the formation of hydroxide ions can also produce hydroperoxyl radicals,

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hydroxyl radicals, and hydrogen peroxide chemical species. That is, during corrosion of metallic biomaterials, the oxidation half-cell can create metal cations and oxide particles, while the reduction reactions can generate ROS that have significant biological affects [28]. In particular, the hydroxide radical is an extremely strong oxidizing agent with an oxidation potential of over 2.5 V [29]. This radical is also one of the most potent oxidizing agents generated by the mononuclear phagocytic cells of the body and can kill invading bacteria, breakdown organic molecules (eg, proteins like collagen) and has significant impact on the inflammatory system of the body. In addition, when such reactive oxygen intermediates are formed in the presence of transition metal cations in solution (eg, Fe2+/Fe3+), then additional reactions, known as Fenton reactions [30–33] can develop. The Fenton and Haber Weiss reactions are utilized by the inflammatory system of the body by way of ferritins which are protein storage sites for iron ions. When significant hydrogen peroxide is generated (either biologically, or by reduction reactions), ferritins release iron ions and create the Fenton catalytic reactions. The Fenton reactions are:



Fe2 + + H 2O2 → Fe3 + + OH• + OH− 2+ Fe3 + + H 2O2 → Fe (3.v) + HO2• + H+ While the Haber Weiss reactions are:



Fe3 + + O2• → Fe2 + + O2 3+ Fe2 + + H 2O2 → Fe (3.vi) + OH− + OH•

It can be seen that when iron ions and hydrogen peroxide are present together both hydroperoxyl and hydroxide radicals are generated and the iron serves as a redox catalyst. Indeed, any transition metal cation that can exist in multivalent states can serve as a Fenton catalyst, including cobalt and chromium. Therefore, when metal ions are present in the electrolyte from oxidation reactions of the metallic biomaterial, and hydrogen peroxide is generated by reduction processes (or inflammation), the Fenton reaction can result in an increase in the amounts of strong oxidizing agents (radicals) in solution. The generation of such highly oxidizing radicals by reduction reactions that arise in conjunction with elevated oxidation reactions associated with MAC may lead to damaging effects to local cells, denature or destroy organic molecules, and may alter the kinetic barrier effects of the oxide

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film itself. That is, the stability and passive character of oxide films may be altered by the presence of the products of these reduction reactions. Some of these effects will be discussed below. Thus, the reduction half-cell in biomaterials corrosion is likely as critical an element of the overall biocompatibility of metals as the toxicity associated with particles and ions.

3.3  IMMUNE CELLS, INFLAMMATION, AND ROS The interactions between metals and the body are undertaken and controlled by a range of immune cells. Upon implantation of the metallic implant, these immune cells react to, interact with, and direct the inflammatory processes. These processes are variable between patients, and depend on the extent and type of damage present and the size (eg, bulk or particulate) and reactivity (eg, ions vs oxides) of the foreign materials. Below is a summary of the cells of the immune system and the ways in which they may interact with the metallic biomaterial. A common feature of these cells is their use of ROS as a primary mechanism of attack of the foreign body, and this will be discussed as well.

3.3.1  Immune Cell Origin The cells of the immune system, or leukocytes (white blood cells), ultimately emerge from populations of multipotent hematopoietic stem cells (HSCs). HSCs produce two common progenitor cells, resulting in either lymphoid or myeloid lineage commitment [34]. Terminal lymphoid cells include T and B lymphocytes as well as natural killer cells. The common myeloid progenitor produces further lineage-restricted progenitors, eventually giving rise to erythrocytes and a host of phagocytes, granulocytes, and dendritic cells [35]. Granulocytes and cells arising from the monocyte are of particular interest when considering biocompatibility as these cells are highly specialized to rid the body of foreign objects. In fact, the same mechanisms used to fight infection may be responsible for excess oxidative stress leading to biomaterial failure [35].

3.3.2 Neutrophil Neutrophils (also known as polymorphonuclear leukocytes) are firstline-of-defense cells armed with potent granules and the ROS producing

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NADPH oxidase [36]. In addition to microbial killing through ROS, neutrophils contain granules which hold digestive proteins that play an important role in eliminating pathogens [37]. These frontline cells in the immune system can generate significant amounts of superoxide anion estimated to be between 1 M and 4 M in phagosomes [38]. This anion is rapidly converted to other ROS molecules either inside the phagosome, or released extracellularly. Neutrophils have long been implicated in causing gross tissue damage through release of extracellular ROS and proteases [39].

3.3.3 Macrophage Macrophages are antigen-presenting cells that arise from monocytes and are responsible for the phagocytosis of microbes, debris, and apoptotic cell remnants [40]. They can be divided into pro- and anti-inflammatory subsets and are designated as M1 and M2 phenotypes, respectively [41]. M1 macrophages are responsible for phagocytosis and regulation of the proinflammatory response through antigen presentation as well as chemokine and cytokine production [41]. The M2 phenotype suppresses pro-inflammatory cytokines and begins work on tissue reconstruction and repair [41]. M2 macrophages can be further sub-classified.

3.3.4  Foreign Body Giant Cell Foreign body giant cells (FBGCs) form from the fusion of macrophages in the presence of a foreign body that is unable to be phagocytosed normally [40]. These cells are large, multinucleated cells that work to breakdown extracellular debris. FBGCs also recruit and regulate fibroblasts to encapsulate the foreign body [42].

3.3.5 Osteoclast Osteoclasts, like FBGCs, are multinucleated cells formed by the fusion of macrophages [43]. These cells differ, however, in their function as osteoclasts are mainly tasked with bone resorption [44]. These cells are often identified by their expression of tartrate-resistant acid phosphatase (TRAP), which is essential in degradation of the protein component of bone due to its ability to produce ROS [45,46]. Additionally, osteoclasts produce hydrochloric acid which is responsible for mineral dissolution [44]. They can be considered inflammatory cells in the context of this text due to their response to certain inflammatory cytokines and their mechanism of action.

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3.3.6  Inflammatory Response/Cascade A general view of host response to an implanted biomaterial (Fig. 3.3) has been well described [47]. Upon initial implantation, the material comes into contact with blood where plasma proteins adhere to the surface. The (a)

Injury, implantation Inflammatory cell infiltration PMNS, Monocytes, Lymphocytes Biomaterial

Exudate/tissue

Acute inflammation Mast cells

IL-4, IL-13

Monocyte adhesion

PMNs

Macrophage differentiation

Chronic inflammation Monocytes Th2: IL-4, IL-13 Lymphocytes Granulation tissue

Macrophage mannose receptor up regulation Macrophage fusion

Fibroblast proliferation and migration Capillary formation Fibrous capsule formation (b) Acute

Chronic

Foreign body giant cell formation

Granulation tissue

Neutrophils Macrophages Neovascularization Intensity

Foreign body giant cells Fibroblasts Fibrosis Mononuclear lekcocytes Time (Minutes, hours, days, weeks)

Figure 3.3  Typical response to an injury or implanted biomaterial which includes (a) an important role for cytokines from T lymphocytes and mast cells, and (b) relative temporal relationship and response intensity of cell types during the healing response. (a) Reproduced with permission from Ref. [47] and (b) adapted from Ref. [48].

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dynamic adsorption and desorption of these proteins is known as the Vroman Effect [49]. This protein layer serves as an active matrix capable of modulating cellular activity in the ensuing immune and wound healing cascade [47]. Acute inflammation is a short-term, aggressive response characterized by neutrophil infiltration [47]. Rapid release of lytic enzymes and ROS causes nonspecific damage to the area immediately surrounding the implant [50]. Neutrophils also release, among other species, CCL2 (monocyte chemotactic protein) and CCL4 (monocyte inflammatory protein) which are monocyte and lymphocyte chemoattractant proteins [51,52]. Recruitment of mononuclear immune cells such as monocytes and lymphocytes marks the onset of the chronic inflammation phase of wound healing [47]. This phase is a complex orchestra of cells, chemokines, and cytokines that revolve around the biomaterial interface [47]. Initially, monocytes differentiate almost exclusively into M1 macrophages in an attempt to clean the wound area by phagocytosing particles, microbes, and cellular debris [50]. In the more advanced stages of chronic inflammation, the macrophage population shifts to an M2 phenotype thereby suppressing inflammation and beginning work toward tissue reconstruction [50]. M2 macrophages release important extracellular matrix components and other products that are involved in recruiting and activating fibroblasts [53]. In addition to this typical view of the wound healing cascade, implanted biomaterials present a unique challenge to these phagocytes because of their size [47]. In fact, macrophages begin to struggle when particle sizes exceed 5 μm [47]. Formation of FBGCs then occurs and they attempt to degrade the surface [47]. Much like an osteoclast, FBGCs create a sealed border between themselves and the surface and release protons, enzymes, and ROS into the confined space [54]. T lymphocytes in the area increase macrophage adhesion and fusion into FBGCs [55]. Similar to the M2 macrophage, the FBGC is attempting to degrade the material; it is also releasing anti-inflammatory cytokines and factors to facilitate collagen deposition for encapsulation by fibroblasts [42,53].

3.3.7  ROS Production As mentioned, there are a wide array of phagocytic cells (mononuclear phagocytes) that rely on ROS generation and utilization to carry out their functions. All share a common mechanism of ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the primary structure that produces ROS for phagocytic activity (see Fig. 3.4 [56–58]). The complex serves as an electron transport chain that

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hyroxyl radical

OH Fenton reaction Fe2+/Fe3+ from Ferritins H2O2 Extracellular or phagosome

SCN–

HOSCN

MPO

2O2

Superoxide anion 2O –

SOD

2

2e–

Cell membrane

hydroperoxyl radical

HO2

NO

NADPHoxidase

R

HOCI hypochlorous acid

ONOO peroxynitrite SOD – Superoxide dismutase MPO – Myeloperoxidase

Intracellular NADPH

NADP+ + H+

Figure 3.4  The generation of ROS in mononuclear phagocytic cells is governed by the NADPH oxidase complex that is bound to the cell membrane in both phagosomes and extracellular membranes. Oxidation of NADPH and transfer of the electrons across the membrane results in reduction of oxygen molecules to superoxide anions, which can then undergo a range of subsequent reactions to generate hydrogen peroxide, hydroxide radicals, and hypochlorous acid among others. This ROS is the principal mechanism phagocytic cells use to attack foreign bodies, degrade bacteria, and even break down bone. In particular, when combined with iron ions, hydrogen peroxide can engage in Fenton reactions (see text) to rapidly increase the generation of hydroxyl radicals which are extremely oxidizing and can adversely affect metallic biomaterial surfaces increasing corrosion by an ICIC process. Schematic adapted from Ref. [56].

results in the reduction of oxygen, O2, to the superoxide anion, O−• 2 , while NADPH is oxidized [57]. NADPH oxidase is located at the membrane of phagocytic vacuole and secretory vesicles (in neutrophils) [59]. Upon activation, neutrophils, for example, mobilize secretory vesicles to fuse with the outer membrane releasing superoxide into the local environment [60]. The superoxide anion can be further processed to produce subsequent ROS which are more active [61]. O−• 2 will undergo dismutation to form hydrogen peroxide, H2O2 when the concentration is high enough, otherwise it can be catalyzed by oxygen dismutase [61]. Hydrogen peroxide can participate in reactions that result in highly reactive species with high oxidative abilities. Myeloperoxidase (MPO) (see Fig. 3.4), primarily located in the azurophilic granules, catalyzes the

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reaction between H2O2 and halides (and pseudohalides) to form hypohalous acid (HOX) [36,62]. Of the many halides present in the body, MPO has the highest affinity for Cl− resulting in the potent oxidant hypochlorous acid (HOCl). Since macrophages lack this peroxidase, they do not typically produce this species [62]. H2O2 can also participate in the Fenton and Haber-Weiss reactions described above, both resulting in the highly active hydroxyl radical (OH•) [63]. In cases of frustrated phagocytosis, where the biomaterial is too large to be phagocytosed, all of these species plus enzymes such as collagenases and gelatinases are released onto the target material [54]. In this way, osteoclasts can be thought of as a specialized cell that acts by frustrated phagocytosis on bone. The cell seals off a resorptive lacuna with a ruffled border creating a small isolated volume on which to act [44]. Osteoclasts seek to acidify this volume by pumping protons created by carbonic anhydrase II and through the release of acidic vesicles into the lacuna [64]. Additionally, the enzymes TRAP and cathepsin K are released and have been shown to work together to produce • OH [65].

3.3.8  Biological Effects of ROS ROS, whether generated and released by cells or from the metal, have been shown to cause damage to specific biological structures. The hydroxyl radical in particular will oxidize both free and membrane-bound lipids which can cause long-term membrane damage [66]. Oxidized lowdensity lipoprotein has been shown to trigger hypertrophy in arterial smooth muscle cells and has been implicated in the pathogenesis of atherosclerosis [67]. ROS easily alters nearby proteins by interacting with the peptide backbone α-carbon, resulting in an α-carbon radical which then continues to an alkylperoxyl radical. This highly active site results in peptide cleavage, peptide linkage, or side chain alteration [68]. In the event that ROS reaches nuclear DNA, mutagenic effects are known to take place [61,69]. The current view is that intracellular H2O2, as is produced in a phagosome, diffuses into the nuclease where it meets available Fe2+ causing the production of • OH [61]. The most common modifications seen occur on guanine [69]. The hydroxyl radical easily associates with the electron-rich π-orbital network to generate 8-oxo-guanine which forms a stable base pair with thymine, a base-pair mismatch resulting in mutagenesis and carcinogenesis [61].

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3.4  METAL IONS AND WEAR DEBRIS EFFECTS ON LOCAL TISSUES A major aspect of the inflammatory response of the body relates to the response of immune cells to the degradation products associated with wear and corrosion of metallic biomaterials surfaces. The effects of these degradation products on the cellular response in the periprosthetic tissue have been the subject of decades of intensive study [37,70,71]. Below is a brief review of the state of knowledge in this area. Biocompatibility of metals has been attributed to the nature and bioactivity of the proteinaceous layer that surrounds wear particles and the complexes formed by released ions [72]. Initial binding by abundant albumins is eventually replaced by larger, more bioactive proteins [72]. Proteins showing high affinity to wear particles include immunoglobulin G (IgG) and lipopolysaccharide [72,73]. IgG coating (or opsonization) is recognized by the monocyte/macrophage integrin, αM/β2 [74]. Integrin binding causes a host of signal transduction and downstream changes [47]. The β2 integrins in particular are crucial for monocyte adhesion and cause cytoskeletal rearrangements in macrophages to form specialized adhesion structures, podosomes [75]. Lipopolysaccharide is recognized by the tolllike receptor pathway in monocytes and macrophages causing cytokine release for cell recruitment and activation [76]. Clearly, specific protein interactions with wear particles are important in the biocompatibility of metals. Studies of the effects of ions on inflammation (mainly Ti, Cr, and Co ions) have revealed release of the cytokines tumor-necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 from macrophages [77]. Other immune cells, such as T lymphocytes and dendritic cells have been shown to increase production of IL-12 and, equally as important, decrease IL-10 production [77]. IL-10 works to modulate the immune response as an anti-inflammatory agent [77]. Ti wear particles have been shown to trigger the macrophage NACHT-, Leucine-rich repeat (LRR)-, and pyrin domain (PYD)containing protein 3 (NALP3) inflammasome leading to escalated inflammation (see Fig. 3.5) [78]. The NALP3 inflammasome initiates the recruitment and cleavage of pro-caspase-1 into caspase-1, which in turn cleaves pro-IL-1β into its active form of IL-1β, recruiting neutrophils to the area [78]. Moreover, macrophages stimulated by metallic particles

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LPS

Wear Ions Ti2+ particles Co2+

TLRs

Cr3+

Co2+ Cr3+

IL-6

NF-κβ

Ti2+

TNF-α

ROS

IFN-γ CCL2

NADPH oxidase

CCL3

NALP3 inflammasome

CCL4 Pro-IL-1β

Pro-caspase-1

Caspase-1

Pro-IL-18 Pro-IL-33

IL-1β IL-18 IL-33

Figure 3.5  Model of cytokine and chemokine release in response to wear particles and ions with a central role for both the NALP3 inflammasome and NF-κβ. These signaling cascades can work in tandem or independently to release many proinflammatory factors, including members of the IL-# and C-C chemokine ligand (CCL#) families as well as the cytokines TNF-α and interferon-γ (IFN-γ). Lipopolysaccharide (LPS) adsorbed to wear particles activate the transcription factor (NF-κβ) through tolllike receptor 4 (TLR4), whereas phagocytosed debris and ions cause NALP3 inflammasome activation by ROS [40,78,79].

increase their production of CCL2, leading to monocyte influx and activation [80]. This chemokine has also been found in increased amounts in tissues surrounding loosening implants implicating its role in failure [80]. Through yet another pathway the production of ROS in response to ions and wear particles triggers the transcription factor nuclear factor-κB (NF-κB), which also results in the production and release of TNF-α, IL-1, and IL-6 [79]. Macrophages that have phagocytosed Ti wear particles also release prostaglandin E2 which causes increased production of receptor activator of nuclear factor-κB-ligand (RANK-L) by osteoblasts encouraging osteoclastogenesis and bone resorption [81].

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3.4.1  Adverse Local Tissue Reaction In a typical injury, multifaceted immune pathways are activated as they attempt to quickly neutralize pathogens and debris, then proceed with normal wound healing, characterized by granulation tissue and finally fibrosis [47]. With regards to metallic biomaterials, however, normal resolution of chronic inflammation may not fully take place. Additionally, continuous release of corrosion products will constantly present new initiation signals for the immune system. The continuous reaction to these products can lead to complications in periprosthetic tissues. Both soft and hard tissues surrounding a metallic implant are subject to damage by the immune response. The response of macrophages and the prolonged release of pro-osteolytic cytokines ultimately leads to bone resorption [76,77]. Soft tissue lesions, typically presenting with cystic properties have been termed “pseudotumors” due to their similarity to necrotic tumors [82]. A reaction similar to type IV delayed hypersensitivity has been observed and has been termed aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) [71]. This reaction is characterized by high degrees of lymphocyte infiltration accompanied by effusion, vascular endothelial swelling, and necrosis [71]. Typically, while foreign body granulomas may exist, they are not the predominant feature of ALVAL suggesting accumulation of wear particles is not the major cause [83]. Analysis of tissues around failed implants has revealed differences in presentation between cases of high and low wear [83]. Cases of both high and low wear were shown to include macrophages and lymphocytes in the surrounding tissue [83]. Those patients with significant pain that underwent revision due to suspected hypersensitivity had predominantly lymphocyte infiltration with far greater tissue damage as compared to the high wear group [83]. Patients with pseudotumors associated with high wear displayed a higher degree of macrophage infiltration but also had healthier soft tissue [83]. There are also cases of necrosis without signs of the hypersensitivity reaction pointing to the involvement of apoptotic macrophages [84]. Phagocytosis of wear particles leads to the release of large quantities of metal ions within the cell, more specifically within the lysosome, which at high enough concentrations can result in macrophage apoptosis or necrosis releasing ROS and ions into the tissue, causing further recruitment and damage [84]. Not only can inflammatory cells affect tissue, but recent evidence from our group suggests cells can directly corrode metallic biomaterial implant surfaces [13].

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In summary, there is an extensive literature related to metal ion and particle effects on periprosthetic tissues and it is not the intent of this chapter to review this aspect of metal biocompatibility. However, within this complex milieu, some common and under-appreciated aspects are present that include the interplay between metals, corrosion, ROS, and immune/inflammatory cells. In the next sections, these interactions are discussed.

3.5  REDUCTION REACTIONS AND CELLULAR VIABILITY Several studies since 1998 have demonstrated the strong effects of reduction reactions and their products on cell viability and behavior. In 1998, Gilbert et al. [15] showed the importance of reduction reactions on cell viability. This was followed in 2002 by Bearinger et al. [16] where the concept was developed that ROS from inflammatory cells may affect the oxides on titanium. Kalbacova et  al. [27,28] showed that when sustained cathodic currents are applied to titanium surfaces, cells exhibit increased ROS generation and demonstrate oxidative stress. Ehrensberger et  al. [8] also demonstrated that bone-like cells (MC3T3-E1 preosteoblasts) were killed rapidly when cultured on titanium surfaces when the potential of the surfaces was potentiostatically held below − 300 mV (vs Ag/AgCl) for 24 h where small sustained cathodic (reduction) currents and reactive oxygen intermediates were generated. More recent work [12] has shown that the threshold potential below which cells are killed is closer to − 400 mV (vs Ag/AgCl) and as the potential is held at more negative values, the killing effects occurred in shorter periods of time. This indicates that − 400 mV (vs Ag/AgCl) is an important potential below which significant amounts of reduced chemical species (eg, reactive oxygen intermediates) begin to be generated and that the farther below this threshold potential, the greater the rate of generation and the more potent the effect on cells cultured on these surfaces [12]. This leads to the idea of a generation-consumption model of interaction whereby the electrode generates reactive oxygen intermediates (or by-products of their generation) which are then “consumed” by cells cultured at the site of generation. When the amounts reach a critical level, the cells rapidly ball up, lose their cytoskeletal structure, and die. As the potentials reach − 1000 mV (vs Ag/AgCl), cells cultured on the surface die in as little as 2 h [12]. Additional insight into this concept can be found by the

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fact that cells that are cultured in the chamber, but remote from the electrode surface, are not killed by this process. Work by Haeri et  al. [9–11] has shown that not only Ti surfaces but also CoCrMo surfaces exhibit similar behavior and that the mechanism of cell death is from activation of the intrinsic apoptosis pathway [9–11]. That is, potentials more cathodic than −400 mV on CoCrMo surfaces induce cellular apoptosis. The progression of this cellular behavior is characterized by the loss of focal adhesion complexes [10], depolymerization of the actin cytoskeleton and loss of cell viability [9–11]. These cellular responses to negatively biased electrodes, below a threshold potential, arise when the cathodic current densities are as small as 0.1 μA/cm2 [9] and appear to be relatively universal in terms of the cells susceptible and in the mechanism of cell death. Indeed, even bacteria [85,86] are susceptible to cathodic electrochemical processes. Haeri’s work [9] also demonstrated that when CoCrMo surfaces are biased to above +300 mV (vs Ag/AgCl), cells cultured on their surface rapidly died as well, however, the process was distinctly different. The cells simply ceased activity and/or lost cell membrane integrity, dying by necrosis. Thus, the concept of a “zone of electrochemical viability” was defined as the potential range (−400< V< +300 mV vs AgAgCl) where cells cultured on CoCrMo remained viable for 24 h or longer (see Fig. 3.6). For titanium, cells remained viable up to +1000 mV (vs Ag/AgCl, the highest investigated) [8] and thus Ti does not have the same killing effect as CoCrMo at positive potentials due to the fact that titanium does not lose its passive oxide film, nor does it release significant metal ions that may be harmful to cells cultured on their surface. CoCrMo, on the other hand, loses its passive oxide layer starting around +300 mV (vs Ag/AgCl) as the oxidation state of Cr in the oxide begins to increase above +3 and the compact, passive nature of the oxide is lost allowing for increased ionic dissolution to occur. Recent efforts have been focused on adoption of this approach (generation of reduction reaction products) to create bimetallic couples in either bulk or particulate form to deliver to the sites of tumors or infection [87]. Early results indicate that bimetallic particles of magnesium and titanium can establish a galvanic couple where the Mg acts as the anode (oxidizes) and titanium as the cathode (where reactive oxygen intermediates are generated). These bimetallic particles have a significant killing effect on MC3T3 cells [87] over and above that seen with Mg particles alone, which also demonstrated cell killing effects.

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Figure 3.6  The concept of an electrochemical zone of viability (cell viability vs static electrochemical potential) is demonstrated in the data of (a) Haeri et  al. and (b) Ehrensberger et al. [8,9] which shows the viability of MC3T3-E1 preosteoblasts cultured on CoCrMo (a) and Ti (b) surfaces, respectively in vitro. For CoCrMo, the zone of viability is between −400 mV and +300 mV (vs Ag/AgCl), while the zone of viability for cells on commercially pure titanium surfaces is above −400 mV (vs Ag/AgCl). Potentials below the viability limit result in generation of reactive oxygen intermediates that appear to cause apoptosis for both alloys, while potentials above +300 mV in CoCrMo induce cellular necrosis. Titanium does not induce cell death at positive potentials up to +1000 mV. Reproduced with permission from (a) Ref. [9] and (b) Ref. [8].

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In summary, reduction electrochemistry at metallic biomaterials, a required set of electrochemical reactions associated with corrosion, has a pronounced effect on the interaction of living cells with the alloy surface. Strong killing effects are generated by relatively small reduction current densities when surfaces engage in reduction reactions of sufficient intensity. These reduction reactions (and products) induce apoptosis of cells (when the voltage is below −400 mV vs Ag/AgCl) as opposed to necrosis, which is seen with CoCrMo alloys when their potential exceeds +300 mV (vs Ag/AgCl). Therefore, there are zones of electrochemical viability established for alloys where cells cultured on their surface will remain viable and beyond which cells will die by apoptosis (if below) and necrosis (if above).

3.6  ICIC OF CoCrMo AND TI ALLOYS: ROS EFFECTS ON CORROSION AND WEAR So far, this chapter has discussed the basic concepts of corrosion (oxidation and reduction) in the context of metallic biomaterials and the biological system, the basic phagocytic cell types involved in immune and inflammatory responses, the effect of metal ions and particles on periprosthetic tissues in the body, and the effects of reduction reactions on cells. In this section, the effect of inflammatory cells on corrosion of implant alloy surfaces is described. This recent work has presented evidence that the cells of the mononuclear phagocytic cell line (ie, monocytes, lymphocytes, neutrophils, macrophages, histocytes, osteoclasts, FBGCs, etc.) are capable of producing local environments that can lead to direct corrosion attack of the CoCrMo and titanium alloy implant surface in vivo. This so-called ICIC is a new observation which is a critical element in the overall interaction between implant and host that has not been previously appreciated. The basic concept here is that these cells use oxidative burst (or respiratory burst) processes to carry out their specific functions. As already described, the products of respiratory burst reactions are superoxide anions and a range of secondary products depending on the cell type and the circumstances that include hydrogen peroxide, hydroxyl and hydroperoxyl radicals, peroxynitrite and hypochlorous acid (bleach) (see Fig. 3.4). It turns out that products of respiratory burst and their secondary products have not been studied in any significant way in terms of their effects on corrosion of metallic biomaterials. All of these ROS molecules

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are electrochemically active and exhibit strong oxidizing power (ie, they are oxidizing agents that drive oxidation of other species). The body evolved these defenses to kill invading bacterial and to breakdown foreign bodies, yet little is known about their effects on corrosion of CoCrMo or Ti alloys. Recent retrieval studies [13] of metallic orthopedic implants have provided evidence that inflammatory cells of the body can attach to metallic implant surfaces made from CoCrMo alloy and titanium alloy [88] and induce direct corrosion attack of the implant surface (see Fig. 3.7, for examples). The corroded surfaces appear to have unique patterns of corrosion damage that reflect the biological nature of the process. The phagocytic cells release granules and vesicles containing ROS into the

Figure 3.7  Digital optical microscopy (DOM) (a) and (c) and scanning electron microscopy (SEM) (b) and (d) images of apparent inflammatory cell induced (ICI) corrosion on retrieved CoCrMo total hip replacement components. The patterns of corrosion attack reflect the phagocytic cell type involved in the corrosion and a range of corrosion morphologies are observed. These include ruffled border-like corrosion, pitting, and disc-shaped crater-like formations that reflect the complex oxidationreduction processes associated with the cell-based attack.

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extracellular space between cell and implant and appear to induce a corrosion pattern that mimics the ruffled border shape of the cell membrane. In fact, if one compares the pattern of corrosion seen in some regions with a transmission electron micrograph (used with permission from Ref. [89]), see Fig. 3.8, there is a significant similarity in the pattern of corrosion attack observed to the intracellular structures seen in the TEM of the neutrophil. The granules and secretory vesicles of the cell appear to have released their contents directly onto the CoCrMo surface and induced corrosion patterns that directly reflect the cellular morphology. There is evidence in these retrievals of iron streaks deposited on to implant surfaces may be the result of immune cells migrating and attacking the implant surface. Fenton reactions, where hydroxyl radicals are generated and iron engages in a cycle of oxidation and reduction and apparently results in reduction of iron to its metallic state on the implant

Figure 3.8  Comparison of ICI corrosion and a transmission electron micrograph of a neutrophil in cross section. Note the similarity in patterns of corrosion damage (SEM micrographs on left) with the structure of the cell (right). Granules, secretory vesicles, and other organelle structures are reflected in the pattern of corrosion attack seen. TEM micrograph reproduced with permission from Ref. [89].

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Figure 3.9  DOM and SEM images of streaks of iron apparently deposited by an inflammatory cell as it migrated across and corroded a CoCrMo implant surface in vivo. These streaks are mostly comprised of metallic iron and are likely a result of reduction reactions during Fenton reactions driven by the phagocytic cell. Note the 5 µm wide weld-like pattern in the SEM micrograph that is the result of the cell-driven process.

surface and appears as if a 10 µm wide weld line has been deposited (see Fig. 3.9). This ICI corrosion may occur on both CoCrMo and Ti alloy surfaces. For the titanium cases observed, in one there was a bacterial infection that appeared to be co-incident with the corrosion attack of the alloy [90], and may have stimulated the inflammatory cells. In addition, during ICI corrosion, patterns of corrosion attack have been documented in the modular taper junction of total hip replacements. In these cases, inflammatory cells appear to be recruited into the crevices formed by modular taper junctions and have engaged in ICIC within these regions. Some of these regions of attack can be so severe that deep pits and fissures are developed from the build-up of ROS species within the crevice itself [90] (see Fig. 3.10). While most studies relate corrosion in modular tapers to MAC alone, the role of ROS and inflammation on

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Figure 3.10  Matched pair of (a) secondary and (b) backscattered SEM micrographs of ICI corrosion in the taper junction region of the CoCrMo liner of an acetabular metalon-metal hip replacement. Evidence of cells (white arrows), cellular remnants, and biological materials are present (dark in the backscattered electron micrograph) entwined with the corroding CoCrMo alloy.

corrosion has been ignored, yet it is clearly a potential element of the overall process. ROS can alter the corrosion behavior of CoCrMo and titanium as can be seen by the increase in corrosion potential of CoCrMo when immersed in physiological solutions containing H2O2 [13]. Increases of upwards of 1000 mV from corrosion potentials in phosphate buffered saline alone can occur. When iron ions are added, additional increases in corrosion susceptibility are seen for CoCrMo. Titanium is also susceptible to increased corrosion attack when the hydrogen peroxide level is increased [16,91]. In both cases, hydrogen peroxide can reduce the barrier effect of the oxide films (ie, make them more susceptible to corrosion attack), and can raise the oxidizing potential of the solution to increase the driving force for corrosion. There have been virtually no systematic studies of the role of other ROS on corrosion of these alloy systems and work is on-going to study factors like HOCl, hydroxyl radicals, and others on corrosion.

3.7  SUMMARY AND CONCLUSIONS This chapter has presented a review of the corrosion interactions of metallic biomaterials with cells of the body. The ability of metallic biomaterial surfaces to generate reactive oxygen intermediates due to reduction reactions which are then able to kill cells on the metal surface

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was described. In addition, immune/inflammatory cells of the body can generate ROS that has the ability to increase the corrosion rate of metals. In fact, evidence of direct corrosion of CoCrMo and titanium alloys by inflammatory cells in retrieved implants shows the ability of the body to attack these most corrosion resistant alloys. While metallic biomaterial compatibility has been focused on the role of wear particles and ions on tissue responses, this chapter has shown that while these issues remain an important aspect of how metals behave in the body, they are by no means the sole effectors of biological response and that the interaction between metal and body is a two-way positive feedback system where corrosion causes biology and biology causes corrosion. These new insights should provide rich opportunities for further exploration of the ways in which the human body and metallic biomaterials interact.

ACKNOWLEDGMENTS This work was supported, in part, by the National Science Foundation IGERT Fellowship (DGE-1068780), and a grant from DePuy Synthes. The lead author would also like to thank the University of Leeds for its support through the Cheney International Faculty Fellowship in the Institute for Medical and Biological Engineering. In addition, we would like to thank Shiril Sivan for use of some electron micrographs from his dissertation work, Katy Pieri, Morteza Haeri, and Mark Ehrensberger for their efforts on different aspects of the work presented.

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[53] Gratchev A, Guillot P, Hakiy N, Politz O, Orfanos CE, Schledzewski K, et  al. Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3. Scand J Immunol 2001;53(4):386–92. [54] Henson PM. The immunologic release of constituents from neutrophil leukocytes. II. Mechanisms of release during phagocytosis, and adherence to nonphagocytosable surfaces. J Immunol 1971;107(6):1547–57. [55] Brodbeck WG, Macewan M, Colton E, Meyerson H, Anderson JM. Lymphocytes and the foreign body response: lymphocyte enhancement of macrophage adhesion and fusion. J Biomed Mater Res A 2005;74A(2):222–9. [56] Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase and bacterial killing. Blood 1996;92(9):3007–17. [57] Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232(1):3–14. [58] Winterbourn CC, Hampton MB, Livesey JH, Kettle AJ. Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome. J Biol Chem 2006;281(52):39860–9. [59] Borregaard N, Tauber AI. Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome, and associated flavoprotein. J Biol Chem 1984;259(1):47–52. [60] Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 1983;97(1):52–61. [61] Bergamini CM, Gambetti S, Dondi A, Cervellati C. Oxygen, reactive oxygen species, and tissue damage. Curr Pharm Design 2004;10(14):1611–26. [62] Podrez EA, Abu-Soud HM, Hazen SL. Myeloperoxidase-generated oxidants and atherosclerosis. Free Radical Bio Med 2000;28(12):1717–25. [63] Liochev SI. The mechanism of “Fenton-like” reactions and their importance for biological systems: a biologist’s view. Metal Ions Biol Syst 1999;36:1–39. [64] Rousselle AV, Heymann D. Osteoclast acidification pathways during bone resorption. Bone 2002;30(4):533–40. [65] Vääräniemi J, Halleen JM, Kaarlonen K, Ylipahkala H, Alatalo SL, Andersson G, et al. Intracellular machinery for matrix degradation in bone-resorbing osteoclasts. J Bone Min Res 2004;19(9):1432–40. [66] Girotti AW. Lipid hydroperoxide generation, turnover, and effector action in biological systems. J Lipid Res 1998;39(8):1529–42. [67] Podrez EA, Poliakov E, Shen Z, Zhang R, Deng Y, Sun M, et  al. A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions. J Biol Chem 2002;277(41):38517–23. [68] Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997;272(33):20313–6. [69] Lee JC, Son YO, Pratheeshkumar P, Shi X. Oxidative stress and metal carcinogenesis. Free Radical Bio Med 2012;53(4):742–57. [70] Willert HG. Reactions of the articular capsule to wear products of artificial joint prostheses. J Biomed Mat Res 1977;11(2):157–64. [71] Willert HG, Buchhorn GH, Fayyazi A, Flury R, Windler M, Köster G, et al. Metalon-metal bearings and hypersensitivity in patients with artificial hip joints: a clinical and histomorphological study. J Bone Joint Surg Am 2005;87A(1):28–36. [72] Hallab NJ, Skipor A, Jacobs JJ. Interfacial kinetics of titanium- and cobalt-based implant alloys in human serum: metal release and biofilm formation. J Biomed Mater Res A 2003;65A(3):311–8. [73] Xing Z, Pabst MJ, Hasty KA, Smith RA. Accumulation of LPS by polyethylene particles decreases bone attachment to implants. J Orthop Res 2006;24(5):959–66.

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[74] McNally AK, Anderson JM. Complement C3 participation in monocyte adhesion to different surfaces. Proc Natl Acad Sci USA 1994;91(21):10119–23. [75] Rose DM, Alon R, Ginsberg MH. Integrin modulation and signaling in leukocyte adhesion and migration. Immunol Rev 2007;218(1):126–34. [76] Greenfield EM, Bi Y, Ragab AA, Goldberg VM, Nalepka JL, Seabold JM. Does endotoxin contribute to aseptic loosening of orthopedic implants? J Biomed Mater Res B 2005;72B(1):179–85. [77] Magone K, Luckenbill D, Goswami T. Metal ions as inflammatory initiators of osteolysis. Arch Orthop Trauma Surg 2015;135(5):683–95. [78] St. Pierre CA, Chan M, Iwakura Y, Ayers DC, Kurt-Jones EA, Finberg RW. Periprosthetic osteolysis: characterizing the innate immune response to titanium wear particles. J Orthop Res 2010;28(1):418–24. [79] Caicedo MS, Desai R, McAllister K, Reddy A, Jacobs JJ, Hallab NJ. Soluble and particulate Co-Cr-Mo alloy implant metals activate the inflammasome danger signaling pathway in human macrophages: a novel mechanism for implant debris reactivity. J Orthop Res 2009;27(7):847–54. [80] Goodman SB, Ma T. Cellular chemotaxis induced by wear particles from joint replacements. Biomaterials 2010;31(19):5045–50. [81] Schwab LP, Marlar J, Hasty KA, Smith RA. Macrophage response to high number of titanium particles is cytotoxic and COX-2 mediated and it is not affected by the particle’s endotoxin content or the cleaning treatment. J Biomed Mater Res A 2011;99A(4):630–7. [82] Kwon YM, Ostlere SJ, McLardy-Smith P, Athanasou NA, Gill HS, Murray DW. “Asymptomatic” pseudotumors after metal-on-metal hip resurfacing arthroplasty. J Arthroplasty 2011;26(4):511–8. [83] Campbell P, Ebramzadeh E, Nelson S, Takamura K, De Smet K, Amstutz HC. Histological features of pseudotumor-like tissues from metal-on-metal hips. Clin Orthop Relat Res 2010;468(9):2321–7. [84] Mahendra G, Pandit H, Kliskey K, Murray D, Gill HS, Athanasou N. Necrotic and inflammatory changes in metal-on-metal resurfacing hip arthroplasties. Acta Orthop 2009;80(6):653–9. [85] Guo J, Gilbert JL. Interactions of Escherichia coli HM22 biofilm with electrochemically active commercially pure titanium surface. In: Transactions of the 37th Annual Meeting of the Society for Biomaterials; 2013 April 10–13; Boston, MA. [86] Niepa THR, Gilbert JL, Ren D. Controlling Pseudomonas aeruginosa persistent cells by weak electrochemical currents and synergistic effects with tobramycin. Biomaterials 2012;33(30):7356–65. [87] Kim J, Gilbert JL. Cytotoxicity of MgTi particles for targeted therapeutic effect. In: Transactions of the 9th World Biomaterials Congress; 2012 Jun 1–5; Chengdu, China. [88] Sivan S, Pieri KG, Gilbert JL. Cell-induced corrosion on titanium alloys. In: Transactions of the 61st Annual Meeting of the Orthopaedic Research Society; 2015 March 28–31; Las Vegas, NV. [89] Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005;23(1):197–223. [90] Gilbert JL, Sivan S. Inflammatory cell-induced corrosion of CoCrMo acetabular liner tapers. In: Transactions of the 38th Annual Meeting of the Society for Biomaterials; 2014 April 16–19; Denver, CO. [91] Gilbert JL, Bai Z, Chandrasakaran N. Method of preparing biomedical surface. United States Patent US 8,012,338. Sep 6, 2011.

CHAPTER FOUR

Oxidative Stress and Biomaterials: The Inflammatory Link Isaac M. Adjei, Glendon Plumton and Blanka Sharma J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States

4.1 INTRODUCTION Biomaterials are used in a broad ranges of clinical devices to monitor physiological conditions, such as glucose levels [1], for controlled drug release [2], and for tissue engineering [3,4] purposes, due to the amenability of their physicochemical and bioactive properties [5,6]. In the course of research and development, several hurdles have been identified that can impact the functionality and longevity of biomaterials in patients. After introduction into patients, a patient’s body most commonly reacts to biomaterials through blood–material interactions, acute and chronic inflammation, formation of foreign body giant cells (FBGCs), and fibrous capsule formation [7]. These responses collectively comprise the foreign body response (FBR) to the material, and can result in pain, and reduced longevity and functionality of the device [8]. The response of the body to a biomaterial is largely dependent on its physicochemical characteristics [9]. For example, the blood residence time of drug delivery systems such as nanoparticles (NPs) and microparticles (MPs) is influenced by their size, surface charge, shape, and chemical composition, which ultimately impacts therapeutic efficacy [10,11]. As such, efforts are focused on understanding biomaterial interactions with tissues and blood in order to develop strategies that will improve their biocompatibility and efficacy. Inflammation, the body’s response to foreign materials and tissue damage mediated by innate immunity, is amongst the first responses of the host to a biomaterial [12]. The process starts with chemical signals produced by the injured tissue upon biomaterial implantation that recruits neutrophils, monocytes, and eosinophils to the site of tissue damage [13,14]. When a chemotactic signal is perceived by these cells, they activate the selectin family of adhesion proteins and integrins that enable Oxidative Stress and Biomaterials. Doi: http://dx.doi.org/10.1016/B978-0-12-803269-5.00004-8

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them to migrate to the damaged tissue. Neutrophils are usually the first to arrive at the injury site and secrete chemokines that recruit monocytes, which differentiate into macrophages [15,16]. Macrophages subsequently secrete growth factors and cytokines that recruit and influence the resulting behavior of epithelial, endothelial, and mesenchymal cells in the tissue to promote repair [17,18]. Reactive oxygen species (ROS) which are normal metabolic byproducts, including superoxide anion (O•− 2 ) and hydrogen peroxide (H2O2), play important roles in the initiation and progression of inflammation as both signaling and effector molecules [19,20]. H2O2 produced at sites of foreign material serves as a chemoattractant to recruit immune cells [21], while O•− 2 produced by immune cells act on the foreign body to neutralize it [22]. This chapter will discuss the inflammatory response to biomaterials and the relationship between oxidative stress and inflammation. The impact of the physicochemical properties and the form in which biomaterials are presented to the body on the induction of both oxidative stress and inflammation will also be reviewed.

4.2  FBR TO BIOMATERIALS The FBR is the body’s response to the implantation of foreign biomaterials in order to repair any tissue damage its implantation may have caused, and to prevent further damage. The FBR is composed of inflammatory and wound healing responses. The FBR is subdivided into the acute response, which is the initial response as immune cells try to degrade the foreign body, and repair any damage the biomaterial itself or process of implantation may have caused. If this is not possible, there is a secondary chronic response, where immune cells form FBGCs, isolate the implanted material in a fibrous capsule to decrease interaction with surrounding tissues, and produce enzymes and ROS in an attempt to degrade the material [8].

4.2.1  Acute Response Immediately following in vivo introduction of a biomaterial, proteins in the blood and extracellular fluid adsorb to its surfaces and are intimately involved in tissue healing and FBR [23]. The specific proteins and the concentrations vary based on the surface properties of the biomaterial,

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and change over time through the Vroman effect, whereby abundant proteins with low affinity desorb and are replaced by proteins with higher affinities to the material’s surface. Proteins adsorbed to the biomaterial provide biochemical signals to attract immune cells to the site of implantation and play critical roles in cellular recognition and adhesion to the material, as well as other responses of inflammatory cells to the biomaterial [24]. Proteins adsorbed to implants play different roles in the FBR, including initiating clotting and recruiting inflammatory cells. Proteins involved in the coagulation cascade minimize bleeding from vascular damage associated with biomaterial implantation, both before and after adsorption to the surfaces of biomaterials. Blood coagulation on the biomaterial surface is initiated by factor XII (FXII) and tissue factor (TF). FXII is activated on contact with negatively charged surfaces and serves as the primer for the subsequent cascade of protein reactions that ends in the release of thrombin. The selectivity of FXII to negatively charged surfaces is due to the displacement of competing proteins between hydrophilic and hydrophobic surfaces [23], and occurs in conjunction with platelet adhesion to the biomaterial surface to induce clot formation [25]. Thrombin generated in the cascade is a serine protease, and cleaves soluble fibrinogen into polymerized, insoluble fibrin (Fig. 4.1). Activated concurrently with the coagulation cascade is the complement system, which plays a role in removing foreign materials by either lysing them or by tagging them for clearance. This occurs in one of two mechanisms, the classical or alternative pathway, and can induce an inflammatory response in the process. The classical pathway requires an antibody, such as IgG, to adsorb to an antigen, which is in turn recognized and bound by C1q protein of the complement systems to produce C1. The C1 protein cleaves the C4 protein into C4b, which eventually leads to the production of C3a and C5a and C5b, forming a recognition complex on the surface of the foreign material. The alternative pathway is initiated when the C3 protein adsorbs to any foreign surface, leading to the hydrolysis of C3 into C3a and C3b. As the complement system proceeds, high levels of C3a and C5a are produced, which activate mast cell degranulation, increase vascular permeability, and attract granulocytes and monocytes [25]. This contributes to the activation of the inflammatory response through the induction of ROS release from granulocytes (Fig. 4.1). Platelet adhesion to the biomaterial surface is then enhanced, which can trigger the coagulation cascade.

Acute inflammation

Chronic inflammation

Fibroblasts

Macrophages Chemoattractants Platelet activation

Frustrated phagocytosis C5a C3a

Factor XII

ROS proteases

C1q Thrombin

Foreign body giant cell Fusion

C3/C4

Coagulation cascade

IgG Complement cascade

Protein adsorption

Biomaterial

ROS H+

Proteases

Fibrous capsule formation

Figure 4.1  Immune response toward biomaterials. The coagulation cascade, the complement response, and protein adsorption all begin simultaneously on the biomaterial, leading to the recruitment and adhesion of macrophages and other inflammatory cells. After adhesion to the biomaterials, inflammatory cells initiate phagocytosis as they attempt to clear the foreign material. If they are unable to phagocytose the biomaterial, frustrated phagocytosis occurs and macrophages fuse into FBGCs that secrete proteolytic enzymes, ROS, and H+, degrading the biomaterial. Unsuccessful frustrated phagocytosis results in the recruitment of fibroblasts that form a fibrous capsule around the biomaterial.

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The coagulation cascade and the complement system interact closely. Many of the components of the coagulation cascade can modulate the activity of the complement system and vice versa. FXIIa is able to cleave C1, initiating the classical complement system, while thrombin can activate C3, C5, and C6. The close relationship between the two pathways is demonstrated in the ability of C1 inhibitors to inactivate FXIIa, thereby slowing the coagulation cascade. On the other hand C3b, a protease in the complement cascade, can mediate the proteolysis of prothrombin. The activities of these two systems are closely linked, and modulating one will potentially affect the other [25]. Products from the activation of the coagulation and complement systems include the chemoattractants and chemokines, such as transforming growth factor (TGF-β), interleukin-1 (IL-1), and platelet-derived growth factor (PDGF), which are released from activated platelets and injured cells around the biomaterial [24,26]. These chemoattractants cause macrophages, granulocytes, and other inflammatory cells to migrate toward the site, where the protein-covered biomaterial provides the infiltrating cells a surface to anchor. The primary adsorbed proteins that have been indicated to play roles in the cell adhesion via integrins are fibrinogen, fibronectin, vitronectin, IgG, and iC3b [8,24]. Macrophages have specialized actin-rich adhesive structures, known as podosomes, that act as sites of attachment and play a role in the adhesion of macrophages to biomaterials [27]. Macrophages undergo cytoskeletal remodeling as they spread to cover the biomaterial surface. After interaction with the biomaterial activating, the FBR, macrophages, and other inflammatory cells activate phagocytic responses in an attempt to endocytose the biomaterial for intracellular degradation. If this is not possible, the inflammatory cells secrete ROS and proteolytic enzymes into the space between the cells and the biomaterial to degrade it extracellularly. This is an attempt to break down the biomaterial into nano- and microsizes that can be phagocytosed. Granulocytes will also secrete chemokines that suppress additional granulocyte migration and promote mononuclear cells migration (Fig. 4.1). Additionally, granulocytes will die off within the first couple days, leaving macrophages as the primary cell type [24].

4.2.2  Chronic Response If a biomaterial is not cleared from the body by the initial acute response due to its size or composition, there is a sustained inflammatory stimulus that results in the onset of chronic inflammation [28]. Characteristics

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of chronic inflammation include an established presence of macrophages, monocytes, and lymphocytes and the formation of connective tissues and blood vessels. Monocytes present undergo phenotypic changes into macrophages, and additional chemokines such as IL-8, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1 (MIP-1) are secreted to continue attracting inflammatory cells [29]. Single macrophages are unable to phagocytose particles larger than 5 µm [30]. In the presence of larger biomaterials, adherent macrophages fuse to form FBGCs when stimulated by IL-4 and IL-13 produced by T-lymphocytes (IL-4 and IL-13) and mast cells (IL-13). Macrophage fusion is highly dependent on the makeup of the adsorbed proteins on the biomaterial surface. High amounts of vitronectin favor both initial macrophage attachment as well as macrophage fusion [31]. Other proteins, such as fibronectin, laminin, and collagen support macrophage attachment, but do not promote fusion. FBGCs express markers, such as gp130, IL-1R, IL-2Rα, IL-2Rγ, IL-6R, TNFR, M-CSFR, and SCFR, which reflect their macrophage origin [32]. They produce IL-1α and TNF-α during the initial time period following their formation, and produce TGF-β as they mature. The presence of these markers can be used to confirm the identity of these cells (Fig. 4.1). After their formation, FBGCs attempt to phagocytose the material. If this fails, they will remain attached to the surface and attempt to degrade the material and/or isolate it from surrounding tissue. They form a closed compartment between their cellular membrane and the material, and release ROS, protons, and enzymes such as matrix metalloproteinases into the compartment to attempt to degrade the biomaterial [33]. In some cases, the acidity of the compartment can reach pH levels as low as pH 4 [34]. The combination of ROS, proteases, and acidic pH is sufficient to degrade many biomaterials. The main source of ROS that are released by cells is from molecular oxygen (O2) reduction using NADPH. This reduction process occurs in mitochondrial respiration and produces oxygen free radicals as a by-product. Inflammatory cells such as macrophages and FBGCs utilize this process as the source of the ROS that they release during inflammation [35]. When a biomaterial is introduced in vivo and induces inflammation, but cannot be cleared by macrophages and FBGCs, the body forms a fibrous capsule around it to isolate it and prevent damage to surrounding tissue. The capsule is a dense fibrous connective tissue shell, consisting of collagen and other glycosaminoglycans produced by fibroblasts [36]. This

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prevents the biomaterial from interacting with surrounding tissues, reducing the functionality of the biomaterial or the device.

4.3  EFFECT OF PHYSICOCHEMICAL PROPERTIES OF BIOMATERIAL ON INFLAMMATION The physical and chemical makeup of biomaterials can play an important role in modulating the inflammatory response. The following describes the manner in which various characteristics can impact inflammation.

4.3.1  Surface Topology Surface features of varying size and shape on biomaterials influence the inflammatory response in different ways. Biomaterial topology influences the type and quantity of proteins that interact with it and, consequently, cell adhesion. Rough surfaces generally show higher protein adsorption than smooth ones, which enhance cellular adhesion because of the increased density of ligand/anchors for cells [37]. Surface features can also affect the morphology and signal transduction in immune cells that can promote or inhibit inflammation. An example is the modulation of macrophage phenotype by controlling cell shape. Macrophages cultured on a micropatterned surface that promoted cellular elongation encouraged macrophage polarization into the M2 phenotype, which represent wound healing and anti-inflammatory macrophages, compared to the M1 phenotype, which is pro-inflammatory. The ability of micropatterned surfaces to affect macrophage polarization is associated with their ability to mediate shape changes in the macrophages by regulating the contractility of the actin cytoskeleton [38,39]. Morphology, proliferation, and differentiation of cells can be affected by geometric features on biomaterial surfaces. Cells align and fill grooves greater than 5 μm, but bridge grooves narrower than 2 μm [40]. With wells and pits smaller than 5 µm, cells become smaller and take on rounded morphology. Patterning on biomaterials also affects proliferation and differentiation of cells that is highly dependent on the cell type and composition of the biomaterial involved. Osteoblasts, fibroblasts, and mesenchymal stem cells can be coaxed to proliferate by modulating the topography of biomaterials. Using different topographies and biomaterials, proliferation can also be decreased in embryonic stem cells, fibroblasts, and osteoblasts. However, for many cell types, there is a little information on

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how they respond to a variety of topographies. Considering the effect of topography on proliferation is also dependent on the chemical nature of the biomaterial and the cell type under consideration, it is difficult to infer the effect of a particular biomaterial on cell growth without experimentation. It is recommended that testing be performed for specific cell type and biomaterial combinations under consideration for use [41]. The effect of nanopatterned surfaces on cell adhesion is dependent on the cell type as well as the specific patterning on the surface; be it grooves, pits, wells, and other patterns. Pits and wells improve adhesion of myocytes and astrocytes, but not of fibroblasts. Adding surface functionalization to the patterned surfaces also influence cellular interactions. ArginineGlycine-Aspartic acid (RGD) biofunctionalized gold dots spaced at 58 nm show enhanced cellular adhesion and spreading, while spacing greater than 73 nm reduced cellular spreading [42]. In a study examining the effects of a grating of three different polymers with line spacing between 250 nm and 2 µm, it was found that macrophages appeared insensitive to nanotopographies less than 500 nm. As the grating size increased to 2 µm, the cellular density was reduced, and macrophage fusion to FBGCs was subsequently reduced.This may be a result of the physical interruption of fusion, and could be an avenue to exploit with regard to the reduction of the inflammatory response [43]. The roughness of nonpatterned biomaterials has been shown to have an effect on cellular adhesion as well. In a test comparing ultrasmooth surfaces versus rough coated surfaces, the ultrasmooth surfaces were found to be inductive of FBGC formation, while the rough samples showed little FBGC presence. This indicates that specific patterning may not be required to gain some of the effects of patterned topography. While it may not allow for controlling the phenotype of the cells adhered, it can reduce the FBGC presence, and consequently provide for a lower level of inflammatory activity [44] (Table 4.1).

4.3.2  Surface Chemistry Surface chemistry of the biomaterial plays an important role in cellular adhesion and the formation of FBGCs. Hydrophobic and charged hydrophilic surfaces have all been shown to promote macrophage adhesion. Surfaces with hydrophobic or cationic hydrophilic surfaces promote similar amounts of macrophage fusion to FBGCs, but at levels lower than that induced by anionic hydrophilic surfaces. Neutral hydrophilic surfaces, on the other hand, are inhibitory to cellular adhesion, which can lead to a reduced FBGC formation. Thus surface charge can regulate macrophage

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Table 4.1 Effects of Surface Properties of Biomaterials on Cellular Behavior Feature Effect Source

Grooves Small grating (