Wound Healing, Tissue Repair, and Regeneration in Diabetes 0128164131, 9780128164136

Table of contents :
Cover
WOUND HEALING,
TISSUE REPAIR, AND
REGENERATION IN
DIABETES
Copyright
Dedication
Contributors
About the authors
Preface
Part 1: Background and overview
1
The diabetic foot
Introduction
Clinical classification
Definition
Risk factors
Etiology
Epidemiology
The complicated diabetic foot
Vascular
Neural
Skin and soft tissue
Bone
Infection
Care and management
Treatment
Other factors
Prevention
Conclusion
References
2
Role of oxidants and antioxidants in diabetic wound healing
Introduction
Oxidative stress and wound healing
Antioxidants and wound healing
Examples of enzymatic antioxidants
SOD, GSTs, GPx, NADPH
Heme-oxygenase 1 (HO-1)
Peroxiredoxins and thioredoxins
Nonenzymatic antioxidants
Vitamin C
Vitamin E
Vitamin D
Alpha-lipoic acid (α-LA) and its reduced form of dihydrolipoic acid (DHLA)
N-acetyl-cysteine (NAC)
Other small molecules
Herbal extracts
Curcumin
Honey
Factor-E2-related factor (Nrf2)
Diabetes, oxidative stress, and impaired/chronic wounds
OS and the triggering of wound chronicity
Conclusions
References
Further reading
3
Chronic infection and inflammation: Hallmarks of diabetic foot ulcers
Background
Chronicity of wound infection
Antibiotic resistance (ABR)
Biofilm infection
Novel approaches for therapeutics
Electroceuticals
Bacteriophage
Dysregulated resolution of wound inflammation
miRNA
Macrophage function and phenotypes
Efferocytosis
Conclusion
References
4
Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics
Introduction
Neuropathy
Peripheral neuropathy
Cranial neuropathy
Autonomic neuropathy
Focal neuropathy
Mononeuropathy
Polyneuropathy
Experimental mouse models for DPN
Diet-induced DPN mouse model
Chemically induced DPN mouse model
Genetically modified DPN mouse model
Nociception assays
Mechanical stimuli
The manual von Frey test
The electronic von Frey
Randall-Selitto test
Heat stimuli
The tail-flick test
Hot plate test
Hargreaves test
Thermal probe test
Cold stimuli
Cold plate test
Acetone evaporation test
Cold plantar assay
Temperature preference test
Nonstimulus evoked nociception
Grimace scales
Burrowing
Weight-bearing and gait analysis
Automated behavioral analysis
Diabetic neuropathy clinical research trials
Nutrition for diabetic neuropathy
Vitamin B12
Alpha-lipoic acid
Acetyl-l-carnitine
Clinical assays for diagnosis of DPN
Neurology exam questions
Neurological function test
Diagnostic testing
Electromyogram
NCV test
Biopsies
Spinal tap
Blood test
Peripheral neuropathy and wound healing
Neuroregeneration in DPN
Gene therapy
NTFs delivery
Stem cell-based strategies
Alternative RNA splicing
Noncoding RNAs
Electrical stimulation
Summary and future perspective
References
Part 2: Molecular mechanisms in diabetic wounds
5
Dysregulated inflammation in diabetic wounds
Introduction
Normal wound healing
Diabetic wound healing
Platelets
Neutrophils
Monocytes/macrophages
T cells
Epigenetics and immune cell function
Macrophage phenotype is epigenetically regulated in diabetic wounds
Neutrophil inflammatory activity is epigenetically regulated
Summary
References
6
Angiogenic response in wound healing
Introduction
Angiogenesis and vasculogenesis
Wound healing
Hemostasis
Inflammation
Neutrophils
Macrophage
Proliferation
Endothelial cells
From bench to bed
Current practice
Future potential therapy
Cell therapy
microRNAs
Conclusion
References
7
Fibrosis and diabetes: Chronic hyperglycemia triggers organ-specific fibrotic mechanisms
Introduction
Normal cutaneous wound-healing pathogenesis
Mechanisms which take place in response to hyperglycemia
Protein glycation
The renin-angiotensin-aldosterone system
Growth factors
Matrix metalloproteinases
Organs
The eye
The kidney
The heart
Blood vessels
The liver
The lung
Concluding remarks
References
Further reading
8
miRNAs in diabetic wound healing
Introduction
miRNAs involved in regular skin function
Expression pattern of miRNAs in diabetic skin
Exploring the functions of miRNAs in diabetic wound healing
Inflammation phase
Angiogenesis phase
Reepithelialization phase
Remodeling (maturation) phase
Advantage of miRNAs as therapeutic target to improve diabetic wound healing
Conclusions
References
9
Epigenetics of diabetic wound healing
Diabetic wounds: Introduction and burden
Epigenetics fundamentals
DNA methylation
DNMTs: Writers for DNA methylation
Ten-eleven translocation (Tet) enzymes: The erasers of DNA methylation
The 5-methylcytosine binding domain (MBD) family: Readers of DNA methylation
Histone modifications
Epigenetics of type 2 diabetes (T2D)
Epigenetic dysregulations in diabetic wound healing impairment
Macrophages
Fibroblasts
Endothelial cells
Therapeutic strategies targeting epigenetics for diabetes and diabetic wound healing impairment
Conclusion
References
10
Role of lipid mediators in diabetic wound healing
Introduction
Fatty acyls
Prostaglandins
Leukotrienes and cytochrome p450 metabolites
Endocannabinoids
Glycerolipids
Glycerophospholipids
Phosphatidylinositol phosphates
Lysophosphatidic acid
Sphingolipids
Ceramides and their phosphates
Sphingosine-1-phosphate
Gangliosides
Sterol lipids
References
11
Role of cytokines and chemokines in wound healing
Introduction
Chemokine classification and function
Cytokine and chemokine expression during acute vs diabetic wound healing
Insult before injury
The AGE of ruin
A spoonful of sugar does not help the medicine
Impact of diabetes on fibroblasts
Impact of diabetes on endothelial and endothelial progenitor cells
Impact of diabetes on keratinocytes
Impact of diabetes on immune cells and cytokines
Hemostasis
Inflammatory stage of wound healing: Damnation by perpetuation
The neutrophil influx
When 2 does not follow 1 (macrophages)
Mast cells and lymphocytes
Proliferation and remodeling stages of wound healing
Granulation tissue deposition
Neovascularization
Re-epithelialization
Remodeling
Diabetic challenges and potential therapeutic opportunities
Biofilms
Cytokines/chemokines that may alleviate diabetic wounds
Mesenchymal stromal cells
Biomaterials
Conclusion
References
12
The wound microbiome
Introduction
From contamination to infection: The wound infection continuum
Culture-based insights into wound microbes
Culture-independent profiling of the DFU microbiome
Who are the members of the DFU microbiome?
Temporal stability of the DFU microbiome
A focus on methodology: High-throughput sequencing approaches to characterize the diabetic wound microbiota
Amplicon-based sequencing approaches
Experimental bias, statistical corrections, and normalization
Current utility and future prospects for amplicon-based microbiome studies of diabetic wound
Whole metagenomic shotgun sequencing
Experimental limitations and computational challenges
Functional characterization of DFU microbiota using gene-level metagenomic profiles
Future directions
Microbial lifestyles: Impact of biofilms
From microbial census to gene expression
Harnessing the therapeutic potential of the microbiome
Summary
References
13
Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing
Introduction
Hemostasis
Inflammation
Proliferation
Remodeling
Influence of diabetes on functions of neutrophils
Hindrance in energy supply
Involvement of polyol pathway
Disrupted bactericidal activity
Inhibition of chemotaxis
Influence of diabetes on functions of keratinocytes
Impaired migration
Impaired angiogenesis
Influence of diabetes on apoptosis
Contribution of AGEs
Apoptosis of fibroblasts
Conclusion
References
Further reading
Part 3: Emerging therapeutics in diabetic wound care
14
Biomaterials for diabetic wound-healing therapies
Introduction
Successes and challenges in the use of biomaterials for the management of diabetic wounds
Biomaterials for dermal tissue engineering and regeneration
Acellular biomaterials
Synthetic polymeric biomaterials
Poly (-caprolactone)
Poly (lactic-co-glycolic acid)
Polylactic acid
Polyurethane
Natural animal-derived polymers used as biomaterials
Collagen
Hyaluronic acid
Gelatin
Chitosan
Alginate
Self-assembling peptides
Cell-based skin substitutes
Cellular skin substitutes based in a combination of natural and synthetic biomaterials
Cellular skin substitutes from human amniotic membrane
Cellular skin substitutes from human cadaver skin
Cellular skin substitutes based on combined human and animal sources
Stem-cell-based substitutes for regenerative healing of diabetic wounds
Induced pluripotent stem cells
Mesenchymal stem cells
Embryonic stem cells
Emerging biomaterial technologies for treatment of diabetic wounds
Conclusions
References
15
Photobiomodulation therapy in diabetic wound healing
Light as a therapeutic intervention
Mechanisms of photobiomodulation therapy
Energizing the powerhouse-The mitochondria: The performance mechanism
Lighting up beacons-Photosensitive membrane receptors and channels: Analgesia mechanism
Harnessing endogenous stem cells for regeneration-TGF-β activation: Healing mechanism
PBM therapy in diabetic wound healing
PBM and redox modulation in diabetic wounds
Optimizing PBM treatments to dysregulated signaling pathways in diabetic wound healing
JAK/STAT signaling
Interleukins, bFGF, and TNFα
VEGF and SDF-1α
TGF-β
PI3K/AKT signaling via mTOR and GSK-3β
Implications for PBM therapy in diabetes wounds and associated fibrosis and cancers
References
16
Therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts
History of placental tissue in wound repair
Preservation techniques of placental membranes for commercial use
Benefits of placental allografts in chronic wound repair
Experimental evidence of wound repair by placental membrane in animal models
Clinical evidence for efficacy of placental membranes in diabetic foot ulcer patients
References
17
Debridement and negative pressure wound therapy
Introduction
Debridement
Medical management and vascular status
Literature review
Types of debridement
Endpoint and biopsies
Applications for the diabetic foot
Negative-pressure wound therapy
Background
Mechanism of action
Description/function
Applications for the diabetic foot
Unique clinical usage and future applications
Safety and conclusion
References
18
Protease technology in wound repair
Diabetes, wound prevalence, and health economics
Risk factors for primary and recurrent DFUs
Dynamics of wound repair and tissue regeneration
Protease therapy
Protease therapy for DFUs
Nonhealing wounds: Chronic inflammation impedes or stalls wound repair and regeneration
Role of protease in wound infection
Mechanisms of action
Proteases as activators of wound healing: Creation of bioactive peptides in situ
Effect of collagenase on cells
Next frontier: Emerging uses of protease therapy
Protease therapy for acute wounds
Effects of plant proteases on inflammation
Angiogenesis and antiinflammation effects of proteases on wound repair and regeneration
Effect on antimicrobial peptides in skin
Emerging protease technologies for treatment of fibrosis
Emerging technologies for treatment of biofilm
Emerging protease-inspired therapies and cancer diagnostics
Conclusions
References
19
Collagen in diabetic wound healing
Introduction
Collagen in diabetic wounds
Diabetic-wound-infection-induced collagen dysfunction
Applications of collagen in wound healing
References
20
Nutrition and diabetic wound healing
Introduction
Dysregulated wound inflammation during diabetes
Nutrition in diabetic wound healing
Carbohydrates
Fats and fatty acids
Proteins and amino acids
Vitamins
Minerals
Other nutritional interventions
Papaya: A natural remedy for diabetic wounds
Conclusion
References
Part 4: Nanotechnology and nanocarriers in wound healing
21
Nanotechnology in diabetic wound healing
Diabetic wounds-An overview
Applications of nanotechnology in wound healing and management
Nanosensors
Nanoparticles
Nanofibers
Nanotransfection
Strategies of nanotechnology applications
Hemostasis
Cell proliferation and skin remodeling
Antiinflammatories
Antimicrobial activity
Concerns regarding nanotechnology in medicine
Future perspectives
References
22
Drug-delivery nanocarriers for skin wound-healing applications
Skin wound healing
Nanotechnology and nanomedicine to address challenges in skin wound healing
Nanotechnology and drug delivery
Nanocarriers for drug delivery in wound healing
Nanomaterials for drug delivery in skin wound healing
Polymeric-based nanomaterials
Dendrimers
Polymeric micelles
Polymeric nanoparticles
Polymersomes
Lipid-based nanocarriers
Liposomes
Niosomes
Transfersomes
Ethosomes
Metallic nanoparticles
Pure metal nanoparticles
Metal oxide nanoparticles
Bimetallic nanoparticles
Carbon-based nanomaterials
Carbon nanotubes
Graphene-based nanomaterials
Fullerene
Nanodiamonds
Conclusion and future directions
References
Part 5: Biomarkers in wound healing
23
Volatile organic compounds: Potential biomarkers for improved diagnosis and monitoring of diabetic wounds
Introduction
How wounds produce VOCs
Role of VOCs in diabetes wound formation
Quantitative collection and processing of VOCs from wounds
Analytical and statistical methods for analyzing VOCs
Bacterial VOCs
In vitro
In vivo
VOCs produced by biofilms
Classification of bacterial VOCs by functional groups
Hydrocarbons
Alcohols
Aldehydes
Acids
Esters
Phenyl groups and other cyclics
Sulfur-containing compounds
VOCs produced in humans due to diabetes
Acetone
Isopropanol
Ethanol
Isoprene
Current status of E-noses and other devices for wound diagnosis and monitoring
Conclusion
References
Further reading
Part 6: Novel concepts in diabetic wound healing
24
Tissue regeneration and reprogramming
Introduction
Cells of tissue regeneration
Platelets
Keratinocytes
Fibroblasts
Immune cells
Bone-marrow-derived stem cells
Epidermal stem cells
Role of ECM in tissue regeneration
Epigenetics of tissue regeneration
Tissue regeneration in diabetes
Conclusion
References
24
Bone marrow monopoiesis and wound healing in diabetes
Introduction
Function of wound Mphi in normal skin wound healing
Mo/Mphi dysregulation and impaired wound healing in diabetes
Ontogeny of tissue mononuclear phagocytes and blood Mo in homeostasis
Origin of wound Mphi
Tissue injury and monopoiesis
Enhanced monopoiesis in diabetes
Diabetes-induced HSPC modifications
Potential mechanisms underlying HSPC alterations in diabetic bone marrow
Signaling pathways involved in obesity and diabetes-associated increases in monopoiesis
Effect of diabetes on skin wounding-induced monocyte expansion in bone marrow
Conclusions, implications, and future directions
References
26
Role of mesenchymal stem cells in diabetic wound healing
Introduction
Mesenchymal stem cells
MSCs and their dysregulation in diabetes
MSCs in normal tissue repair
Use of MSCs in tissue regeneration and wound healing
MicroRNAs in diabetes
MSC modulation of miRNA in diabetic complications
Diabetic neuropathy
MSCs and diabetic nephropathy
MSCs and diabetic retinopathy
MSCs and diabetic cardiomyopathy
MiRNA in skin physiology
Application of MSCs in correcting dysregulated wound healing
Inflammation
Angiogenesis
MSC correction of ECM remodeling
MSC correction of re-epithelialization
Future directions
References
Further reading
27
Fetal wound healing
Introduction
Fetal wound healing: Historical perspective
Contributions of the local wound environment
Inflammation
Extracellular matrix
Collagen
Hyaluronic acid
Fibromodulin
Importance of fetal fibroblasts
Fetal wound healing and diabetes
Conclusions
References
Index
Back Cover

Citation preview

WOUND HEALING, TISSUE REPAIR, AND REGENERATION IN DIABETES

WOUND HEALING, TISSUE REPAIR, AND REGENERATION IN DIABETES Edited by

DEBASIS BAGCHI Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX, United States

AMITAVA DAS Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

SASHWATI ROY Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2020 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-816413-6 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Elizabeth Brown Editorial Project Manager: Samantha Allard Production Project Manager: Punithavathy Govindaradjane Cover Designer: Matthew Limbert Typeset by SPi Global, India

Dedicated to respected Mr. Biji K. Kurien, ex-Berger, a great mentor, philosopher, and guide. Debasis Bagchi Dedicated to my family members, friends, relatives and The Almighty. Amitava Das Dedicated to my mentors who helped me learn the science of wound healing and to my beloved parents, friends, and family. Sashwati Roy

Contributors

Motaz Abas Ross University School of Medicine, Bridgetown, Barbados Mangilal Agarwal Integrated Nanosystems Development Institute (INDI); Department of Mechanical and Energy Engineering, Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States Alessandro Ajo Department of Chemical Engineering; Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Praveen Arany Oral Biology and Biomedical Engineering, University at Buffalo, Buffalo, NY, United States Said A. Atway Department of Orthopedics, The Ohio State University Wexner Medical Center, Columbus, OH, United States Mark Azeltine Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver–Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States Debasis Bagchi Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX, United States Swathi Balaji Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Jaideep Banerjee Osiris Therapeutics (now part of Smith & Nephew), Columbia, MD, United States Pijus K. Barman Center for Wound Healing and Tissue Regeneration, Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, United States Rubinder Basson Division of Musculoskeletal & Dermatological Sciences, University of Manchester, Manchester, United Kingdom Ardeshir Bayat Division of Musculoskeletal & Dermatological Sciences, University of Manchester, Manchester, United Kingdom

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Contributors

Nirupam Biswas Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Amy Campbell Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Yuk Cheung Cyrus Chan Biomedical Technology Cluster (IncuBio), The Hong Kong Science and Technology Park, Hong Kong, China Andrew Clark Department of Surgery, IU Health Comprehensive Wound Center, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States Ali Daneshkhah Integrated Nanosystems Development Institute (INDI), Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States Amitava Das Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Aaron D. denDekker Vascular Surgery Division, University of Michigan, Ann Arbor, MI, United States Karishma Desai Oral Biology and Biomedical Engineering, University at Buffalo, Buffalo, NY, United States Sandeep Dhall Osiris Therapeutics (now part of Smith & Nephew), Columbia, MD, United States Nicholas V. DiMassa Department of Orthopedics, The Ohio State University Wexner Medical Center, Columbus, OH, United States Dibyendu Dutta Bengal College of Pharmaceutical Sciences and Research, Durgapur, India Mohamed El Masry Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States Haytham Elgharably Department of Thoracic and Cardiovascular Surgery, Cleveland Clinic, Cleveland, OH, United States Katherine A. Gallagher Vascular Surgery Division, University of Michigan, Ann Arbor, MI, United States

Contributors

Subhadip Ghatak Department of Surgery, IU Health Comprehensive Wound Center, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States Nandini Ghosh Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Elizabeth A. Grice Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Komel Grover Swiss American, Carrollton, TX, United States Ira M. Herman Program in Cell, Molecular and Developmental Biology, Center for Innovations in Wound Healing Research, Tufts University School of Medicine, Boston, MA, United States Aditya Kaul Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Imran Khan Division of Plastic Surgery, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States Puneet Khandelwal Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States Savita Khanna Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States Dolly Khona Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Timothy J. Koh Center for Wound Healing and Tissue Regeneration, Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, United States Hui Li Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Kenneth W. Liechty Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver-Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States

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Contributors

Amanda E. Louiselle Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver-Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States Amit Kumar Madeshiya Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Sayantan Maitra Government of West Bengal, Department of Health and Family Welfare, Institute of Pharmacy, Jalpaiguri, India Manuela Martins-Green Molecular, Cell and Systems Biology Department, University of California, Riverside, CA, United States Leighanne Masri Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Shomita S. Mathew-Steiner Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States David Medina-Cruz Department of Chemical Engineering; Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Qi Miao Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Jennifer Mohnacky Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Rodrigo Mosca Oral Biology and Biomedical Engineering, University at Buffalo, Buffalo, NY, United States Daria A. Narmoneva Department of Biomedical Engineering, College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, United States Colby Neumann Division of Plastic Surgery, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States

Contributors

Stephen M. Niemiec Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver-Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States Priya Niranjan Swiss American, Carrollton, TX, United States Durba Pal Tissue Engineering and Regenerative Medicine Lab, Indian Institute of Technology Ropar, Rupnagar, Punjab, India Umang Parikh Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Dhamotharan Pattarayan Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Sasikumar Ponnusamy Oral Biology and Biomedical Engineering, University at Buffalo, Buffalo, NY, United States Atul Rawat Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Nava P. Rijal Department of Biomedical Engineering, College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, United States Amit K. Roy Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Sashwati Roy Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Shayan Saeed Molecular, Cell and Systems Biology Department, University of California, Riverside, CA, United States Bahram Saleh Department of Chemical Engineering; Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Suman Santra Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

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Contributors

Swetha Saravanan Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Abhishek Sen Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Chandan K. Sen Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Amanda P. Siegel Integrated Nanosystems Development Institute (INDI); Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States Kanhaiya Singh Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States Mithun Sinha Division of Plastic Surgery, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States Harrison Strang Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States Aayushi Uberoi Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Ada Vernet-Crua Department of Chemical Engineering; Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Thomas J. Webster Department of Chemical Engineering; Nanomedicine Science and Technology Center, Northeastern University, Boston, MA, United States Dayanjan S. Wijesinghe Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Richmond, VA, United States Traci A. Wilgus Department of Pathology, The Ohio State University, Columbus, OH, United States

Contributors

Junwang Xu Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver-Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States Carlos Zgheib Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver–Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States

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About the authors Debasis Bagchi, PhD, MACN, CNS, MAIChE, received his Ph.D. in medicinal chemistry in 1982. He is the Director of Scientific Affairs, VNI Inc., Lederach, PA; a Professor in the Department of Pharmacological and Pharmaceutical Sciences at the University of Houston, College of Pharmacy, Houston, TX; and, an Adjunct Faculty in Texas Southern University, Houston, TX. He served as the Chief Scientific Officer at Cepham Inc, Belmont, NJ, from June 2013 till December 2018. He served as the Senior Vice President of Research & Development of InterHealth Nutraceuticals Inc, Benicia, CA, from 1998 till February 2011, and then as Director of Innovation and Clinical Affairs, of Iovate Health Sciences, Oakville, ON, till June 2013. Dr. Bagchi received the Master of American College of Nutrition Award in October 2010. He is the Past Chairman of International Society of Nutraceuticals and Functional Foods (ISNFF), Past President of American College of Nutrition, Clearwater, FL, and Past Chair of the Nutraceuticals and Functional Foods Division of Institute of Food Technologists (IFT), Chicago, IL. He is serving as a Distinguished Advisor on the Japanese Institute for Health Food Standards (JIHFS), Tokyo, Japan. Dr. Bagchi is a Member of the Study Section and Peer Review Committee of the National Institutes of Health (NIH), Bethesda, MD. Dr. Bagchi has 368 papers in peer-reviewed journals, 40 books, and 20 US patents. Dr. Bagchi is also a Member of the Society of Toxicology, Member of the New York Academy of Sciences, Fellow of the Nutrition Research Academy, and Member of the TCE stakeholder Committee of the Wright Patterson Air Force Base, OH. Dr. Bagchi is the Associate Editor of the Journal of Functional Foods, Journal of the American College of Nutrition, and Archives of Medical and Biomedical Research, and serving as Editorial Board Member of numerous peer-reviewed journals. Dr. Bagchi has a US patent on role of edible Berries in Wound Healing and numerous publications on Wound Healing.

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About the authors

Amitava Das, PhD, is a Postdoctoral Researcher at Indiana Center for Regenerative Medicine and Engineering (ICRME), Indiana University School of Medicine. Dr. Das earned his Bachelor’s and Master’s in Pharmacy and Pharmaceutical Chemistry, respectively, from PES College of Pharmacy, Bangalore, India. He earned his PhD degree in Human Nutrition from The Ohio State University in 2016 after which he joined as a Postdoctoral Researcher in Department of Surgery at The Ohio State University Medical Center, and then moved to Indiana University in 2018. His research interests are wound healing, tissue repair and regeneration in diabetes with an emphasis on Nutraceuticals. He has published over 20 research papers and numerous book chapters. Since 2017, Dr. Das is a member of the Editorial Board of Journal of American College of Nutrition. Dr. Das also serves as a reviewer for multiple prestigious journals. He has presented at various national and international conferences and is the recipient of several awards. Sashwati Roy, PhD, is a Professor of Surgery and Director of Clinical Research at IUH Comprehensive Wound Center. She is an expert in inflammation and macrophage biology in chronic wounds. She did her PhD from University of Kuopio, Finland, and postdoc from University of California, Berkeley. Her research interests include wound inflammation, mechanisms of resolution of diabetic wound infection, tissue repair, and cellular plasticity. Dr. Roy has over 225 peer-reviewed publications. Her work has been cited over 18,300 times. Dr. Roy’s research program is funded by National Institute of Health (NIDDK & NINR) and Department of Defense (DoD). Dr. Roy is a permanent member of the NIH urgery Anesthesia Trauma (SAT) study section. In addition, she routinely serves as a reviewer for multiple other NIH, VA study sections, International grant review panels as well as for prestigious journals. She has served as committee chairs, secretary, and executive board member at the national Wound Healing Society where she is currently serving as the immediate past President.

Preface

Around 415 million adults worldwide are affected by diabetes mellitus, and the count is estimated to soar as high as 642 million by 2040. As per the National Diabetes Statistics Report, 2014, nearly 30.3 million people in the United States, representing 9.4% of the US population, suffer from diabetes. Persistent hyperglycemia in T2DM cases gives rise to the development of secondary complications, including diabetic neuropathy, nephropathy, retinopathy, peripheral vascular diseases, cerebrovascular diseases, and chronic wounds. Chronic wounds affect  6.5 million patients only in the United States with an excess of US$25 billion being spent annually on their treatment. The burden is increasing rapidly because of increasing health care costs, an aging population, and a sharp world-wise increase in the incidence of diabetes and obesity. A survey of the Medicare beneficiaries exhibited that approximately 8.2 million people survive with wounds with or without infection, while Medicare costs for the treatment of acute and chronic wounds fluctuated between $28.1 and $96.8 billion. Surgical wounds and diabetic foot ulcers are the most expensive category. This book, written by leading experts in the field, addresses a wide range of topics related to the concepts and pathophysiology of tissue repair and regeneration, especially for diabetic wounds. Novel molecular/cellular pathways and complications involved in the process of diabetic wound healing have been discussed. A salient feature of this book is the novel management strategies for diabetic wound inflammation and healing. In this book, a total of 27 chapters are divided into six major thematic sections. The introductory theme is the general information entitled Background and overview of the concept and the process of wound healing, specifically diabetic foot ulcers (DFU). This section is a compilation of four chapters reviewing the salient features of pathophysiology of diabetic foot, acute and chronic inflammation and infections. The role of oxidants and antioxidants in diabetic wound healing and the intricate aspects of the peripheral diabetic nephropathy in wound repair have been highlighted. The second section has nine chapters highlighting Molecular mechanisms in diabetic wounds. The concept of dysregulated inflammation in diabetic wounds has been discussed in the first chapter, which is followed by a chapter emphasizing the critical aspect of angiogenic factors in wound healing. The third chapter discusses important features of the role of fibrosis in diabetes and how chronic inflammation triggers organ-specific fibrotic mechanisms, the fourth chapter elaborates the novel role of miRNAs in diabetic wounds and therapeutics, while the subsequent chapter discusses the role of epigenetic alterations of diabetic wound healing. The subsequent two chapters discuss the role of lipid mediators in diabetic wound healing, and the role of cytokines and chemokines

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in wound healing. The eighth chapter extensively discusses the aspects of wound microbiome, and the ninth chapter elaborates how downregulation of hexose sugar metabolism in diabetes diminishes the rate of wound healing. The third section entitled Emerging therapeutics in diabetic wound care comprises seven chapters. The first chapter discusses the use of novel biomaterials for diabetic wound healing. The next chapter demonstrates the aspects of innovative photomodulation therapy for diabetic wounds. The therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts have been discussed in the third chapter. The fourth chapter highlights the salient features of clinical interventions such as debridement and negative pressure wound therapy in the care of DFU, and the fifth chapter discusses the protease technology in wound repair and healing. The sixth chapter discusses the role of collagen in diabetic wound healing, and the seventh extensively discusses the role of appropriate nutrition in diabetic wound healing. The fourth section comprises two chapters that discuss the novel aspects of nanotechnology and nanocarriers in wound healing, while the fifth section discusses the significance of biomarkers in wound healing and highlights the use of volatile organic compounds as promising and prospective biomarkers for improved diagnosis and monitoring of diabetic wounds. In the final section, Novel concepts in diabetic wound healing have been discussed. The first chapter demonstrates the salient aspects of tissue regeneration and reprogramming, while the second chapter elaborates the significance of bone marrow monopoiesis in the process of diabetic wound healing. The third chapter discusses critical role of mesenchymal stem cells in diabetic wound healing and the fourth chapter discusses the critical features of fetal wound healing. In summary, this book covers a broad range of topics related to Wound Healing, Tissue Repair, and Regeneration in Diabetes by renowned experts in the field. This volume will be invaluable resource for professionals interested in understanding the pathophysiology of diabetic wound healing. Our sincere gratitude and sincere thanks to all our eminent contributors and scientists. Our special thanks to Megan Ball and Samantha Allard for their continued support, cooperation, and assistance.

CHAPTER 1

The diabetic foot Shomita S. Mathew-Steiner, Dolly Khona, Chandan K. Sen

Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

1. Introduction Diabetic foot ulcers (DFU) are a common reason for hospitalization of diabetic patients and frequently result in amputation of lower limbs. Of the one million people who undergo nontraumatic leg amputations annually worldwide, 75% are performed on people who have type 2 diabetes mellitus (T2DM) [1,2]. The risk of death at 10 years for a diabetic with DFU is twice as high as the risk for a patient without a DFU [3]. The amputation rate in patients with DFU is 38.4% [4]. Infection is a common (>50%) complication of DFU [1,5–13]. Emerging evidence underscores the importance of biofilm infection in the progression of nonhealing DFU [12,14]. Eighty-five percent of amputations in diabetic patients is attributed to DFUs made chronic by infections [15]. DFUs occur due to a combination of mechanical changes in the foot, peripheral neuropathy and peripheral arterial disease [16,17]. In the United States, DFU management alone is estimated to cost somewhere between $9 and $13 billion [18]. DFU management includes the use of therapeutic footwear to offload the wound [19,20] together with maintenance of a moist wound environment [21]. Debridement together with aggressive antibiotic therapy is necessitated for infected wounds [22,23]. Additionally, the maintenance of optimal blood glucose and treatment of vascular insufficiencies are key elements of wound management. This chapter provides a brief overview of the pathology of the diabetic foot. Details of the component elements are presented in other chapters within this book.

2. Clinical classification 2.1 Definition A diabetic foot is characterized by ulceration that is driven by neuropathy and/or peripheral artery disease of the lower limb and is a complex, frequent, and expensive complication in diabetic patients.

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00001-0

© 2020 Elsevier Inc. All rights reserved.

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2.2 Risk factors Several direct and indirect risk factors are associated with the development of a DFU. These include (a) lifestyle factors such as smoking, uncontrolled diabetes, poor nutrition, immobility, age, etc.; (b) physiological factors such as neuropathy (loss of sensation), vasculopathy (insufficient oxygen availability), shear stress and trauma, and bone deformities (c) genetic and ethnic factors have been implicated in the development of diabetes and diabetic complications [24,25]. DFU is of particular concern among Latinos African Americans, and in Native Americans, who have the highest risk for diabetes worldwide [26].

2.3 Etiology DFUs develop due to a combination of neuropathy [27], arterial disease [28], pressure [19], and plantar deformity [6]. Diabetic neuropathy is found in 80% of diabetic persons with foot ulcers and is a key factor in development of DFUs. Biomechanical factors, such as tissue stiffness and thickness, may contribute to DFUs. In a study involving 39 subjects, the heel pad of the foot without an ulcer was found to be stiffer than that with an ulcer [29]. In patients with neuropathy, the plantar soft tissue is thicker and less stiff in specific parts of the foot that are more prone to developing into ulcers. Therefore, mechanical properties of plantar soft tissue could have predictive value for DFU prognosis [30].

2.4 Epidemiology According to the Centers for Disease Control, 30 million Americans are estimated to have diabetes [31]. DFUs, in particular, are associated with increased hospitalizations [18]. Fifteen percent of diabetic patients develop a foot ulcer, and 38.4% of these require amputation. Indeed, in the United States, most nontraumatic lower-extremity amputations are associated with diabetes [32]. Diabetes and complications such DFUs have been reported globally and are indicators of the growing and profound impact of this pervasive disease [8,33–41].

3. The complicated diabetic foot The diabetic foot develops as a consequence of several independent and interdependent complications discussed briefly below.

3.1 Vascular Diabetes is associated with microvascular and macrovascular etiologies, including cerebrovascular, cardiovascular, and peripheral arterial disease. DFU patients have higher premature mortality due to cardiovascular complications [42,43]. Diabetic adults are

The diabetic foot

2–4 times more at risk for heart disease and stroke than their nondiabetic counterparts. In fact, 65% of deaths in diabetics are associated with vascular complications [31]. In addition to the association of ischemic heart disease and mortality, chronic ulceration could induce chronic inflammation, which could promote the development of atherosclerosis [44]. Furthermore, the ischemia caused by poor blood flow promotes nonhealing ulceration of the foot, leading to amputation. The key impact from this complication is the lack of oxygen supply to the foot and wound and therefore, aggressive treatment of limb ischemia is vital to managing the wound and preventing amputation [45,46].

3.2 Neural It is believed that 45%–60% of all DFUs are purely neuropathic, while up to 45% have neuropathy combined with ischemia [47,48]. Peripheral sensory neuropathy (PSN) is a major factor leading to DFUs [27,47,49,50]. PSN, even with adequate arterial perfusion, will often promote infection progression primarily due to a lack of sensation in the foot [51,52]. Unnoticed excessive temperature exposures, pressure from ill-fitting shoes, or trauma may cause blistering and ulceration. Motor neuropathy results in muscle atrophy causing foot deformities such as hammertoe, foot drop, etc. [53,54]. Neuropathy induced muscular atrophy together with vascular deficiencies are significant risk factors for limb loss in diabetic patients. Autonomic neuropathy leads to skin breakdown creating a portal for entry of microbes that cause infection [55,56]. In diabetic people with neuropathy [57], the wound recurrence rate is 66% with an associated increase in amputation rate by 12%. Cardiovascular autonomic neuropathy (CAN) is another complication of diabetes that is associated with increased mortality and silent myocardial ischemia [58]. Somatic neuropathy has been implicated in the calcification of the arterial wall leading to cardiovascular mortality [59]. Sensory and motor neuropathy in the foot together with bone and joint deformities and associated vascular changes cause a progressive condition called Charcot foot (neuropathic osteoarthropathy), which physically manifests as a convex foot with a rockerbottom appearance [60–62]. It is thought to affect about 2% of diabetic persons. Excessive inflammation and vascular calcification common in diabetic patients are implicated in the etiology of this condition [61,63–66]. Neuropathic feet with low intrinsic muscle volume and plantar aponeurosis dysfunction may contribute to the development of claw toes, manifesting as extension and flexion of the metatarsophalangeal and interphalangeal joints, respectively [67].

3.3 Skin and soft tissue The diabetic skin is mechanically less competent due to glycation of fibrillar collagen and may have defects in the epidermal barrier [68]. The defective vascular supply and

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neuropathy affects the skin resulting in poor skin nutrition and regenerative capacity and increased vulnerability to cutaneous injuries [1,13,69,70].

3.4 Bone Bone disease is a complication in diabetes resulting in impaired bone quality that contributes to increased vulnerability to fracture [71]. Underlying mechanisms include modification of bone cells by advanced glycation end products, changes in the incretin hormone response, and microvascular deficiencies. The diabetic Charcot foot shows increased bone resorption and inflammation stimulating osteoclastic hyperactivity leading to gross deformities [72].

3.5 Infection DFUs are prone to infections that could be more severe than those found in nondiabetic patients, affecting not only the skin and soft tissue but underlying bone [73]. The incidence of DFU infection (DFI) ranges from 25% in all persons with the diagnosis, to 4% yearly in patients undergoing treatment. The combined effect of hyperglycemia, poor vascularization (poor oxygenation), neurological problems, and immunological disturbances contribute to sustaining DFIs [74–77]. DFIs are polymicrobial in nature and complicated by the biofilm mode of growth [5,9,11,52,78–84]. Uncontrolled diabetes impairs the ability of host immune defenses to control microbial pathogens. Ischemia promotes infection by decreasing oxygenation of tissue and impairs the delivery of immune cells and systemic antibiotics to the site of infection. Biofilms are estimated to account for 60% of chronic wound infections [85]. Recent studies demonstrated a role for biofilm infection in compromising the functionality of repaired skin. Biofilm infection promoted the down-regulation of epithelial junctional proteins, resulting in wounds that appeared to close visually, but lacked barrier function. There are no reliable point-of-care diagnostic methods for direct biofilm detection in wounds, but, a promising indirect alternative could be the measurement of transepidermal water loss (TEWL) using a commercial handheld device [86–89].

4. Care and management 4.1 Treatment The treatment of DFUs involves at minimum surgical interventions (debridement, revascularization etc.), wound coverage (dressings and maintenance of moisture) [21], antibiotic therapy (infection treatment) [22,23], the use of appropriate therapeutic footwear [19,20], optimal control of blood glucose, and evaluation and correction of vascular insufficiency [90].

The diabetic foot

Surgical interventions include debridement of infected tissue and sometimes bone, removal of excess callus, skin grafting, and revascularization. Debridement may not eliminate microbial biofilms from the wounds and could possibly drive biofilm debris deeper into the wound, aiding in recurrent infection [89]. The use of cultured human cells [91, 92], recombinant growth factors [93–95], placenta or grafts for coverage, and hyperbaric oxygen treatments may promote wound healing, if vascular supply is adequate. Hyperbaric oxygen therapy (HBOT) increases the supply of oxygen to wounds [96,97] and has proven to be beneficial in the treatment of ischemic wounds as seen in DFUs [98]. Multiple hyperbaric oxygen treatments (85 min daily, 5 d/wk for 8 wk) were shown to result in the complete healing of 52% of patients with DFU in the treatment group compared to 29% in the placebo group [99]. The dosing of oxygen needs to cater to the specific needs of the wound to be most effective [100–102]. 4.1.1 Other factors The management of systemic and local factors [103–106] such as sugar control, limited activity (offloading and bed rest) and physiological parameters such as obesity, hypertension, hyperlipidemia, heart disease, and smoking cessation are key elements in preventing and treating DFU [107, 108]. Ongoing and completed clinical trials (https://clinicaltrials.gov/) on DFU treatments include over 500 studies testing therapies, such as maggot debridement, oxygen, platelet gel, human amniotic membrane, laser, shockwave, vacuum-assisted closure (VAC) combined with various dressings, erythropoietin hydrogels, pulsed radiofrequency energy, phage therapy (infection related), surfactants, mesenchymal stromal cells and derivatives, fish skin grafts, phototherapy among others.

4.2 Prevention Routine preventive podiatric care, appropriate shoes, and patient education are key factors in preventing ulcer formation and amputation [75]. Eighty-five percent of DFUs are estimated to be preventable with appropriate medical evaluation, supportive care, including the use of well-fitting shoes and prompt treatment of wounds. Additionally, physical activity and exercise decreased ulcer occurrence significantly better than no physical activity (0.02 vs 0.12, respectively). DFU patients who had more activity also had improved peripheral neurological parameters [109].

5. Conclusion DFUs are a complicated manifestation of several interconnecting pathologies that may not be managed by any single intervention or therapeutic. A productive solution would

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involve interdisciplinary approaches that seamlessly integrate medical interventions, community outreach, nutritional support, and consideration of socioeconomic and cultural factors.

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[46] Gibbons GW. Lower extremity bypass in patients with diabetic foot ulcers. Surg Clin North Am 2003;83:659–69. https://doi.org/10.1016/S0039-6109(02)00199-8. [47] Reiber GE, et al. Causal pathways for incident lower-extremity ulcers in patients with diabetes from two settings. Diabetes Care 1999;22:157–62. https://doi.org/10.2337/diacare.22.1.157. [48] Edmonds ME, et al. Improved survival of the diabetic foot: the role of a specialized foot clinic. Q J Med 1986;60:763–71. [49] Boyko EJ, et al. A prospective study of risk factors for diabetic foot ulcer. The Seattle Diabetic Foot Study. Diabetes Care 1999;22:1036–42. https://doi.org/10.2337/diacare.22.7.1036. [50] Abbott CA, et al. The north-west diabetes foot care study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet Med 2002;19:377–84. https://doi. org/10.1046/j.1464-5491.2002.00698.x. [51] Gibbons GW, Habershaw GM. Diabetic foot infections. Anatomy and surgery. Infect Dis Clin N Am 1995;9:131–42. [52] Eneroth M, Larsson J, Apelqvist J. Deep foot infections in patients with diabetes and foot ulcer: an entity with different characteristics, treatments, and prognosis. J Diabetes Complicat 1999;13:254–63. https://doi.org/10.1016/s1056-8727(99)00065-3. [53] Frykberg RG. Diabetic foot ulcers: pathogenesis and management. Am Fam Physician 2002;66:1655–62. [54] Frykberg RG, et al. Role of neuropathy and high foot pressures in diabetic foot ulceration. Diabetes Care 1998;21:1714–9. https://doi.org/10.2337/diacare.21.10.1714. [55] Shaw JE, Boulton AJ. The pathogenesis of diabetic foot problems: an overview. Diabetes 1997;46 (Suppl. 2):S58–61. https://doi.org/10.2337/diab.46.2.s58. [56] Flynn MD, Tooke JE. Aetiology of diabetic foot ulceration: a role for the microcirculation? Diabet Med 1992;9:320–9. https://doi.org/10.1111/j.1464-5491.1992.tb01790.x. [57] Galkowska H, Olszewski WL, Wojewodzka U, Rosinski G, Karnafel W. Neurogenic factors in the impaired healing of diabetic foot ulcers. J Surg Res 2006;134:252–8. https://doi.org/10.1016/j. jss.2006.02.006. [58] Vinik AI, Ziegler D. Diabetic cardiovascular autonomic neuropathy. Circulation 2007;115:387–97. https://doi.org/10.1161/circulationaha.106.634949. [59] Jeffcoate WJ, Rasmussen LM, Hofbauer LC, Game FL. Medial arterial calcification in diabetes and its relationship to neuropathy. Diabetologia 2009;52:2478–88. https://doi.org/10.1007/s00125-0091521-6. [60] Armstrong DG, Peters EJ. Charcot’s arthropathy of the foot. J Am Podiatr Med Assoc 2002;92:390–4. https://doi.org/10.7547/87507315-92-7-390. [61] Jeffcoate WJ. Abnormalities of vasomotor regulation in the pathogenesis of the acute charcot foot of diabetes mellitus. Int J Low Extrem Wounds 2005;4:133–7. https://doi.org/ 10.1177/1534734605280447. [62] Stevens MJ, Edmonds ME, Foster AV, Watkins PJ. Selective neuropathy and preserved vascular responses in the diabetic Charcot foot. Diabetologia 1992;35:148–54. https://doi.org/10.1007/ bf00402547. [63] Jeffcoate W. Vascular calcification and osteolysis in diabetic neuropathy-is RANK-L the missing link? Diabetologia 2004;47:1488–92. https://doi.org/10.1007/s00125-004-1477-5. [64] Jeffcoate WJ, Game F, Cavanagh PR. The role of proinflammatory cytokines in the cause of neuropathic osteoarthropathy (acute Charcot foot) in diabetes. Lancet 2005;366:2058–61. https://doi.org/ 10.1016/S0140-6736(05)67029-8. [65] Hofbauer LC, Schoppet M. Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases. JAMA 2004;292:490–5. https://doi.org/10.1001/jama.292.4.490. [66] Hofbauer LC, Kuhne CA, Viereck V. The OPG/RANKL/RANK system in metabolic bone diseases. J Musculoskelet Neuronal Interact 2004;4:268–75. [67] Ledoux WR, Schoen J, Lovell M, Huff E. Clawed toes in the diabetic foot: neuropathy, intrinsic muscle volume, and plantar aponeurosis thickness. J Foot Ankle Res 2008;1:O2. https://doi.org/ 10.1186/1757-1146-1-S1-O2. [68] Quondamatteo F. Skin and diabetes mellitus: what do we know? Cell Tissue Res 2014;355:1–21. https://doi.org/10.1007/s00441-013-1751-2.

The diabetic foot

[69] Schramm JC, Dinh T, Veves A. Microvascular changes in the diabetic foot. Int J Low Extrem Wounds 2006;5:149–59. https://doi.org/10.1177/1534734606292281. [70] Ngo BT, et al. Manifestations of cutaneous diabetic microangiopathy. Am J Clin Dermatol 2005;6:225–37. https://doi.org/10.2165/00128071-200506040-00003. [71] Hygum K, Starup-Linde J, Langdahl BL. Diabetes and bone. Osteoporos Sarcopenia 2019;5:29–37. https://doi.org/10.1016/j.afos.2019.05.001. [72] Jansen RB, Svendsen OL. A review of bone metabolism and developments in medical treatment of the diabetic Charcot foot. J Diabetes Complicat 2018;32:708–12. https://doi.org/10.1016/j. jdiacomp.2018.04.010. [73] Shah BR, Hux JE. Quantifying the risk of infectious diseases for people with diabetes. Diabetes Care 2003;26:510–3. https://doi.org/10.2337/diacare.26.2.510. [74] Uc¸kay I, Arago´n-Sa´nchez J, Lew D, Lipsky BA. Diabetic foot infections: what have we learned in the last 30 years? Int J Infect Dis 2015;40:81–91. https://doi.org/10.1016/j.ijid.2015.09.023. [75] Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA 2005;293:217–28. https://doi.org/10.1001/jama.293.2.217. [76] Lipsky BA, et al. Diagnosis and treatment of diabetic foot infections. Clin Infect Dis 2004;39:885–910. https://doi.org/10.1086/424846. [77] Lipsky BA, International Consensus Group on, Diagnosing and Treating the Infected Diabetic Foot. A report from the international consensus on diagnosing and treating the infected diabetic foot. Diabetes Metab Res Rev 2004;20(Suppl 1):S68–77. https://doi.org/10.1002/dmrr.453. [78] Kalan L, et al. Redefining the chronic-wound microbiome: fungal communities are prevalent, dynamic, and associated with delayed healing. MBio 2016;7(5). https://doi.org/10.1128/ mBio.01058-16. [79] Percival SL, Malone M, Mayer D, Salisbury AM, Schultz G. Role of anaerobes in polymicrobial communities and biofilms complicating diabetic foot ulcers. Int Wound J 2018;15:776–82. https://doi. org/10.1111/iwj.12926. [80] Buch PJ, Chai Y, Goluch ED. Treating polymicrobial infections in chronic diabetic wounds. Clin Microbiol Rev 2019;32. https://doi.org/10.1128/cmr.00091-18. e00091-18. [81] Rahim K, et al. Bacterial contribution in chronicity of wounds. Microb Ecol 2017;73:710–21. https://doi.org/10.1007/s00248-016-0867-9. [82] Lipsky BA, et al. Diagnosis and treatment of diabetic foot infections. Plast Reconstr Surg 2006;117:212S–238S. https://doi.org/10.1097/01.prs.0000222737.09322.77. [83] Gardner SE, Hillis SL, Heilmann K, Segre JA, Grice EA. The neuropathic diabetic foot ulcer microbiome is associated with clinical factors. Diabetes 2013;62:923–30. https://doi.org/10.2337/ db12-0771. [84] Kalan LR, et al. Strain- and species-level variation in the microbiome of diabetic wounds is associated with clinical outcomes and therapeutic efficacy. Cell Host Microbe 2019;25:641–655.e645. https:// doi.org/10.1016/j.chom.2019.03.006. [85] James GA, et al. Biofilms in chronic wounds. Wound Repair Regen 2008;16:37–44. https://doi.org/ 10.1111/j.1524-475X.2007.00321.x. [86] Ganesh K, et al. Chronic wound biofilm model. Adv Wound Care (New Rochelle) 2015;4:382–8. https://doi.org/10.1089/wound.2014.0587. [87] Barki KG, et al. Electric field based dressing disrupts mixed-species bacterial biofilm infection and restores functional wound healing. Ann Surg 2019;269:756–66. https://doi.org/10.1097/ sla.0000000000002504. [88] Roy S, et al. Staphylococcus aureus biofilm infection compromises wound healing by causing deficiencies in granulation tissue collagen. Ann Surg 2019; https://doi.org/10.1097/sla.0000000000003053. [89] Roy S, et al. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol 2014;233:331–43. https://doi.org/10.1002/path.4360. [90] Everett E, Mathioudakis N. Update on management of diabetic foot ulcers. Ann N Y Acad Sci 2018;1411:153–65. https://doi.org/10.1111/nyas.13569. [91] Brem H, Balledux J, Bloom T, Kerstein MD, Hollier L. Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent: a new paradigm in wound healing. Arch Surg 2000;135:627–34. https://doi.org/10.1001/archsurg.135.6.627.

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[92] Veves A, Falanga V, Armstrong DG, Sabolinski ML, Apligraf Diabetic Foot Ulcer Study. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Diabetes Care 2001;24:290–5. https://doi.org/ 10.2337/diacare.24.2.290. [93] Bennett SP, Griffiths GD, Schor AM, Leese GP, Schor SL. Growth factors in the treatment of diabetic foot ulcers. Br J Surg 2003;90:133–46. https://doi.org/10.1002/bjs.4019. [94] Guzman-Gardearzabal E, et al. Treatment of chronic ulcers in the lower extremities with topical becaplermin gel .01%: a multicenter open-label study. Adv Ther 2000;17:184–9. https://doi.org/ 10.1007/bf02850294. [95] Jirkovska A, Boucek P, Woskova V, Bartos V, Skibova J. Identification of patients at risk for diabetic foot: a comparison of standardized noninvasive testing with routine practice at community diabetes clinics. J Diabetes Complicat 2001;15:63–8. https://doi.org/10.1016/s1056-8727(00)00141-0. [96] Stoekenbroek RM, et al. Hyperbaric oxygen for the treatment of diabetic foot ulcers: a systematic review. Eur J Vasc Endovasc Surg 2014;47:647–55. https://doi.org/10.1016/j.ejvs.2014.03.005. [97] Kranke P, Bennett MH, Martyn-St James M, Schnabel A, Debus SE. Hyperbaric oxygen therapy for chronic wounds. Cochrane Database Syst Rev 2012;2:CD004123, https://doi.org/ 10.1002/14651858.CD004123.pub3. [98] Roeckl-Wiedmann I, Bennett M, Kranke P. Systematic review of hyperbaric oxygen in the management of chronic wounds. Br J Surg 2005;92:24–32. https://doi.org/10.1002/bjs.4863. [99] Londahl M, Katzman P, Nilsson A, Hammarlund C. Hyperbaric oxygen therapy facilitates healing of chronic foot ulcers in patients with diabetes. Diabetes Care 2010;33:998–1003. https://doi.org/ 10.2337/dc09-1754. [100] Sen CK, et al. Oxygen, oxidants, and antioxidants in wound healing: an emerging paradigm. Ann N Y Acad Sci 2002;957:239–49. https://doi.org/10.1111/j.1749-6632.2002.tb02920.x. [101] Gordillo GM, Sen CK. Revisiting the essential role of oxygen in wound healing. Am J Surg 2003;186:259–63. https://doi.org/10.1016/s0002-9610(03)00211-3. [102] Sen CK. Wound healing essentials: let there be oxygen. Wound Repair Regen 2009;17:1–18. https://doi.org/10.1111/j.1524-475X.2008.00436.x. [103] Muha J. Local wound care in diabetic foot complications. Aggressive risk management and ulcer treatment to avoid amputation. Postgrad Med 1999;106:97–102. https://doi.org/10.3810/ pgm.1999.07.603. [104] Pinzur MS, et al. Guidelines for diabetic foot care: recommendations endorsed by the Diabetes Committee of the American Orthopaedic Foot and Ankle Society. Foot Ankle Int 2005;26:113–9. https:// doi.org/10.1177/107110070502600112. [105] Edmonds M. Diabetic foot ulcers: practical treatment recommendations. Drugs 2006;66:913–29. https://doi.org/10.2165/00003495-200666070-00003. [106] Bello YM, Phillips TJ. Recent advances in wound healing. JAMA 2000;283:716–8. https://doi.org/ 10.1001/jama.283.6.716. [107] Frykberg RG, et al. Diabetic foot disorders. A clinical practice guideline. For the American College of Foot and Ankle Surgeons and the American College of Foot and Ankle Orthopedics and Medicine. J Foot Ankle Surg 2000;39(5 Suppl):1–60. [108] Margolis DJ, Kantor J, Santanna J, Strom BL, Berlin JA. Risk factors for delayed healing of neuropathic diabetic foot ulcers: a pooled analysis. Arch Dermatol 2000;136:1531–5. https://doi.org/ 10.1001/archderm.136.12.1531. [109] Matos M, Mendes R, Silva AB, Sousa N. Physical activity and exercise on diabetic foot related outcomes: a systematic review. Diabetes Res Clin Pract 2018;139:81–90. https://doi.org/10.1016/j. diabres.2018.02.020.

CHAPTER 2

Role of oxidants and antioxidants in diabetic wound healing Manuela Martins-Green, Shayan Saeed

Molecular, Cell and Systems Biology Department, University of California, Riverside, CA, United States

1. Introduction Healing of cutaneous wounds involves a series of complex processes that occur sequentially with each phase overlapping the previous one, leading to a partial regeneration of the dermal tissue and reestablishment of the epithelial barrier. Upon wounding, hemostasis occurs with formation of a clot that stops the bleeding and releases factors that stimulate the inflammatory phase, which involves the chemoattraction of various types of leukocytes to the site of wounding. The first type of leukocytes to appear are the neutrophils that kill bacteria by producing reactive oxygen species (ROS), and in this manner prevent wound infection. Following the influx of neutrophils, monocytes arrive at the wound site and become proinflammatory macrophages. These cells are involved in cleaning the damage caused by neutrophils when phagocytosing the bacteria. At the same time antiinflammatory macrophages secrete growth factors and cytokines that promote healing. Furthermore, keratinocytes, fibroblasts, and endothelial cells also migrate to the wound site to close the wound. While keratinocytes form the epidermis, fibroblasts produce the extracellular matrix molecules (ECM) that confer structural and biochemical properties to the wound tissue and endothelial cells that contribute to the development of new blood vessels. These processes form the so-called granulation tissue named so because of its granular appearance. In the final phase of wound healing, remodeling, the surplus cells undergo apoptosis and the excess ECM produced is removed by phagocytes that remodel the wound tissue, ultimately resulting in scar tissue. Acute wound healing follows this path of events. However, when the processes involved in acute healing are either stalled or fail to occur in the proper order, impaired healing occurs, the wounds do not close properly, skin barrier is not established, and the granulation tissue does not evolve into a scar tissue. Furthermore, when impaired wound healing occurs and is accompanied by chronic inflammation and infection with biofilmforming bacteria, the wounds become chronic. Therefore, chronic wounds develop as a result of defective regulation of the complex cellular and molecular processes involved in proper healing (e.g., [1–4]). They impact 6.5 M people and cost $25B/year in the United States alone (e.g., [5]). Although a number of studies have been performed to Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00002-2

© 2020 Elsevier Inc. All rights reserved.

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address processes involved in wound chronicity, until now the scientific community in the wound healing field has been unable to crack the complex and multidimensional processes involved in initiation and development of chronic wounds, in particular of diabetic chronic wounds. This is primarily because it is virtually impossible to study initiation and development of wound chronicity in humans because by the time these wounds present in the clinic, the initial stages of development are long gone. In addition, we cannot experiment in humans with chronic wounds. The critical need for a cure of diabetic chronic wounds is underlined by the continuous increase in type II diabetes (which accounts for 90%–95% of all diabetes). It has been reported that diabetes affects 387 M people globally and 28 M in the United States and prediabetes affects 316 M more globally and 86 M more in the United States [6]. These statistics are daunting, considering that a significant number of these patients will go on to develop foot ulcers and that about 12% of them will require amputation (e.g., [5]); the 5-year survival after amputation is 50%. Animal models for study of the genesis and development of nonhealing wounds are critical to elucidate the processes involved in wound chronicity. Although a number of such animal models have been developed previously to study chronic wounds, these models only mimic some of the critical elements of wound chronicity in humans [7–26]. Recently, the TallyHo Polygenic Mouse Model of Diabetes was developed as a model with potential to study diabetic chronic wounds. However, only males were hyperglycemic and the authors needed to introduce biofilm-forming bacteria for the wounds to become infected [27,28]. A more recent model used Wister rats fed with high-fat diet and treated with multiple injections of streptozotocin (STZ) to develop type II diabetes. This treatment caused noticeable insulin resistance, persistent hyperglycemia, moderate degree of insulinemia, as well as high serum cholesterol and high triglyceride levels. Furthermore, the rat wounds showed excessive wound inflammatory response, excessive and prolonged ROS production, excessive production of MMPs, and delayed healing [29]. However, these wounds did not become chronic nor did they develop biofilm much like chronic wounds in diabetic patients. We have recently developed a novel chronic wound model using db/db / , a type II diabetic mice, which has all the characteristics present in human chronic wounds. What makes it a powerful model is the fact that this model develops biofilm naturally and without having the need to introduce extraneous bacteria. To develop our model, we took advantage of the fact that chronic wounds in humans contain toxic levels of oxidative stress (OS) and biofilm-producing bacteria [20–23]. High levels of OS lead to deregulation of gene expression, damage to DNA, proteins, and lipids, a hostile proteolytic environment, and cell death [30–32]. OS also leads to impaired keratinocyte migration in vitro, potentially inhibiting re-epithelialization and leading to poorly developed granulation tissue [11,29]. To create chronic wounds in our db/db / mouse model, we increase the OS to high levels immediately after wounding. The wounds go on to become chronic 100% of the time [20–23]. All of the defects found in human chronic

Role of oxidants and antioxidants in diabetic wound healing

wounds are present in our model including the presence of naturally formed biofilm. Therefore, we now have a mouse model that can be used to understand the basic cell and molecular mechanisms of initiation and development of wound chronicity under diabetic conditions.

2. Oxidative stress and wound healing Oxygen is essential for just about all cellular functions (e.g., cell proliferation, migration, differentiation, death), and it is a prerequisite for successful wound healing. Sufficient oxygenation of the wound tissue is needed for adequate energy levels, which are essential for proper cellular function during the healing process. Under normoxic conditions, oxygenation of the wound is sufficient for proper healing. Under hypoxic conditions oxygenation of the wound tissue is insufficient for healing and under hyperoxic conditions, oxygenation is excessive, which can lead to increased ROS adversely affecting healing. Oxidative stress is present in tissues/cells when there is an imbalance between the levels of ROS and the ability of antioxidants in the tissues/cells to remove these species and repair the damage they cause. Balanced levels of ROS kill pathogens, stimulate immune cells, keratinocyte migration, granulation tissue formation, angiogenesis, and collagen synthesis. Imbalanced levels of ROS cause DNA damage, lipid peroxidation, protein nitration, and eventually cell death. ROS are highly reactive forms of O2-derived radicals or atoms/molecules that contain one or more unpaired electrons [33]. Examples are superoxide (O2• ), hydroxyl (•OH) and peroxyl (LOO•) radicals. Nonradical reactive forms of O2 can also occur in tissues such hydrogen peroxide (H2O2), hypochlorous acid (HClO), and peroxynitrite (ONOO). O2• are produced when NADPH oxidases (NOX) oxidize NADPH to NADP+ and in the process give one electron to O2 making O2• . O2• is produced primarily in the mitochondrial electron transport chain as small amounts of O2 leak from this system during the oxidative-phosphorylation reactions and by phagocytic cells, such as neutrophils, in the process of killing bacteria [34]. Oxygen-derived radicals are not the only chemical species that damage tissues. Nitrogen and sulfur-derived radicals are also damaging. Nitric oxide (NO) is generated enzymatically by nitric oxide synthetases (NOS). In the presence of NO, O2• will form ONOO , which causes peroxidation of lipid molecules and oxidation of aromatic amino acids in proteins, therefore damaging their function [33]. O2• can also be dismutated to H2O2, spontaneously or by superoxide dismutase (SOD), which in turn, in the presence of ferrous ions can produce both hydroxyl anions (OH ) and •OH, which are highly damaging of proteins and DNA. However, H2O2 can be broken down into H2O by glutathione peroxidase (GPx) and into H2O + O2 by catalase, two very effective antioxidant enzymes present in cells and tissues. If this system of antioxidants does not function correctly, excessive oxidative stress builds in the tissue.

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A balanced redox state is critical for proper healing. During the hemostasis phase of healing, ROS contribute to blood vessel constriction to stop bleeding at the same time that thrombin activation leads to polymerization of fibrin fibers to which platelets and collagen adhere contributing to the formation of the clot [35]. As platelets degranulate, they release growth factors as well as proinflammatory cytokines and chemokines that are important in attracting neutrophils to initiate the inflammatory phase of healing. These leukocytes are very effective in killing bacteria via the respiratory/oxidative burst, a defense strategy that involves the production of high levels of O2• and H2O2 via NADPH oxidase, into the bacteria-containing phagosome leading to the killing of these infectious agents [36]. Simultaneously, some of these ROS are released into the microenvironment causing tissue damage. At low concentrations (10 μM), H2O2 attracts leukocytes to the site of injury, whereas at concentrations 10  higher, it induces levels of basic fibroblast growth factor (bFGF) that stimulate fibroblast proliferation and migration, increase vascular endothelial growth factor (VEGF), which stimulates angiogenesis. H2O2 also stimulates signaling by transforming growth factor beta 1 (TGFβ1), which leads to increased chemotaxis for keratinocytes and stimulation of matrix production [36–38]. H2O2 is the major signaling ROS during wound healing; it is easily synthesized and degraded, it moves readily through cell membranes and tissues because it is uncharged, it can have a long half-life and is selective in the molecules it reacts with.

3. Antioxidants and wound healing The redox state of tissues and cells is maintained by a balance between oxidant and antioxidant molecules. The latter molecules remove the deleterious effects of the reactive species by donating their electrons to these species and preventing them from capturing electrons from other important molecules, such as DNA, proteins, and lipids. In the presence of excessive levels of ROS and/or RNS and when the levels of antioxidant molecules are not sufficient to detoxify the cells and tissues, damage occurs. There are several types of antioxidants, those that perform enzymatic reactions and those that are nonenzymatic in their effects (Table 1). Some of these reactions occur in the extracellular microenvironment, others occur intracellularly in the cytosol and/ or in organelles such as the mitochondria [39].

3.1 Examples of enzymatic antioxidants Examples of enzymatic antioxidants are SOD, glutathione S-transferases (GSTs), glutathione peroxidases (GPx), NADP(H), catalase, heme-oxygenase 1 (HO-1), peroxiredoxins (Prdx), thioredoxin-1 (Trx-1) and -2 (Trx-2).

Role of oxidants and antioxidants in diabetic wound healing

Table 1 Types of antioxidants for wound healing. Categories

Examples

Functions

Enzymatic antioxidants

Heme oxygenase 1 (HO-1)

Catalyzes degradation of heme into CO, iron, and biliverdin, which is subsequently converted into bilirubin, a potent antioxidant. Is highly induced during oxidative stress. Expressed in keratinocytes and in inflammatory cells. Catalyze reduction of H2O2 and other peroxides. Prdx 6 is expressed in higher levels than other Prdx and detoxifies cells from peroxynitrite. Transgenic mice overexpressing Prdx display accelerated wound closure. Facilitate reduction of other proteins by cysteine thiol-disulfide exchange. Transgenic mice shown to increased resistance to inflammation. Cofactor for hydroxylation of prolines and lysines in fibrillar collagen. Essential for stabilization of triple helix in collagen and in restoration of strength and integrity of connective tissue. Shown to decrease lipid peroxidation and improve healing. Both diabetic and aged mice exhibit decreased levels of Vit E. Evidence of antimicrobial properties in perturbing biofilm structure and formation. Role in normalizing inflammatory cytokines and oxidative stress levels and in other wound-healing responses. NAC administration on wounds improve healing Shown to chelate metals, scavenge for reactive oxygen species, regenerate endogenous antioxidants, and repair oxidative damage. Decreases oxidative stress in the wound tissue. Demonstrates improved wound healing by favorably modulating various growth factors and cytokines, increased neovascularization, and improving collagen deposition. Excellent free radical scavenger. Found to enhance healing properties of endothelial cells, keratinocytes, and fibroblast. Treatment of the wounds with this analog showed accelerated wound healing and elevated elemental antioxidants levels.

Peroxiredoxins (Prdx)

Thioredoxins (Trx)

Nonenzymatic antioxidants

Vitamin C

Vitamin E

N-acetyl-cysteine (NAC)

α-Lipoic acid Other small molecules

Bilirubin

6,8 dithio-UA (Analog of uric acid)

Continued

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Table 1 Types of antioxidants for wound healing—cont’d Categories

Examples

Functions

Herbal antioxidants

Curcumin

Treated irradiated rats showed improved collagen content and wound contraction than untreated groups. It remains difficult to apply. Accelerates wound closure mainly during the inflammatory phase and demonstrates affective antibacterial properties for wound healing. Manuka honey, as opposed to others, shows improved wound healing and tissue regeneration. Lacks sufficient evidence as a topical treatment for wounds. Shown to participate in improving healing under oxidative stress conditions in impaired wounds. Nrf2 deficient mice show markers of poor healing. Cofactor for the GPx, essential for H2O2 breakdown and detoxification. Reduced levels of Glutathione during wound healing cause decreased wound strength.

Honey

Factor-E2-related factor (Nrf2) Glutathione

3.1.1 SOD, GSTs, GPx, NADPH The mechanisms of action of SOD, GSTs, GPx are well coordinated during normal wound healing, they are altered in chronic wounds. SOD + O2• + 2H+ produces H2O2, which is then broken down by either catalase that takes 2H2O2 molecules to produce H2O + O2 or by GPx that takes one molecule of H2O2 with 2 glutathione (GSH) molecules to produce 2H2O plus one molecule of glutathione disulfide (GSSG). The latter in turn will regenerate GSH for the GPx reaction by reacting with the coenzyme NADPH + H+ to produce 2GSH + NADP+, a reaction that is catalyzed by glutathione reductase (GRX) (Fig. 1). 3.1.2 Heme-oxygenase 1 (HO-1) HO-1 is primarily known for its ability to catalyze degradation of heme into CO and/or iron in the presence of O2 and NADPH giving rise to biliverdin that is converted into bilirubin, a very strong antioxidant, by biliverdin reductase in the presence of NADPH (Fig. 2A) [40,41]. CO is a vasodilator, antiinflammatory, antithrombotic, and antiapoptotic agent, whereas Fe++ through ferritin is an antioxidant and has cytoprotective properties. It was shown several decades ago that HO-1 is also highly induced by a variety of agents causing oxidative stress [42,43]. HO-1 is elevated during the first day after wounding, whereas HO-2 is not. HO-1 is expressed in keratinocytes of the wound epithelium and in inflammatory cells. Knockout mice for HO-1 showed delayed wound closure and decreased angiogenesis resulting in impaired wound healing.

Role of oxidants and antioxidants in diabetic wound healing

Fig. 1 Generation and breakdown of H2O2. Superoxide dismutase (SOD) catalytically converts the superoxide radical (O2• ) in the presence of 2 protons to one molecule of hydrogen peroxide (H2O2). H2O2 is catalytically convert to water and oxygen by catalase, or combines with the antioxidant glutathione (GSH) and catalytically forms water and glutathione disulfide (GSSH) by the enzyme glutathione peroxidase (GPx). Upon the depletion, glutathione is regenerated by interacting GSSG with NADPH and hydrogen by the enzyme glutaredoxin (GRx).

3.1.3 Peroxiredoxins and thioredoxins Peroxiredoxins and thioredoxins are antioxidant enzymes that function in various ways to reduce oxidative stress [44,45]. Prx(s) catalyze the reduction of H2O2 as well as a broad range of peroxides [46,47] (Fig. 2B). Prx 6 can also detoxify tissues and cells from peroxynitrite [48,49]. This enzyme is expressed at higher levels than other Prdx(s) in the wound tissue of mouse excisional wounds. In transgenic mice overexpressing Prdx(s) in keratinocytes, wound closure occurs more rapidly [49,50]. Trx(s) constitute a family of small redox proteins that function as antioxidant enzymes that facilitate the reduction of other proteins by cysteine thiol-disulfide exchange (Fig. 2B). These proteins contain 2 cysteines in a CXXC motif, which are critical for their ability to reduce other proteins such as insulin, the glucocorticoid receptor, and coagulation factors. Mice that are transgenic for Trx(s) are more resistant to inflammation [51].

3.2 Nonenzymatic antioxidants Nonenzymatic antioxidants are low-molecular-weight molecules that are themselves used during the antioxidant process and are many times called “sacrificial” antioxidants [33,52]. Molecules such as vitamin C (ascorbic), vitamin E (α-tocopherol), vitamin D, glutathione, N-acetyl cysteine (NAC), alpha lipoic acid (αLA), carotenoids (e.g., lycopenes), bilirubin, and uric acid all belong to this class of antioxidant molecules. Several of these small molecules are found depleted after wounding indicating that they have been consumed to reduce oxidative stress in the wounded tissue and then recover to normal levels within 2 weeks of injury [53].

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(A)

(B) Fig. 2 Antioxidant cycles for hemet oxygenase 1 (HO-1), thioredoxin (Trx), and peroxiredoxin (Prx). (A) The protein NRF2 stimulates expression of HO-1, which catalytically degrades the hemet complex, with the addition of oxygen and NADPH, to form biliverdin with the release of iron (Fe2+) and carbon monoxide (CO). Biliverdin is then reduced to bilirubin in the presence of NADPH by biliverdin reductase. (B) The oxidation of NADPH catalyzes the reduction of cysteines in Trx proteins. As Trx becomes oxidized, Prx is reduced and in the presence of H2O2, one oxygen is transferred to one of the SH groups in a cysteine of the Prx releasing water and regenerating reduced Prx.

3.2.1 Vitamin C Is a very important cofactor for the hydroxylation of prolines and lysines in fibrillar collagen. This hydroxylation is essential for the stabilization of the triplex helix in these collagens and providing integrity and strength to the connective tissue. It is, therefore, logical to think that Vit C is critical for scar formation where fibrillar collagens, in particular Col I, are important to confer strength to the tissue. However, two clinical

Role of oxidants and antioxidants in diabetic wound healing

trials that used Vit C as a supplement for the treatment of patients with significant wounds show contradictory results [54–56]. This outcome is most likely due to the lack of standardization on dose, time of application, and type of patients chosen. This is not an uncommon outcome of clinical trials in particular when the patients have different comorbidities. 3.2.2 Vitamin E Both tocopherols and tocotrienols have long been known to have antioxidant properties. It has been shown that wounds of aging rats have much lower levels of glutathione and Vit E than those of young rats [57]. This is also true for wounds of diabetic mice when compared to those in nondiabetic mice. [53]. Glutathione is a particularly important antioxidant as it serves as cofactor for the GPx that as described earlier are involved in the breakdown of H2O2 and detoxification of other toxic substances. When glutathione is reduced during wound healing, it has been found that the strength of the wound tissue is significantly decreased [58], whereas topical treatment of wounds in diabetic mice improved healing [59]. It has also been shown that supplementing diabetic mice with Vit E (α-tocopherol) decreased lipid peroxidation and improved healing [60]. We have recently shown that chronic wounds in db/db / mice that are fully diabetic and obese improve with treatment of the animals with α-tocopherol systemically and N-acetyl-cysteine locally in the wound [31–33]. 3.2.3 Vitamin D Is best known for its function as a steroid hormone that activates nuclear receptors and regulates phosphorus and calcium homeostasis in skeletal tissue maintenance and repair, but it is now clear that it has additional roles in human health in particular in cancer prevention and inhibition of inflammation related to its antioxidant properties [61]. Vit D3 has been shown to stimulate proliferation and migration of porcine endothelial cells when delivered by the three-dimensional hydrogel matrix, Epigel B, depleted of growth factors, to monolayer cultures of these cells [62]. However, these observations were made in vitro, and therefore the properties of Vit D in vivo need to be studied to determine whether this vitamin plays a role in angiogenesis as an antioxidant that improves wound healing. 3.2.4 Alpha-lipoic acid (α-LA) and its reduced form of dihydrolipoic acid (DHLA) Both have four antioxidant properties. They can chelate metals, scavenge reactive oxygen species (ROS), regenerate endogenous antioxidants, and repair oxidative damage. As a metal chelator, α-lipoic acid was shown to provide antioxidant activity by chelating many toxic heavy metal ions, including Fe2+ and Cu2+. Fe2+ can react with H2O2 to produce Fe3+ + OH• + OH– (Fenton reaction), which can cause protein modification, lipid peroxidation, and DNA damage. As scavengers of OS species, α-LA has

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Fig. 3 Antioxidant network of Vitamin E and C with glutathione and lipoic acid. Oxidation of α-tocopherol (vitamin E) to form the α-tocopheroxyl radical causes the reduction of oxidative species to unreactive species. Simultaneously, the presence of glutathione and vitamin C is needed to transform the α-tocopheroxyl radical back to α-tocopherol. DHLA is reduced to lipoic acid by taking an electron from a dehydroascorbate radical that in the process is regenerated to ascorbate or Vit C. Simultaneously, DHLA takes GSSG and reduces it to GSH thereby regenerating glutathione.

the capacity to act as an antioxidant against the pro-oxidant activity of DHLA [63]. The latter can regenerate Vit E, Vit C, coenzyme Q10, and glutathione. DHLA is reduced to lipoic acid by taking an electron from a dehydroascorbate radical that in the process is regenerated to ascorbate or Vit C. Simultaneously, DHLA takes GSSG and reduces it to GSH thereby regenerating glutathione. In the process, these two reactions remove the electron from the tocopherol radical and regenerate α-tocopherol, an effective antioxidant for the prevention of lipid peroxidation (Fig. 3). 3.2.5 N-acetyl-cysteine (NAC) Has been safely used in the clinic for years as an efficient agent to treat bronchopulmonary disorders, chronic obstructive pulmonary disease, and acetaminophen overdose [64,65]. Emerging evidence indicates that NAC has antimicrobial properties interfering with biofilm formation and perturbing the structure of the biofilm in a variety of bacteria propagated in laboratory settings, such as Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae [66–69]. NAC is not only a precursor in humans for the synthesis of the antioxidant glutathione (GSH), but it also modulates granulocyte function, increases IL-12 secretion, activates NF-κB pathway, decreases metalloproteinase-9, IL-8, IL-6, and brings inflammatory cytokines and oxidative stress to normal levels [70–76]. In recent studies, 0.1%–3.0% of NAC promoted burn wound healing in rats via PKC/Stat3 pathway [77].

Role of oxidants and antioxidants in diabetic wound healing

Administration of NAC to incisional wound of diabetic and nondiabetic mice appears to improve the healing process by downregulating inducible nitric oxide synthase expression and upregulating VEGF expression [78]. We have recently shown that NAC, when applied to chronic wounds in a diabetic mouse model that we have developed, dramatically improved healing within 20 days of initiation of treatment [30–33].

3.3 Other small molecules Other small molecules such as carotenoids, in particular lycopenes, are potent antioxidants, which are abundant in tomatoes, but their connection to improved healing is yet to be made. Bilirubin has also been used to treat wounds created in rats that were made diabetic by treatment with streptozotocin. Wounds were treated with 0.3% bilirubin ointment, wound area measured over time, histologic assessments made, and levels of wound healing markers such as HIF 1α, VEGF, MMP9, TGFβ, TNFα, and IL-10 and 1β measured. It was found that bilirubin improved healing by modulating these growth factors and cytokines in the direction of better healing, increased neovascularization, and improved collagen deposition [79]. This same model was used to measure/evaluate the antioxidant properties of bilirubin. It was found that the levels of GSH and of GPx, SOD, and CAT activities were significantly increased during the early stages of healing, whereas MDA levels (a molecule that damages DNA) were decreased during the late stages of healing suggesting that bilirubin reduces oxidative stress in wound tissues [80]. Uric acid, the final byproduct of urine metabolism that is excreted in the urine, although an excellent radical scavenger, has low solubility in water and can accumulate in tissues, making it detrimental to health. However, a synthetic analog of uric acid (6,8 dithio-UA) has been developed with high solubility in water making it an excellent free radical scavenger with great potential to protect neural cells from oxidative damage. This same analog has been used to treat endothelial cells, keratinocytes, and fibroblast in culture and found to enhance the wound-healing properties of these cells. Furthermore, when applied daily onto full-thickness wounds in mice it accelerated wound healing. One of the effects observed was that the levels of Cu/Zn SOD in the wound tissue was elevated suggesting that the uric acid analog has antioxidant properties [81,82]. Finally, elemental antioxidants are also present in the wound tissue. For a discussion and relevance to human wound healing, see [56].

3.4 Herbal extracts Herbal extracts can also be strong antioxidants. There are several herbal medicines that have been used as antioxidants in wound healing. However, most of those that have effects in wound healing are difficult to evaluate because mechanisms of action are not known. A full review of herbal antioxidants is beyond the scope of this chapter. Here we will discuss the effects of the two most commonly used herbal medicines, curcumin and honey.

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3.4.1 Curcumin It is a potent antioxidant and antiinflammatory component of turmeric [83], a spice that has been used for centuries by the Indian Ayurvedic medicine system, which has been in existence for 6 thousand years. Because radiation stimulates the production of oxygenderived free radicals, the effects of curcumin were studied in radiation-exposed rats with full-thickness wounds. Prior to radiation treatment rats received an oral dose of 10 mg/kg of curcumin or they received placebo. In the curcumin-treated rats the collagen content measured by hydroxyproline levels was higher than that in the placebo-treated animals, but it did not reach the levels of the sham-exposed animals. The wounds also contracted better in the irradiated animals treated with curcumin [84]. Curcumin has also been used to treat wounds locally in rats with excision wounds. Daily application of 40 mg/kg of curcumin to the wounds for 12 days showed that at the end of one week of treatment SOD and catalase levels were lower, whereas levels of GPx were higher indicating a reduction in oxidative stress in the wound tissue of the treated animals. However, these effects disappeared by 12 days of treatment [85]. Although the toxic effects of curcumin are low, it has still not been studied in clinical trials of human wound healing. One of the reasons may be because curcumin has low aqueous solubility, poor tissue absorption, rapid metabolism, and short plasma half-life making it difficult to apply systemically or even locally for improvement of healing. Therefore, in recent years, there have been a number of studies developing films, nanofibers, nanoconjugates, emulsions, and hydrogels to deliver curcumin more effectively [86,87]. 3.4.2 Honey It is composed primarily of glucose, fructose, amino acids, vitamins, and minerals. This composition may vary based on differences in season and geographic location of the plants producing the nectar, and how the nectar is stored. In the past, honey has been used widely for the treatment of various ailments but more recently, with the increase in understanding of how it works it has been used as antioxidant, antimicrobial, antiinflammatory, for cardiovascular problems, as an antitumor agent and as a wound dressing and healing substance [88]. Recently a metanalysis was performed to examine the effectiveness of the antimicrobial properties of honey compared to those of silver, the most commonly used antimicrobial in wound healing. The analysis of data from seven databases showed that honey was a more effective antibacterial agent than silver for wound healing of burns, as measured in the number of days needed for wounds to heal [89]. Studies using several different types of honey embedded in a hydrocolloid gel showed that all honeys caused faster wound closure during the inflammatory phase, but the healing process stalled during re-epithelialization, granulation tissue formation, and collagen deposition [90]. Early improvement of the healing process is probably due to the fact that its acidity increases the release of oxygen from hemoglobin making the microenvironment less favorable for the activity of destructive proteases. Interestingly the high

Role of oxidants and antioxidants in diabetic wound healing

osmolarity of honey draws fluid out of the wound bed to create an outflow of lymph as occurs with negative pressure wound therapy [91]. In most honeys the antimicrobial activity is due to the production of H2O2, but this is quickly inactivated by catalase in the wound tissues. However, in manuka honey, the antimicrobial activity is due to methylglyoxal, which is not inactivated by the antioxidant enzymes. Because of that, the manuka honey used in wound-care products can withstand better the adverse effects of the wound microenvironment [92]. In addition to inhibiting bacterial growth, the methylglyoxal from manuka honey can improve wound healing and tissue regeneration due to its immunomodulatory property [93,94]. A Cochrane database reviewed 26 eligible trials with three trials for acute wounds, 11 trials for burns, and 10 trials for chronic wounds of various kinds (two trials in people with venous leg ulcers, two trials in people with diabetic foot ulcers, and single trial in infected postoperative wounds, pressure injuries, cutaneous Leishmaniasis, and Fournier’s gangrene). From this study, it was difficult to make conclusions about the effects of honey as a topical treatment for wounds because of the heterogeneous nature of the patient populations and the low quality of the evidence. Honey appears to heal partial thickness burns more quickly than conventional treatment such as polyurethane film, paraffin gauze, soframycin-impregnated gauze, sterile linen and by leaving the burns exposed. It was also found that infected postoperative wounds healed more quickly with honey than with antiseptics and gauze. Beyond these comparisons it is difficult to determine whether honey is more effective than current treatments of wounds.

3.5 Factor-E2-related factor (Nrf2) Factor-E2-related factor (Nrf2) has been well documented to mediate antioxidant responses; in particular, it has been shown to participate in improving healing under oxidative stress conditions in impaired wounds [95,96]. It has also been shown that Nrf2 / rats treated with streptozotocin to induced diabetes show delayed wound healing. It was found that there was a higher levels of DNA damage, lower levels of TGFβ1, and higher levels of MMP9, all makers of poor healing. Furthermore, increasing the levels of Nrf2 by treating the wound with sulforaphane (SF) and cinnamaldehyde (CA), reversed these effects, improving healing. The activity of Nrf2 is primarily regulated by the Kelch ECH associating protein 1 (Keap1). Under nonstressed conditions, Keap1binds to Nrf2 in the cytosol promoting its ubiquitination by Cullin3 (CUL3)-containing ubiquitin 3 ligase, sending Nrf2 to the proteasome to be degraded. Under oxidative stress conditions in acute wound healing, the Nrf2-Keap1 interaction is disrupted allowing Nrf2 to translocate to the nucleus to activate gene expression of antioxidant proteins [97,98]. Simultaneously, Keap1 inhibits the NFκB proinflammatory pathway and healing proceeds in the proper way (Fig. 4). It is clear that the Nrf2 effects are critical for reduction of oxidative stress in the wound tissue and that SF and CA are both well-characterized

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Fig. 4 Nrf2 and response to oxidative stress. Under normal conditions, Nrf2 forms a complex with Keap-1 (Nrf2-Keap1), is ubiquitinated in the cytoplasm, and sent to the proteosome for degradation. However, in the presence of oxidative stress Nrf2 disassociates from Keap1 is phosphorylated and translocated into the nucleus to activate antioxidant gene expression. At the same time, Keap1 inhibits NF-κB proinflammatory pathways.

Nrf2 activators, which could potentially be used therapeutically because they are not toxic at the doses required to activate Nrf2 [96,99,100]. The question now remains on whether this transcription factor, and its downstream activated factors, function the same way in truly chronic wounds in diabetic patients. If so, this would be a very powerful tool for the treatment of these chronic wounds.

4. Diabetes, oxidative stress, and impaired/chronic wounds Clinically, chronic wounds are a big burden worldwide. Patients with chronic wounds require treatment over time making it difficult and painful for the patient and expensive for the society. The microenvironment in the wound tissue of chronic wounds is marked by the presence of elevated levels of ROS and numerous inflammatory cells, which release excessive levels of proinflammatory cytokines and proteolytic enzymes, such as matrix metalloproteinases. Therefore, chronic wounds have high levels of inflammation that do not resolve and are associated with inhibition of cell proliferation, cell migration, development of microvessels and with stimulation of cell death.

Role of oxidants and antioxidants in diabetic wound healing

Currently, the Food and Drug Administration has only approved two treatments for chronic wounds: Platelet-derived growth factor (PDGF) and TGFβ1. This is primarily because most studies have focused on stages of healing that involve growth factor functions, which occurs largely during granulation tissue formation. However, by the time this phase of healing occurs, it becomes very difficult to control the events that lead to chronicity. It is therefore important to study early healing events so that the treatments can focus on the new margins of the wound after debridement in order to set the wound on a course toward proper healing. Low levels of ROS are essential in stimulating normal wound healing, whereas elevated and sustained ROS in vivo have been associated with impaired wound repair as well as nonhealing and chronic wound development. It has been known for some time that chronic wounds in humans have high levels OS and that hyperglycemia is typically associated with exacerbated levels of OS. One of the important sources of ROS in diabetes is the mitochondria. In hyperglycemia, increases in pyruvate oxidation during the Krebs cycle lead to elevated production of NADH and FADH2, both of which are electron donors that provide electrons to the electron transport chain during oxidative phosphorylation. As the electron transfer increases, the proton gradient generated in the inner membrane of the mitochondria, and shuffled to the intermembrane space increases, which disrupts the function of Complex III in the inner membrane of the mitochondria leaving only Coenzyme Q to handle the electron transfer. This leads to inefficient transfer of electrons to O2 resulting in high levels of O2• production, ultimately impacting ROS levels. It has also been shown that, under these conditions, pores in the mitochondrial membranes facilitate the exit of O2• into the cytosol, which increases ROS levels in the intermembrane space [101,102] and leads to inhibition of glyceraldehyde-3phosphate dehydrogenase (GAPDH), an enzyme critical in glycolysis, the cytosolic process that provides pyruvate to the Kreb’s cycle in the mitochondrial matrix and that function under hypoxic conditions to produce ATP. These events lead to further ROS production, which overcomes antioxidant levels and increases tissue damage. In diabetic patients, the levels of SOD, CAT, and GPx are decreased in the blood resulting in the concomitant reduction of antioxidants. Also, the function of Nrf2, the master regulator of antioxidant expression, is impaired in fibroblasts exposed to high glucose; Keap1 retains Nrf2 in the cytosol, which leads to its ubiquitination and degradation [103]. Simultaneously, the NFκB pathway is activated leading to an increase in oxidative stress that causes further damage to the wound tissue. Persistent inflammation in chronic wounds is also associated with the sustained activity of the NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome complex. This complex of proteins has the ability to sense and be activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These are molecules known as alarmins that are released by stressed cells undergoing necrosis and that act as “danger” molecules triggering excessive inflammation.

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In addition, the NLRP3 inflammasome can be activated by mitochondrial-derived ROS suggesting that the use of mitochondrial-targeted antioxidants decreases NLRP3-induced inflammation. Once activated, NLRP3 associates with adapter proteins involved in apoptosis, including procaspase 1, and stimulate the increase in the proinflammatory proteins IL1β and IL-18. This inflammasome complex is also associated with an antimicrobial response [35]. In the latter case, gasdermin D (GSDMD) is activated, inserts into the membrane, and forms pores that lead to pyroptosis. Modulation/inhibition of NLRP3 activity has been shown to improve impaired healing [104,105] (Fig. 5). Formation of advanced glycation end (AGEs) products also occurs in diabetic patients as the excess glucose circulating in the blood reacts nonenzymatically with the NH2 group in an amino acid of a protein altering its function (Fig. 6). This is particularly important for cell surface receptors because AGEs will inhibit/change the binding of the receptor with the ligand and subsequently the signal transduction pathways in the

Fig. 5 NLRP3 inflammasome complex function. Inactive NLRP3 (nucleotide-binding domain and leucine-rich repeat containing protein 3) oligomerizes with caspase 1 and ASC (apoptosisassociated speck-like protein) to form the NLRP3 inflammasome complex. Oxidative stress causes an influx of PAMPs, DAMPs, and ROS, all of which activate the inflammasome complex, which in turn activates caspase 1. Activated caspase 1 cleaves pro-IL-1β and pro-IL-18, leading to their release into the extracellular space as inflammatory cytokines. Gasdermin D (GSDMD) is also cleaved by caspase 1 and forms pores in the cell membrane that lead to cell death by pyroptosis.

Role of oxidants and antioxidants in diabetic wound healing

Fig. 6 Advanced glycation end (AGE) product formation and impairment of healing. Excess glucose, especially found in diabetic patients, glycates proteins by reacting with their amine groups (NH2), forming advance glycation end products (AGEs). AGEs disrupt many signal transduction pathways important for proper wound healing by altering the interaction of the signaling proteins with their receptors. During inflammation, AGE increases reactive oxygen species, further resulting in increased inflammation, which affects endothelial cell function resulting in poor oxygenation, decreased neutrophil chemotaxis and activation, and delayed healing. AGE interaction with its receptor (RAGE) causes impairment in wound closure by the keratinocytes, matrix deposition by fibroblasts and angiogenesis, processes vital for proper healing.

cell that lead to specific functions. One of the ways AGE products impair wound healing is by affecting endothelial cell function, and hence vascular function, which leads to poor oxygenation, decreases in neutrophil influx to the wounds resulting in delayed microbial killing. However, when inflammation ensues, AGE can elicit an increase in ROS levels, which in turn stimulate more inflammation, which creates a vicious circle. Moreover, interaction of AGE with its receptor (RAGE) leads to changes in interaction of cells with the ECM, causing changes in fibroblast migration that occur during wound healing to help build the wound tissue, keratinocytes migration to close the wound and endothelial cell migration to form microvessels [35]. Blocking RAGE, however, was successful in reversing these effects [106,107]. When RAGE was inhibited either by application of anti-RAGE antibodies or soluble RAGE in competition experiments, granulation tissue formation and vascularization improved [108]. In humans’ chronic wounds, AGE/ RAGE interactions are elevated, contributing to all the perturbation we have described here that occur when chronic wounds develop (Fig. 6).

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5. OS and the triggering of wound chronicity Under normal conditions, the response to injury is adaptive, designed to restore homoeostasis and to protect the cells from further injury (Fig. 7). Because injury creates a hypoxic state due to the lack of blood circulation to delivering of O2, the tissue turns to producing ATP by anaerobic glycolysis in order to maintain sufficient levels of oxygen to fulfill the needs of the cell. Therefore, levels of glycolytic enzymes such as GAPDH are elevated as are stress response proteins to minimize cell death. Under these circumstances, activation of NOX is controlled, the level of O2• remains low, and SOD is able to effectively dismutate O2• to H2O2, avoiding the formation of the more damaging ROS. H2O2, in turn, is broken down effectively by catalase and GPx to H2O + O2 and H2O, respectively. Simultaneously, the transcription factor Nrf2 is released from Keap1, becomes activated, and translocates to the nucleus to turn on expression of genes for antioxidant proteins, cytokine/chemokine production is controlled, and persistent inflammation is minimized. Keap1, on the other hand, inhibits the proinflammatory pathway induced by NFκB and normal wound healing ensues with re-epithelialization, granulation tissue formation,

s

g

r

Fig. 7 Normal oxidative stress response. NADPH oxidase oxidizes NADPH to form NADP+, concurrently reducing oxygen (O2) to form the superoxide radical (O2• ). Superoxide radical reacts with hydrogen and is catalyzed by SOD to form hydrogen peroxide (H2O2). In some cases, it is then catalytically converted to water and oxygen by catalase. Other times, hydrogen peroxide oxidizes glutathione (GSH) to form GSSG by GSH peroxidase (GPX). Oxidized GSSG in turn allows for the reduction of NADPH. Under normal oxidative stress conditions, the Nrf2-Keap1 complex dissociates and Nrf2 is activated by phosphorylation goes to the nucleus and activates antioxidant gene expression, while Keap1 inhibits NF-κB proinflammatory pathway.

Role of oxidants and antioxidants in diabetic wound healing

including new blood vessel development, and finally remodeling. However, if the need for ATP is not met due to excessive or continuous damage and/or decreased levels of GAPDH, the ATP-dependent ion pumps will fail, membrane integrity will be lost, cell swelling will occur, intracellular Ca++ will be released, and cell death will ensue. This results in release of “danger” molecules (DAMPs and PAMPs) that, in turn, result in strong activation of NOX, which produce elevated levels of O2• that SOD is unable to fully dismutate O2• to H2O2. In addition, excess O2• in the presence of. NO results in formation of excess ONOO . Furthermore, if H2O2 is not fully processed by catalase or GPx, in the presence of ferrous ions (Fe2+), •OH radicals are made by the Fenton reaction. Both ONOO and •OH cause extensive damage to many molecules, including DNA damage, nitration of tyrosines on proteins, and peroxidation of lipids. The damage is further compounded by insufficient Nrf2 levels, or Nrf2 itself is inactivated, to effectively activate antioxidant genes/proteins with subsequent increase in oxidative stress and the inability of Keap1 to inactivate cytokine/chemokine production, which results in increased inflammation, increased cell death, and impaired healing (Fig. 8).

6. Conclusions As established by others it has been extremely difficult to study the evolution of the development of chronic wounds because they do not contain a single defect but rather have numerous defects such as excess oxidative stress, inflammation, ischemia, presence of microbial biofilms, lack of granulation tissue formation, inhibition of keratinocyte proliferation and migration, and lack of ECM deposition. Studies in humans are limited to analysis of wound fluids, limited biopsies from wound tissues and observation through time after debridement, antibiotic treatment, topical application of growth factors and other classical products such as colloidal silver and silver nitrate. Treatments such as offloading, hyperbaric oxygen exposure, and negative pressure have also been used. Experimental studies to understand how chronic wounds initiate and develop have been difficult because of the lack of models that truly mimic wound chronicity in humans. This has led to very limited understanding of the cell and molecular mechanisms leading to chronicity and consequently in advancement to discover new treatments to prevent wound chronicity. However, a recently developed model of chronic wounds in db/ db / mice may shine light on the molecular triggers involved in initiation and development of chronicity [20–23]. Effective treatments are likely to emerge from simultaneously studying several different measurable parameters in the newly exposed tissue after debridement with the goal of determining which of these parameters will need to be addressed for the treatment of chronic wounds in general but also potentially of the wounds of a particular individual. Identification of molecular networks that contribute to initiation of chronicity will have a major impact on how a wound that is not chronic becomes chronic. The focus needs to be at the level of genes, proteins, lipids, metabolites, and microbes because all of them

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Fig. 8 Response to high levels of oxidative stress. As seen in normal oxidative stress response, NADPH oxidase oxidizes NADPH to form NADP+, concurrently reducing oxygen (O2) to form the superoxide radical (O2• ). Superoxide radical reacts with hydrogen and is catalyzed by SOD to form H2O2. High levels of oxidative stress result in excess O2• that in in the presence of NO results in formation of excess ONOO , an anion that modifies proteins inhibiting their normal functions. In the presence of ferrous ions (Fe2+), H2O2 forms •OH radicals by the Fenton reaction. These radicals cause peroxidation of lipids and modification of DNA and proteins resulting in lack of function or malfunction. The Nrf2-Keap1 complex dissociates during high levels of oxidative stress, but it is not effective in decreasing inflammation and increasing expression of antioxidant genes. The dysregulation of all of these pathways leads to impaired/chronic wounds.

contribute significantly to the development of chronic wounds. OS plays a critical role in these processes [109]. Low levels of ROS are important for initiation and progression of proper healing, whereas high levels of ROS derail the cell and molecular mechanisms involved in healing, leading to cell death and paralyzes of the healing process. A better understanding of how wounds become chronic is critical for future success in the treatment of such wounds after debridement. Given that high levels of OS are present in chronic wounds and are critical for chronic wound initiation and development [109], we speculate that treatment of human chronic wounds with antioxidants both systemically and locally after debridement coupled with other treatments such as antibiotics, would be effective in resolving chronicity.

Role of oxidants and antioxidants in diabetic wound healing

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[23] Martins-Green M, Riverside SD, Do D, Schiller N. United States Patent No No. 15/492,649 issued August 13th 2014. Riverside: University of CA; 2014. [24] Roy S, Elgharably H, Sinha M, Ganesh K, Chaney S, Mann E, Miller C, Khanna S, Bergdall VK, Powell HM, Cook CH, Gordillo GM, Wozniak DJ, Sen CK. Mixedspecies biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol 2014;233:331–43. [25] Gould L, Abadir P, Brem H, Carter M, Conner-Kerr T, Davidson J, DiPietro L, Falanga V, Fife C, Gardner S, Grice E, Harmon J, Hazzard WR, High KP, Houghton P, Jacobson N, Kirsner RS, Kovacs EJ, Margolis D, Horne FM, Reed MJ, Sullivan DH, Thom S, Tomic-Canic M, Walston J, Whitney J, Williams J, Zieman S, Schmader K. Chronic wound repair and healing in older adults: current status and future research. J Am Geriatr Soc 2015;63(3):427–38. https://doi.org/ 10.1111/jgs.13332. [26] Rahim K, Saleha S, Zhu X, Huo L, Basit A, Franco O. Bacterial contribution in chronicity of wounds. Microb Ecol 2016;73:710–21. https://doi.org/10.1007/s00248-016-0867-9. [27] Buck II D, Jin DP, Geringer M, Hong SH, Galiano RD, Mustoe TA. The TallyHo polygenic mouse model of diabetes: implications in wound healing. Plast Reconstr Surg 2011;128:427–37. [28] Nguyen KT, Seth AK, Hong SJ, Geringer MR, Xie P, Leung KP, Mustoe TA, Galiano RD. Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm-containing chronic wounds. Wound Repair Regen 2013;21:833–41. [29] Yang P, Pei Q, Yu T, Chang Q, Wang D, Gao M, Zhang X, Liu Y. Compromised wound healing in ischemic type 2 diabetic rats. PLoS ONE 2016;11:e0152068. https://doi.org/10.1371/journal. pone.0152068. [30] Sen CK, Roy S. Redox signals in wound healing. Biochim Biophys Acta 2008;1780:1348–61. [31] Wlaschek M, Scharetter-Kochanek K. Oxidative stress in chronic venous leg ulcers. Wound Repair Regen 2005;13:452–61. [32] Loo AEK, Wong YT, Ho R, Wasser M, Du T, Ng WT, Halliwell B. Effects of hydrogen peroxide on wound healing in mice in relation to oxidative damage. PLoS ONE 2012;7(11):e49215. [33] Taylor R, James T. The role of oxidative stress in the development and persistence of pressure ulcers. In: Pressure ulcer research: current and future perspectives. Berlin: Springer; 2005. p. 205–32. https:// doi.org/10.1007/3-540-28804-X_13. [34] Babior BM. Phagocytes and oxidative stress. Am J Med 2002;109:33–44. [35] Sanchez MC, Lancel S, Boulanger E, Neviere R. Targeting oxidative stress and mitochondrial dysfunction in the treatment of impaired wound healing: a systematic review. Antioxidants 2018;7:98. https://doi.org/10.3390/anitox7080098. [36] Hoffmann MH, Griffiths HR. The dual role of ROS in autoimmune and inflammatory diseases: evidence from preclinical models. Free Radic Biol Med 2018;125:62–71. https://doi.org/10.1016/j. freeradbiomed.2018.03.016. [37] Loo AE, Halliwell B. Effects of hydrogen peroxide in a keratinocyte-fibroblast coculture model of wound healing. Biochem Biophys Res Commun 2012;423(2):253–8. https://doi.org/10.1016/j. bbrc.2012.05.100. [38] Dunnill C, Patton T, Brennan J, Barrett J, Dryden M, Cooke J, Leaper D, Georgopoulos NT. Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROSmodulating technologies for augmentation of the healing process. Int Wound J 2015;14:89–96. https://doi.org/10.1111/iwj.12557. [39] Marczin N, El-Habashi N, Hoare GS, Bundy RE, Yacoub M. Antioxidants in myocardial ischemiareperfusion injury: therapeutic potential and basic mechanisms. Arch Biochem Biophys 2003;420:222–36. [40] Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci U S A 1968;61:748–55. [41] Baranano DE, Rao M, Ferris CD, Snyder SH. Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A 2002;99:16093–8. [42] Keyse SM, Tyrrell RM. Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts. J Biol Chem 1987;262:14821–5. [43] Keyse SM, Tyrrell RM. Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts. J Biol Chem 1987;262:14821–5.

Role of oxidants and antioxidants in diabetic wound healing

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[88] Khan RU, Naz S, Abudabos AM. Towards a better understanding of the therapeutic applications and corresponding mechanisms of action of honey. Environ Sci Pollut Res Int 2017;24(36):27755–66. https://doi.org/10.1007/s11356-017-0567-0. [89] Lindberg T, Andersson O, Palm M, Fagerstr€ om C. A systematic review and meta-analysis of dressings used for wound healing: the efficiency of honey compared to silver on burns. Contemp Nurse 2015;51(2–3):121–34. https://doi.org/10.1080/10376178.2016.1171727. [90] Mukai K. Effectiveness of changing the application of Japanese honey to a hydrocolloid dressing in between the inflammatory and proliferative phases on cutaneous wound healing in male mice. Wounds 2017;29(1):1–9. [91] Molan P, Rhodes T. Honey: a biologic wound dressing. Wounds 2015;27(6):141–51. [92] Alvarez-Suarez JM, Gasparrini M, Forbes-Herna´ndez TY, Mazzoni L, Giampieri F. The composition and biological activity of honey: a focus on manuka honey. Foods 2014;3(3):420–32. https://doi.org/ 10.3390/foods3030420. [93] Niaz K, Maqbool F, Bahadar H, Abdollahi M. Health benefits of Manuka honey as an essential constituent for tissue regeneration. Curr Drug Metab 2017;18(10):881–92. https://doi.org/ 10.2174/1389200218666170911152240. [94] Jull AB, Cullum N, Dumville JC, Westby MJ, Deshpande S, Walker N. Honey as a topical treatment for wounds. Cochrane Database Syst Rev 2015;3:CD005083https://doi.org/10.1002/14651858. CD005083.pub4. [95] Long M, de la Vega MR, Wen Q, Bharara M, Jiang T, Zhang R, Zhou S, Wong PK, Wondrak GT, Zheng H, Zhang DD. An essential role of NRF2 in diabetic wound healing. Diabetes 2016;65:780–93. https://doi.org/10.2337/db15-0564. [96] Zheng H, Whitman SA, Wu W, Wondrak GT, Wong PK, Fang D, Zhang DD. Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy. Diabetes 2011;60:3055–66. [97] Suzuki T, Yamamoto M. Stress-sensing mechanisms and the physiological roles of the Keap1-Nrf2 system during cellular stress. J Biol Chem 2017;292(41):16817–24. https://doi.org/10.1074/jbc. R117.800169. [98] Uruno A, Yagishita Y, Yamamoto M. The Keap1–Nrf2 system and diabetes mellitus. Arch Biochem Biophys 2015;566:76–84. https://doi.org/10.1016/j.abb.2014.12.012. [99] Jiang T, Tian F, Zheng H, Whitman SA, Lin Y, Zhang Z, et al. Nrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-kappaB mediated inflammatory response. Kidney Int 2014;85:333–43. [100] Wu W, Qiu Q, Wang H, Whitman SA, Fang D, Lian F, Zhang DD. Nrf2 is crucial to graft survival in a rodent model of heart transplantation. Oxidative Med Cell Longev 2013;2013:919313. [101] Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev 2014;94:909–50. https://doi.org/10.1152/physrev.00026.2013. [102] Banerjee M, Vats P. Reactive metabolites and antioxidant gene polymorphisms in type 2 diabetes mellitus. Redox Biol 2014;2:170–7. https://doi.org/10.1016/j.redox.2013.12.001. [103] Ambrozova N, Ulrichova J, Galandakova A. Models for the study of skin wound healing. The role of Nrf2 and NF-kappaB. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2017;161:1–13. https://doi.org/10.5507/bp.2016.063. [104] Hughes MM, O’Neill LAJ. Metabolic regulation of NLRP3. Immunol Rev 2018;281:88–98. https:// doi.org/10.1111/imr.12608. [105] He Y, Hara H, Nu´n˜ez G. Mechanism and regulation of NLRP3 inflammasome activation. Trends Biochem Sci 2016;41(12):1012–21. https://doi.org/10.1016/j.tibs.2016.09.002. [106] Wear-Maggitti K, Lee J, Conejero A, Schmidt AM, Grant R, Breitbart A. Use of topical sRAGE in diabetic wounds increases neovascularization and granulation tissue formation. Ann Plast Surg 2004;52:519–21. https://doi.org/10.1097/01.sap.0000122857.49274.8c. [107] Wautier MP, Guillausseau PJ, Wautier JL. Activation of the receptor for advanced glycation end products and consequences on health. Diabetes Metab Syndr 2017;11:305–9. https://doi.org/10.1016/j. dsx.2016.09.009. [108] Niu Y, Xie T, Ge K, Lin Y, Lu S. Effects of extracellular matrix glycosylation on proliferation and apoptosis of human dermal fibroblasts via the receptor for advanced glycosylated end products. Am J Dermatopathol 2008;30:344–51. https://doi.org/10.1097/DAD.0b013e31816a8c5b.

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[109] Kim JH, Yang B, Tedesco A, Lebig E, Ruegger PM, Xu K, Borneman J, Martins-Green M. High levels of oxidative stress and skin microbiome are critical for initiation and development of chronic wounds in diabetic mice. Sci Rep 2019;9:19318.

Further reading Claudio M, Rizzib M, Squarzantia DF, Pittarellab P, Vaccaa G, Reno`b F. 1α,25-Dihydroxycholecalciferol (Vitamin D3) induces NO-dependent endothelial cell proliferation and migration in a three-dimensional matrix. Cell Physiol Biochem 2013;31:815–22. https://doi.org/10.1159/000350099.

CHAPTER 3

Chronic infection and inflammation: Hallmarks of diabetic foot ulcers Suman Santra, Atul Rawat, Dhamotharan Pattarayan, Sashwati Roy

Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

1. Background Diabetes is one of the 21st-century chronic disease epidemics, and it is estimated that over 10% of the global population is at high risk of developing this disease [1]. About 20% of the people with diabetes develop chronic skin wounds on the lower extremities, known as diabetic foot ulcers (DFUs). These DFUs are very difficult to treat and frequently result in hospitalization and amputation of lower limbs. Of the one million people who undergo nontraumatic leg amputations annually worldwide, 75% have type 2 diabetes (T2DM) [2,3]. Innervation and vasculopathy are well-known factors that are involved in the chronicity of DFUs. Emerging evidence underscores the seminal role of chronic persistent infection and immune dysfunction leading to unresolved inflammation, which is among the key factors leading to complications in the healing of DFUs. The objective of this chapter is to elucidate novel concepts in the understanding of chronic infection and unresolved inflammation that may play a critical role in the pathogenesis of nonhealing DFUs.

2. Chronicity of wound infection Infection is a frequent cause of the chronicity of diabetic ulcers, leading to a large economic burden on the healthcare system [4]. The risk of hospitalization greatly increases in people with an infected DFU (155 times) as compared to those with a DFU and no infection (56%) [5]. An estimated 85% of lower-limb amputations in diabetic patients are preceded by an infected DFU [6]. Despite extensive education of patients, the incidence of infection in DFU has been reported to be quite high. About 9% of the subjects in a 2-year follow-up study developed infection involving mostly soft tissue; however, 20% of these also developed bone infection. The International Working Group on the Diabetic Foot (IWGDF) and the Infectious Diseases Society of America (IDSA) have developed clinical criteria for recognizing and classifying the severity of DFU infection [7].

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00003-4

© 2020 Elsevier Inc. All rights reserved.

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Primarily, diabetes impairs host immune defenses, enabling chronic wound infection complications [8]. Infections resulting from antimicrobial-resistant strains of bacteria as well as biofilm-forming bacteria represent major threats of chronicity in wounds.

2.1 Antibiotic resistance (ABR) Overuse of antibiotics resulting in selective pressure on human infectious microbes drives these organisms to develop antibiotic resistance (ABR) [9]. The polymicrobial nature of chronic wounds promoting genetic exchange between bacterial species and the overuse of antibiotics have led to ABR being a major threat in open chronic wounds [10]. In fact, the first two cases of vancomycin-resistant Staphylococcus aureus in the United States were isolated from chronic wound patients [11]. In most cases, development of a severe wound infection will result in hospitalization, resulting in transmission of multidrug-resistant bacteria to further complicate the wounds. In a study of 110 patients with chronic vascular origin wounds, the most frequently isolated bacteria were S. aureus, E. coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis. A significant predominance (P < .05) of gram-negative bacteria (55.1%) was noted. The spectrum of ABR strains included 36% beta-lactams, 20% macrolides (20%), 9% tetracyclines, 8% aminoglycosides, and 4.5% fluoroquinolones, indicating a wide spectrum of ABR bacteria present in chronic wounds. Methicillin-resistant S. aureus (MRSA) is considered a major health threat in both the hospital and community. High frequency of isolation of MRSA has been reported from DFUs [12]. In a clinical study of DFUs, the risk factors involved in infection with multidrug resistance (MDR) were evaluated. The study determined that poor glycemic control, duration of infection, and ulcer size were independently associated with the risk of MDR organism infection in DFUs [13].

2.2 Biofilm infection Emerging evidence underscores the significant risk that biofilm infection poses to the nonhealing DFU [14,15]. As per estimates, over 60% of chronic wounds show biofilm infection [16]. Biofilm refers to a unique state of microbial infection in which bacteria are encased in a thick film-like extracellular polymeric substance (EPS) produced by these microbes. In their clinically presented forms, biofilms are host-interactive and polymicrobial, often including fungi, viruses, and/or protozoa in addition to multispecies bacterial communities. In the biofilm form, bacteria are in a dormant metabolic state and are inherently ABR. Thus, standard clinical techniques like the colony forming unit (CFU) assay to detect infection may not detect biofilm infection [17]. In the broader discipline of wound infection, polymicrobial biofilm aggregates have emerged as a major threat, because it is in this form that microbes acquire more resistance to host defenses and antibiotics, as well as acquire more pathogenicity [18–21]. We have extensively studied the effect of chronic biofilm infection in healing of wounds. Skin barrier function is of

Chronic infection and inflammation: Hallmarks of diabetic foot ulcers

extraordinary significance to overall human health in general, as evident by its direct impact on lung and immune function [22–25]. We have demonstrated that biofilm infection causes faulty reepithelialization, compromising barrier function at the closed wound site [26,27]. Such defects are caused by biofilm-inducible miRs which silence junctional proteins necessary for skin barrier function [26,27]. Furthermore, biofilm infection degraded the extracellular matrix, thus weakening tensile strength of the repaired skin and making it vulnerable to wound recurrence [28]. Thus biofilm infection may be viewed as a silent threat in wound healing and clinical care.

2.3 Novel approaches for therapeutics Both ABR and biofilm infection are recalcitrant to standard antimicrobial treatment or procedures for wound care, such as wound debridement [29]. In this section, we discuss novel approaches to mitigate ABR and biofilm infection. 2.3.1 Electroceuticals Electroceuticals broadly encompass all bioelectronic medicines that employ electrical stimulation to affect and modify functions of the body [30,31]. Clinical implants such as cochlear implants, retinal implants, or cardiac pacemakers are conventional examples of electroceuticals [30]. More recently, electrical stimulation of the vagus nerve has been used to modulate immune system rheumatoid arthritis relief and prevent epileptic seizures [31]. Electrical principles influence fundamental processes in bacterial biology that may influence biofilm development; these include adhesion to surfaces (electrostatic interactions) for initial establishment of biofilms [32] and interbacterial communication (ion channels) [33]. This inherent dependence on the flow of electric charge for integral biological activities, if perturbed via an externally applied low-level electric current/field, could disrupt biofilm infection. The fact that an electric field (1.5 V/cm) may have an antibiofilm property was first reported in 1992 [34,35]. Our studies demonstrated that bacterial biofilms can be disrupted by a wireless electroceutical dressing (WED), which is available commercially (Procellera) with FDA clearance for burn care dressings [26,36–39]. We reported that the electric field generated by the WED due to its silver-zinc (Ag/Zn) redox chemistry is effective as prohealing [36] and antibiofilm [37,40]. More recently we reported that a second-generation disposable patterned electroceutical dressing (PED-10) is safe for treatment of open clinical chronic wounds [41]. 2.3.2 Bacteriophage At the beginning of the 20th century, the use of bacteriophages for treatment of clinical bacterial infection was proposed by Felix d’Herelle. While not popular then in the West, the approach was widely practiced in the Soviet Union. With the advent of antibiotics as a “magic bullet,” the interest in phage therapy gradually faded. Now, several decades later, with the current menace of ABR and biofilm, phage therapy is gaining attention

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[42–45]. Recently, the use of phage therapy in infected DFUs has been reported in a case report in which the authors describe the successful treatment, with a long-term followup, of a 63-year-old diabetic female with distal phalangeal osteomyelitis using bacteriophage [44]. The therapy is not yet FDA approved in the United States; however, the area has high potential for mitigating ABR/biofilm infection [43]. Initial reports warrant a proper randomized controlled trial (RCT) using this therapy for DFUs.

3. Dysregulated resolution of wound inflammation Inflammation is a physiological response triggered by the body via unleashing the immune response to unwanted stimuli. This response is required as part of an adequate adult wound-healing response. The innate immune cells are the first responders to an inflammatory response and are primarily the cells derived from common myeloid progenators, including neutrophils and macrophages. The switch-off of inflammation in a timely manner is as crucial as the initiation of this response [46–48]. We demonstrated that mechanisms that control switching off of inflammation are dysfunctional in diabetic wounds [46]. A persistent inflammation resulting in stalled wound healing ultimately results in the loss of organ function. Prolonged inflammation has been identified as a significant contributor to the development of various disorders, such as atherosclerosis, obesity, cancer, chronic obstructive pulmonary disease, asthma, neurodegenerative disease, and arthritis. We discuss some critical mechanisms that control resolution of wound inflammation and how dysregulation of these in diabetic wounds may lead to chronic inflammatory nonhealing DFUs.

3.1 miRNA The microRNAs (miRNAs) are an established class of well-conserved, short singlestranded noncoding RNAs, with approximately 21 nucleotides being known to play significant roles in most, if not all, biological processes by influencing the stability and translation of mRNAs by regulating gene expression at a posttranscriptional level. The transcription of miRNA genes occurs through RNA polymerase II catalysis to form primary precursors (pri-miRNAs), which are subjected to cleavage by nuclear RNase III Drosha-DGCR8 to shorter hairpin-shaped precursors (pre-miRNAs, approximately 60 nt) and exported from the nucleus to the cytoplasm [49–53]. About 40% of miRNAs are derived from the introns and 10% from the exons of other coding or noncoding transcripts, sharing transcriptional regulation with the host genes. miRNA genes are often clustered together in the genome, transcribed as a multicistronic primary transcript and then further processed, even though the expression pattern of these miRNAs may differ within the cluster due to complicated posttranscriptional regulation. To mediate their biological function, mature miRNAs are loaded to the Argonaute (AGO) protein to form the RNA-induced silencing complex (RISC) and bring this enzyme

Chronic infection and inflammation: Hallmarks of diabetic foot ulcers

complex to the target messenger RNA (mRNA); miRNAs downregulate the expression of target genes by repressing translation, reducing stability, or degrading their mRNAs. Moreover, the RISC can also silence the target gene at the genomic level by formation of heterochromatin or via DNA elimination. Aberrant expression and function of several miRNAs have been reported in chronic wounds [54,55]. We have shown that TNF-α neutralization led to miR-200b downregulation that supported wound closure by improving angiogenesis in diabetic mice [56]. A plasma miRNA profile in type 2 diabetic (T2DM) patients with and without chronic wounds identified 41 deregulated miRNAs, among which decreased miR-191 and miR-200b levels were validated [57]. Furthermore, the authors showed that miR-191 secreted by endothelial cells or platelets was either taken up by dermal endothelial cells to reduce angiogenesis, or taken by fibroblasts to inhibit migration, thereby causing delay in wound repair [57]. MiR-146a has been found attenuated in diabetic mouse wounds, and application of miR-146a conjugated with cerium oxide nanoparticles improved wound healing of diabetic mice without impairing biomechanical properties of the healed skin [58]. Disruption of miRNA biogenesis has a profound impact on the overall immune system. We have demonstrated that miRNAs, especially miR-21, switch a macrophage to a proresolving reparative phenotype, thus regulating the resolution of the inflammatory response [47,59]. We have reviewed in detail the role of miRNAs in macrophage and inflammatory responses in the context of wound healing [53].

3.2 Macrophage function and phenotypes The central role of cells of myeloid origin, specifically macrophages, in controlling all phases of wound healing is well established. These cells are highly sensitive to environmental cues and molecular mediators and acquire adequate form and function to drive the healing process. The heterogeneity of monocytes/macrophages is well described. We have reviewed the underlying mechanisms that determine the phenotype and function of macrophages in the wound environment [60]. We have recently reported that injuryinduced immune cell plasticity is driven by extracellular vesicles (EVs) in the wound environment and that is an inherent component of physiological tissue repair. This immune cell plasticity is lost in diabetic wounds.

3.3 Efferocytosis Among leukocytes, the neutrophils serve as the first line of defense to reach the site of injury, with the primary role of disinfection by clearing invading pathogens and releasing proinflammatory signals. Upon the culmination of this initial immune response, the neutrophils undergo programmed cell death and macrophages descend on the inflammatory site to clear the area in a process called efferocytosis [47]. This process involves the removal of dead cells and debris and is a critical component of wound healing and a

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Wound healing, tissue repair, and regeneration in diabetes

key function of macrophages in the wound environment. An adequate macrophage differentiation response facilitates progress of a wound microenvironment from a proinflammatory to a proresolution state. A primary role for macrophages is the clearance of neutrophils after they begin the immune response, and as the effectiveness of neutrophils is impaired in diabetic patients, the wound is unable to progress from a proinflammatory environment. Studies have shown impaired efferocytosis in diabetic wounds, leading to an accumulation of apoptotic cells at the wound site [46]. The impairment of immune cell function and the continual presence of proinflammatory cytokines prevented the wound environment from resolving in a timely fashion. The removal of apoptotic cells via efferocytosis prevents exposure to toxic cellular remains and avoids further damage to the surrounding tissues [46]. To have timely resolution of inflammation, triggering of adequate efferocytosis [47,61] is thus critical. Milk-fat globule EGF factor 8 (MFG-E8) is a glycoprotein that aids wound macrophages in the clearance of apoptotic cells [62]. Studies have shown that animals and patients suffering from sustained hyperglycemia and the presence of glycated end products deactivate MFG-E8, which attenuates wound healing via persistent inflammation and the development of new blood vessels. Finally, delivery of MFG-E8 to diabetic mice showed significantly improved wound closure as a result of the resolution of the inflammatory response [62].

4. Conclusion Chronic infection and inflammation are major threats that complicate the healing of diabetic ulcers. Lack of adequate solutions for management of chronic wound infections such as ABR and biofilm infections leads to serious consequences, such as poor quality of life and amputations in patients with diabetes. Novel solutions that can counter these threats are urgently needed. A thorough understanding of the principles of immune dysfunction and insufficient resolution of inflammation will lead to innovative interventions that may drive a diabetic wound towards resolution of inflammation, which is conducive to healing.

References [1] WHO. Global report on diabetes. Geneva: WHO; 2016. [2] Caravaggi C, Ferraresi R, Bassetti M, Sganzaroli AB, Galenda P, Fattori S, De Prisco R, Simonetti D, Bona F. Management of ischemic diabetic foot. J Cardiovasc Surg (Torino) 2013;54:737–54. [3] Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;366:1736–43. [4] Prompers L, Huijberts M, Schaper N, Apelqvist J, Bakker K, Edmonds M, Holstein P, Jude E, Jirkovska A, Mauricio D, Piaggesi A, Reike H, Spraul M, Van Acker K, Van Baal S, Van Merode F, Uccioli L, Urbancic V, Ragnarson Tennvall G. Resource utilisation and costs associated with the treatment of diabetic foot ulcers. Prospective data from the Eurodiale study. Diabetologia 2008;51:1826–34. [5] Lavery LA, Armstrong DG, Wunderlich RP, Mohler MJ, Wendel CS, Lipsky BA. Risk factors for foot infections in individuals with diabetes. Diabetes Care 2006;29:1288–93.

Chronic infection and inflammation: Hallmarks of diabetic foot ulcers

[6] Adler AI, Boyko EJ, Ahroni JH, Smith DG. Lower-extremity amputation in diabetes. The independent effects of peripheral vascular disease, sensory neuropathy, and foot ulcers. Diabetes Care 1999;22:1029–35. [7] Peters EJ, Lipsky BA. Diagnosis and management of infection in the diabetic foot. Med Clin North Am 2013;97:911–46. [8] Ahmed AS, Antonsen EL. Immune and vascular dysfunction in diabetic wound healing. J Wound Care 2016;25(Suppl 7):S35–46. [9] Bell M. Antibiotic misuse: a global crisis. JAMA Intern Med 2014;174:1920–1. [10] Tzaneva V, Mladenova I, Todorova G, Petkov D. Antibiotic treatment and resistance in chronic wounds of vascular origin. Clujul Med 2016;89:365–70. [11] Howell-Jones RS, Wilson MJ, Hill KE, Howard AJ, Price PE, Thomas DW. A review of the microbiology, antibiotic usage and resistance in chronic skin wounds. J Antimicrob Chemother 2005;55:143–9. [12] Cavanagh PR, Lipsky BA, Bradbury AW, Botek G. Treatment for diabetic foot ulcers. Lancet 2005;366:1725–35. [13] Zubair M, Malik A, Ahmad J. Clinico-microbiological study and antimicrobial drug resistance profile of diabetic foot infections in North India. Foot (Edinb) 2011;21:6–14. [14] Neut D, Tijdens-Creusen EJ, Bulstra SK, van der Mei HC, Busscher HJ. Biofilms in chronic diabetic foot ulcers—a study of 2 cases. Acta Orthop 2011;82:383–5. [15] Davis SC, Martinez L, Kirsner R. The diabetic foot: the importance of biofilms and wound bed preparation. Curr Diab Rep 2006;6:439–45. [16] James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, Costerton JW, Stewart PS. Biofilms in chronic wounds. Wound Repair Regen 2008;16:37–44. [17] Hall-Stoodley L, Stoodley P, Kathju S, Hoiby N, Moser C, Costerton JW, Moter A, Bjarnsholt T. Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunol Med Microbiol 2012;65:127–45. [18] Roilides E, Simitsopoulou M, Katragkou A, Walsh TJ. How biofilms evade host defenses. Microbiol Spectr 2015;3. https://doi.org/10.1128/microbiolspec.MB-0012-2014. [19] Martinez JL, Baquero F. Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol Rev 2002;15:647–79. [20] Pestrak MJ, Chaney SB, Eggleston HC, Dellos-Nolan S, Dixit S, Mathew-Steiner SS, Roy S, Parsek MR, Sen CK, Wozniak DJ. Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments. PLoS Pathog 2018;14: e1006842. [21] Roy S, Elgharably H, Sinha M, Ganesh K, Chaney S, Mann E, Miller C, Khanna S, Bergdall VK, Powell HM, Cook CH, Gordillo GM, Wozniak DJ, Sen CK. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol 2014;233:331–43. [22] Clausen M-L, Agner T, Thomsen SF. Skin barrier dysfunction and the atopic march. Curr Treat Options Allergy 2015;2:218–27. [23] Lee SH, Jeong SK, Ahn SK. An update of the defensive barrier function of skin. Yonsei Med J 2006;47:293–306. [24] Elias PM. Skin barrier function. Curr Allergy Asthma Rep 2008;8:299–305. [25] Bouwstra JA, Ponec M. The skin barrier in healthy and diseased state. Biochim Biophys Acta 1758;2006:2080–95. [26] Barki KG, Das A, Dixith S, Ghatak PD, Mathew-Steiner S, Schwab E, Khanna S, Wozniak DJ, Roy S, Sen CK. Electric field based dressing disrupts mixed-species bacterial biofilm infection and restores functional wound healing. Ann Surg 2019;269:756–66. [27] Roy S, Elgharably H, Sinha M, Ganesh K, Chaney S, Mann E, Miller C, Khanna S, Bergdall VK, Powell HM. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol 2014;233:331–43. [28] Roy S, Santra S, Das A, Dixith S, Sinha M, Ghatak S, Ghosh N, Banerjee P, Khanna S, Mathew-Steiner S, Ghatak PD, Blackstone BN, Powell HM, Bergdall VK, Wozniak DJ, Sen CK. Staphylococcus aureus biofilm infection compromises wound healing by causing deficiencies in granulation tissue collagen. Ann Surg 2019;.

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[29] Roy S, Elgharably H, Sinha M, Ganesh K, Chaney S, Mann E, Miller C, Khanna S, Bergdall VK, Powell HM, Cook CH, Gordillo GM, Wozniak DJ, Sen CK. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J Pathol 2014;233:331–43. [30] Famm K, Litt B, Tracey KJ, Boyden ES, Slaoui M. Drug discovery: a jump-start for electroceuticals. Nature 2013;496:159–61. [31] Moore SK. The vagus nerve: a back door for brain hacking. IEEE Spectr 2015;. [32] Renner LD, Weibel DB. Physicochemical regulation of biofilm formation. MRS Bull 2011;36:347–55. [33] Prindle A, Liu J, Asally M, Ly S, Garcia-Ojalvo J, Suel GM. Ion channels enable electrical communication in bacterial communities. Nature 2015;527:59–63. [34] Blenkinsopp SA, Khoury AE, Costerton JW. Electrical enhancement of biocide efficacy against Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 1992;58:3770–3. [35] Khoury AE, Lam K, Ellis B, Costerton JW. Prevention and control of bacterial infections associated with medical devices. ASAIO J 1992;38:M174–8. [36] Banerjee J, Das Ghatak P, Roy S, Khanna S, Sequin EK, Bellman K, Dickinson BC, Suri P, Subramaniam VV, Chang CJ, Sen CK. Improvement of human keratinocyte migration by a redox active bioelectric dressing. PLoS ONE 2014;9:e89239. [37] Banerjee J, Das Ghatak P, Roy S, Khanna S, Hemann C, Deng B, Das A, Zweier JL, Wozniak D, Sen CK. Silver-zinc redox-coupled electroceutical wound dressing disrupts bacterial biofilm. PLoS ONE 2015;10:e0119531. [38] Ghatak S, Chan YC, Khanna S, Banerjee J, Weist J, Roy S, Sen CK. Barrier function of the repaired skin is disrupted following arrest of dicer in keratinocytes. Mol Ther 2015;23:1201–10. [39] FDA-NIH BWG. BEST (Biomarkers, EndpointS, and other Tools) Resource [Internet]. Diagnostic Biomarker. Silver Spring, MD/Bethesda, MD: Food and Drug Administration/National Institutes of Health; 2016. [40] Barki KG, Das A, Dixith S, Ghatak PD, Mathew-Steiner S, Schwab E, Khanna S, Wozniak DJ, Roy S, Sen CK. Electric field based dressing disrupts mixed-species bacterial biofilm infection and restores functional wound healing. Ann Surg 2019;269(4):756–66. [41] Roy S, Prakash S, Mathew-Steiner SS, Das Ghatak P, Lochab V, Jones TH, Mohana Sundaram P, Gordillo GM, Subramaniam VV, Sen CK. Disposable patterned electroceutical dressing (PED-10) is safe for treatment of open clinical chronic wounds. Adv Wound Care (New Rochelle) 2019;8:149–59. [42] Golkar Z, Bagasra O, Pace DG. Bacteriophage therapy: a potential solution for the antibiotic resistance crisis. J Infect Dev Ctries 2014;8:129–36. [43] Fish R, Kutter E, Wheat G, Blasdel B, Kutateladze M, Kuhl S. Compassionate use of bacteriophage therapy for foot ulcer treatment as an effective step for moving toward clinical trials. Methods Mol Biol 1693;2018:159–70. [44] Fish R, Kutter E, Bryan D, Wheat G, Kuhl S. Resolving digital Staphylococcal osteomyelitis using bacteriophage—a case report. Antibiotics (Basel) 2018;7. https://doi.org/10.3390/antibiotics7040087. [45] Donlan RM. Preventing biofilms of clinically relevant organisms using bacteriophage. Trends Microbiol 2009;17:66–72. [46] Khanna S, Biswas S, Shang Y, Collard E, Azad A, Kauh C, Bhasker V, Gordillo GM, Sen CK, Roy S. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS ONE 2010;5:e9539. [47] Das A, Ganesh K, Khanna S, Sen CK, Roy S. Engulfment of apoptotic cells by macrophages: a role of microRNA-21 in the resolution of wound inflammation. J Immunol 2014;192:1120–9. [48] Das A, Datta S, Roche E, Chaffee S, Jose E, Shi L, Grover K, Khanna S, Sen CK, Roy S. Novel mechanisms of collagenase santyl ointment (CSO) in wound macrophage polarization and resolution of wound inflammation. Sci Rep 2018;8:1696. [49] Shilo S, Roy S, Khanna S, Sen CK. Evidence for the involvement of miRNA in redox regulated angiogenic response of human microvascular endothelial cells. Arterioscler Thromb Vasc Biol 2008;28:471–7. [50] Sen CK, Roy S. miRNA: licensed to kill the messenger. DNA Cell Biol 2007;26:193–4.

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[51] Roy S, Sen CK. miRNA in wound inflammation and angiogenesis. Microcirculation 2012;19:224–32. [52] Roy S, Sen CK. MiRNA in innate immune responses: novel players in wound inflammation. Physiol Genomics 2011;43:557–65. [53] Roy S. miRNA in macrophage development and function. Antioxid Redox Signal 2016;25:795–804. [54] Li X, Li D, Wang A, Chu T, Lohcharoenkal W, Zheng X, Grunler J, Narayanan S, Eliasson S, Herter EK, Wang Y, Ma Y, Ehrstrom M, Eidsmo L, Kasper M, Pivarcsi A, Sonkoly E, Catrina SB, Stahle M, Xu Landen N. MicroRNA-132 with therapeutic potential in chronic wounds. J Invest Dermatol 2017;137:2630–8. [55] Ramirez HA, Liang L, Pastar I, Rosa AM, Stojadinovic O, Zwick TG, Kirsner RS, Maione AG, Garlick JA, Tomic-Canic M. Comparative genomic, MicroRNA, and tissue analyses reveal subtle differences between non-diabetic and diabetic foot skin. PLoS ONE 2015;10:e0137133. [56] Chan YC, Khanna S, Roy S, Sen CK. miR-200b targets Ets-1 and is down-regulated by hypoxia to induce angiogenic response of endothelial cells. J Biol Chem 2011;286:2047–56. [57] Dangwal S, Stratmann B, Bang C, Lorenzen JM, Kumarswamy R, Fiedler J, Falk CS, Scholz CJ, Thum T, Tschoepe D. Impairment of wound healing in patients with type 2 diabetes mellitus influences circulating microRNA patterns via inflammatory cytokines. Arterioscler Thromb Vasc Biol 2015;35:1480–8. [58] Zgheib C, Hilton SA, Dewberry LC, Hodges MM, Ghatak S, Xu J, Singh S, Roy S, Sen CK, Seal S, Liechty KW. Use of cerium oxide nanoparticles conjugated with MicroRNA-146a to correct the diabetic wound healing impairment. J Am Coll Surg 2019;228:107–15. [59] Sinha M, Sen CK, Singh K, Das A, Ghatak S, Rhea B, Blackstone B, Powell HM, Khanna S, Roy S. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue. Nat Commun 2018;9:936. [60] Das A, Sinha M, Datta S, Abas M, Chaffee S, Sen CK, Roy S. Monocyte and macrophage plasticity in tissue repair and regeneration. Am J Pathol 2015;185:2596–606. [61] Elgharably H, Mann E, Awad H, Ganesh K, Ghatak PD, Gordillo G, Sai-Sudhakar CB, Roy S, Wozniak DJ, Sen CK. First evidence of sternal wound biofilm following cardiac surgery. PLoS ONE 2013;8:e70360. [62] Das A, Ghatak S, Sinha M, Chaffee S, Ahmed NS, Parinandi NL, Wohleb ES, Sheridan JF, Sen CK, Roy S. Correction of MFG-E8 resolves inflammation and promotes cutaneous wound healing in diabetes. J Immunol 2016;196:5089–100.

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CHAPTER 4

Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics Puneet Khandelwal, Savita Khanna

Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States

1. Introduction Diabetes has become one of the most challenging global health concerns of the modern world, with an affected population of around 463 million adults between 20 and 79 years of age in 2019, which is expected to increase to 700 million by the year 2045 (Fig. 1). Although diabetes affects people of all ages, the most affected age group is between 60 and 69 years (Fig. 2). Approximately half of the total number of people with diabetes, which is around 232 million, are undiagnosed and contribute to the deaths of about 4.2 million due to diabetes worldwide. Around 10% of overall health expenditures of adults, approximately $760 billion, is due to diabetes globally. Numbers of deaths and expenses per person due to diabetes are even worse in low and middle-income countries, which can be observed by the fact that about 87% of the diabetes-related deaths occurred in low and middle-income countries, while only 35% of diabetes-related health expenditures occurred there. About 1 in 8 adults in the United States have diabetes. Among 233 million adults, approximately 31 million adults have diabetes, and about 0.2 million of them died due to diabetes in the United States. (These data were obtained from the International Diabetes Federation. IDF Diabetes Atlas. 9th ed. Brussels, Belgium; 2019.) Two types of diabetes are most common in the population: (a) type 1 diabetes, and (b) type 2 diabetes (Table 1). Type 1 diabetes is an immune-mediated disorder associated with the destruction of pancreatic cells, resulting in low insulin and high blood glucose level [1]. The high blood glucose level can damage nerves and blood vessels in kidneys, eyes, and heart, which can lead to diabetic neuropathy, retinopathy, and cardiovascular diseases. It is reported that more than 1.1 million children and adolescents are living with type 1 diabetes worldwide. Among them, the United States has the highest number of children and adolescents with type 1 diabetes, nearly 0.2 million. Type 2 diabetes is a progressive disorder in which the body is not able to make enough insulin and the cells also become resistant to insulin and no longer respond to insulin to maintain blood sugar

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00004-6

© 2020 Elsevier Inc. All rights reserved.

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Fig. 1 The number of adults (20–79 years) with diabetes worldwide in the year 2019. (The data have been obtained from the International Diabetes Federation. IDF Diabetes Atlas. 9th ed. Brussels, Belgium; 2019.)

Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics

Fig. 2 The relationship of diabetes with age. Diabetes affects people of all age groups but shows the most prevalence in the age group of 60–69 years. (The data have been obtained from the International Diabetes Federation. IDF Diabetes Atlas. 9th ed. Brussels, Belgium; 2019.) Table 1 The major differences between type 1 diabetes mellitus and type 2 diabetes mellitus. S. No.

Type 1 diabetes

Type 2 diabetes

1

Also known as insulin-dependent or juvenile-onset diabetes mellitus Categorized as an autoimmune disease Mostly occurs in children (adolescents)

Also known as noninsulin dependent or maturity-onset diabetes mellitus Categorized as a metabolic lifestyle disease Mainly occurs in adults of middle ages (>35–40 years) Accounts for >80% of all diabetes cases. Mostly connected with excess body weight or blood pressure, or cholesterol It can be treated with diet and lifestyle interventions and medication may not be required. However, metformin may be given often and insulin also sometimes. A relative deficiency of insulin due to the dysfunction of pancreatic β-cells or insulin resistance

2 3 4 5 6

7

Accounts for
10% of all diabetes cases Generally not related to excess body weight or blood pressure or cholesterol It can be treated with insulin injections and cannot be controlled without any medication. Absolute deficiency of insulin caused by autoimmune destruction of pancreatic β-cells.

level. The primary causes behind type 2 diabetes are factors such as diet and lifestyle. The proportion of type 2 diabetes is increasing in most countries, with around 374 million people at increased risk of developing type 2 diabetes. Diabetic peripheral neuropathy (DPN) is the most common issue in both type 1 and type 2 diabetes [2]. It is found that around 12%–50% of people suffering from diabetes have some degree of peripheral neuropathy characteristics. DPN is one of the major

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reasons for disability due to foot ulceration, amputation, gait instability, and fall-related injury. DPN significantly lowers quality of life and substantially increases medical costs: for example, the total annual average medical cost for diabetes is $6632 per patient, while this amount is almost doubled in the case of DPN, to $12,492 [3–5]. Despite the long history of research, there is still no medical treatment available that can cure inherited peripheral neuropathy. However, there are a few therapies available for many other forms of neuropathies, which can reduce the physical and emotional effects of peripheral neuropathies. These therapies include a healthy lifestyle (for example, maintaining optimal weight, avoiding exposure to toxins, carrying out physiciansupervised exercise, eating a balanced diet, removing vitamin deficiencies, and avoiding alcohol consumption and smoking), and controlling blood sugar [6].

2. Neuropathy Neuropathy can be defined as the malfunction of nerves at any location in the body. It may lead to symptoms like loss of feeling, tingling, numbness, and pain. Neuropathy can be the result of different types of diseases (such as cancer and diabetes), side effects of therapies (such as chemotherapy), deficiencies (such as vitamin B12 deficiency), infections (such as HIV), and trauma/injury (Fig. 3). Depending on the location of malfunctioned nerves, neuropathy can be classified into four different categories:

2.1 Peripheral neuropathy Damage to the peripheral nerves, which are outside of the brain and spinal cord, affects the extremities.

2.2 Cranial neuropathy If any of the 12 cranial nerves are damaged, cranial neuropathy occurs. There are two specific types of neuropathies: optic neuropathy and auditory neuropathy. As the name suggests, when the optic nerve (which transmits the visual signal from the retina to the brain) is damaged, it is known as optic neuropathy. When the auditory nerve (transmits the signal from the inner ear to the brain) is damaged, it is known as auditory neuropathy [7].

2.3 Autonomic neuropathy In this type of neuropathy, the nerves of the involuntary nervous system are damaged. These nerves are involved in the heartbeat, blood circulation, digestion, bladder function, sexual response, and perspiration [7].

Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics

Fig. 3 Neuropathy can be triggered by many factors, including diabetes, vitamin deficiency, autoimmune diseases (rheumatoid arthritis, systemic lupus, and Guillain-Barr e syndrome), infections (HIV, varicella-zoster virus, leprosy, Lyme disease, and syphilis), excessive alcohol consumption, genetic disorders (Friedreich’s ataxia and Charcot-Marie-Tooth disease), amyloidosis (aggregation and deposition of protein molecules), uremia, toxins (lead, arsenic, mercury), drugs for cancer therapy, antibiotics, trauma, and tumor.

2.4 Focal neuropathy This type of neuropathy occurs when the damaged nerves affect one particular area of the body. In this type, peripheral neuropathy is the most common, and it can be further classified into the following categories, based on the cause. 2.4.1 Mononeuropathy When a single peripheral nerve is damaged, such as in the case of physical injury or trauma, it is called mononeuropathy. One of the examples of mononeuropathy is carpal tunnel syndrome, where the nerve of the wrist is repeatedly compressed. Ulnar nerve palsy, radial nerve palsy, and peroneal nerve palsy are a few other examples of mononeuropathy. 2.4.2 Polyneuropathy Polyneuropathy occurs when multiple peripheral nerves in the body are damaged at the same time. This is the most common type of peripheral neuropathy. There can be a wide

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variety of causes, including excessive alcohol consumption, poor nutrition (such as vitamin B deficiency), and certain diseases such as cancer, diabetes, or kidney failure. Among these, diabetes is the most common cause of peripheral neuropathy, known as DPN, as previously defined. DPN is the most common kind of peripheral neuropathy in the Western world and it affects more than 50% of diabetic patients, leading to a wide range of clinical manifestations [8]. As the name suggests, DPN is the most common consequence of high blood sugar and diabetes. It can be one of three types depending on the type of nerve damaged: sensory neuropathy (sensory nerves), motor neuropathy (motor nerves), and autonomic neuropathy (autonomic nerves).

3. Experimental mouse models for DPN Among all the animal models, rodents (mice and rats) are the only animals that have been used widely for the study of mechanism, pathogenesis, and treatment of DPN because of their advantages, including cost, housing and handling, breeding time, and ethical considerations [9–12]. DPN can be induced in mice by any of three methods: (1) diet induced, (2) chemically induced, and (3) genetically modified.

3.1 Diet-induced DPN mouse model The type 2 diabetic neuropathy model has been established using a diet-induced method whereby a high-fat diet is given to experimental animals to induce diabetes. When C57BL/6 mice were fed a high-fat diet including 24% fat from soybean oil and lard, 24% protein, and 41% carbohydrates for 12 weeks, these mice developed the signs of prediabetes and presented the indications of neuropathy, such as reduced density of intraepidermal nerve fibers, decreased sensory nerve conduction velocity (NCV), and thermal hypoalgesia [10]. However, it has been found that the diabetic neuropathy is more robust when the high-fat diet is fed to C57BL/6 db/db mice [13,14]. Several factors, such as sex and age of the mice, duration of the high-fat diet, type of fat, content of the fatty diet, and percentage of fat in the diet, have also been reported to have an effect on the degree of neuropathy in these mice models. It has been observed that the sex of the mice also plays an important role in the induction of diabetes; for example, male mice are more susceptible to diet-induced diabetes [10,11].

3.2 Chemically induced DPN mouse model Streptozotocin (STZ) is a potent alkylating agent that selectively destroys pancreatic β cells by interfering with glucose transport and glucokinase function, and by introducing DNA strand breaks. STZ-induced diabetes is a commonly used model for the study of DPN. The dose of STZ is a crucial parameter in the development of neuropathy [9]. A higher dose injection of STZ destroys the pancreatic β-cells and leads to the

Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics

development of type 1 diabetes, while the administration of an intermediate dose of STZ causes partial damage of pancreatic β-cells, which ends up as type 2 diabetes [15]. This is an inexpensive method and optimization is also easy in comparison to other methods.

3.3 Genetically modified DPN mouse model Both type 1 and type 2 diabetic neuropathy models have been developed by genetic modifications. Two mouse models have been developed for type 1 diabetes, namely nonobese diabetic (NOD) and B6Ins2Akita mice models [14,16]. Due to a heritable polygenic immunodeficiency, NOD mice develop autoimmune T cell-mediated insulindependent diabetes mellitus spontaneously. The B6Ins2Akita mouse model was developed by a point mutation in the Ins2 insulin gene of C57BL/6 mice, which spontaneously develop type 1 diabetes at the age of 7 weeks [16]. On the other hand, a type 2 diabetes model was developed from a mutation in leptin (ob/ob mice) and its receptor (db/db mice) [17]. A decrease in the density of IENF and motor and sensory neuron conduction velocities suggests that the ob/ob mice are a good model of hypoalgesia. However, again, the choice of mouse strain affects the development of neuropathy: for example, C57BKS db/db mice have more stable hyperglycemia and more severe neuropathy in comparison to C57BL/6 db/db [13,14].

4. Nociception assays Nociception cannot be measured directly in rodents, but many indirect methods can be used. These methods can be divided into two categories: (a) stimulus-evoked nociception, which includes manual and electronic von Frey, Randall-Selitto, and the Hargreaves test; (b) nonstimulus-evoked nociception, comprising grimace scales, burrowing, and weight-bearing and gait analysis [18]. The stimulus-evoked nociception methods can be further categorized based on stimulus modalities such as mechanical stimulus, heat stimulus, and cold stimulus (Fig. 4) [19].

4.1 Mechanical stimuli The behaviors in response to mechanical stimuli can be measured by manual or the electronic von Frey or Randall-Selitto tests. 4.1.1 The manual von Frey test The manual von Frey test is used to measure mechanical allodynia in rodents. This test is still a gold standard even after the development of an electronic von Frey test. In this assay, a monofilament is applied perpendicular to the plane of the hind paw until it buckles [20]. The test is considered positive if the mice show any nocifensive behaviors, including quick paw withdrawal, licking, or shaking of the paw during the test or just

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Filament is applied until it buckles

von Frey filament

Glass floor

(D)

(F)

Force

(A)

Mesh or barred floor

Acetone Heat source

Time

Mesh or barred floor

(H)

Glass floor

Stick of ice

(I)

(B)

54°C 50°C

(C)

(E)

(G)

(J)

Reference

Test

(K)

Temperature gradient

Fig. 4 Methods used to assess stimulus-evoked pain-like behaviors: (A) manual von Frey, (B) electronic von Frey, (C) Randall-Selitto, (D) tail-flick test, (E) hot plate test, (F) Hargreaves test, (G) thermal probe test, (H) acetone evaporation test, (I) cold plantar assay, temperature preference assays. (J) two-temperature choice assay, (K) continuous temperature gradient assay. (From Deuis JR, Dvorakova LS, Vetter I. Methods used to evaluate pain behaviors in rodents. Front Mol Neurosci 2017;10:284.)

Diabetic peripheral neuropathy: An insight into the pathophysiology, diagnosis, and therapeutics

after the removal of the filament. Though the hind paw is most commonly used for the von Frey test, other body parts can be used, including the dorsal surface of the hind paw and the abdomen. 4.1.2 The electronic von Frey The electronic von Frey assay is the same in principle as the manual von Frey test, with only a single difference: it uses a nonbending filament. The filament is applied with an increasing force to the hind paw until the paw withdrawal response is elicited. The force at which the paw withdrawal response is found is recorded by the instrument automatically and referred to as the paw withdrawal threshold [21]. Moreover, a continuous scale of paw withdrawal threshold can be measured as increasing force is applied continuously. 4.1.3 Randall-Selitto test The Randall-Selitto test, also known as the paw pressure test, is used to measure the response threshold to mechanical pressure stimulation [22]. In this test, increasing mechanical pressure is applied to the surface of the paw until withdrawal or vocalization occurs. This test measures nociceptive thresholds and is mostly useful for rats, as mice cannot tolerate the heavy physical restraint required for the test. This test can be performed using bench-top or hand-held devices with animals restrained in a hammock, towel, or a plastic cone or cylinder, which provides access to the hind paws [23]. Before performing the test, the animals must be adapted to the restraint method and the apparatus in order to obtain reliable data, which makes the method time-consuming. Moreover, the threshold measurement is entirely dependent on the researcher’s visualization. Some researchers use vocalization as an endpoint, but it should be kept in mind that rodents don’t vocalize in audible range unless the pain is severe, making it ethically limited.

4.2 Heat stimuli 4.2.1 The tail-flick test The tail-flick test requires a heat stimulus, preferably a focused light beam or hot water, applied to the tail of the mouse or rat, and measurement of the time needed for the tail to “flick” or twitch. It has been often observed that the tail-flick response is mostly a spinal reflex rather than requiring the involvement of higher brain centers, which is the indication of pain behaviors. However, the heating slope and temperature play an important role [24]. The stimuli that lead to the delayed withdrawal response show a higher central nervous system response necessary to process pain. 4.2.2 Hot plate test A hot plate test is used to measure the heat threshold in mice and rats. In the conventional hot plate method, the hot plate temperature is maintained between 50°C and 55°C, and the unrestrained animals allow place on the plate until a nocifensive behavior is

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observed [24]. The nocifensive response includes forepaw withdrawal or licking, hind paw withdrawal or licking, stamping, leaning, or jumping. Among all these behaviors, hind paw withdrawal is more reliable as the forepaw is involved in other activities like grooming, due to which the forepaw will not be in constant contact with the hot plate. If the animal shows no reactivity, then it should be removed after a certain time point, designated by the researcher, before any tissue damage to the animal occurs. In contrast, a dynamic hot plate uses an increasing temperature ramp, starting from the lowest, nonnoxious, temperature ( T genotypes and wound severity. Int J Low Extrem Wounds 2014;13:94–102. [5] Singh K, Agrawal NK, Gupta SK, Mohan G, Chaturvedi S, Singh K. Decreased expression of heat shock proteins may lead to compromised wound healing in type 2 diabetes mellitus patients. J Diabetes Complicat 2015;29:578–88. [6] Singh K, Agrawal NK, Gupta SK, Mohan G, Chaturvedi S, Singh K. Increased expression of endosomal members of toll-like receptor family abrogates wound healing in patients with type 2 diabetes mellitus. Int Wound J 2016;13:927–35. [7] Singh K, Agrawal NK, Gupta SK, Sinha P, Singh K. Increased expression of TLR9 associated with proinflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing. J Diabetes Complicat 2016;30:99–108. [8] Singh K, Agrawal NK, Gupta SK, Mohan G, Chaturvedi S, Singh K. Genetic and epigenetic alterations in toll like receptor 2 and wound healing impairment in type 2 diabetes patients. J Diabetes Complicat 2015;29:222–9. [9] Sen CK. Human wounds and its burden: an updated compendium of estimates. Adv Wound Care 2019;8:39–48. [10] Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK, Gottrup F, Gurtner GC, Longaker MT. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen 2009;17:763–71. [11] Singh K, Singh K. Carcinogenesis and diabetic wound healing: evidences of parallelism. Curr Diabetes Rev 2015;11:32–45. [12] Singh K, Pal D, Sinha M, Ghatak S, Gnyawali SC, Khanna S, Roy S, Sen CK. Epigenetic modification of MicroRNA-200b contributes to diabetic vasculopathy. Mol Ther 2017;25:2689–704. [13] Mazzio EA, Soliman KF. Basic concepts of epigenetics: impact of environmental signals on gene expression. Epigenetics 2012;7:119–30. [14] Esteller M. CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 2002;21:5427–40. [15] Ling C, Ronn T. Epigenetics in human obesity and type 2 diabetes. Cell Metab 2019;29:1028–44. [16] Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010;28:1057–68. [17] Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology 2013;38:23–38. [18] Wu X, Zhang Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 2017;18:517–34. [19] Dakhlallah DA, Wisler J, Gencheva M, Brown CM, Leatherman ER, Singh K, Brundage K, Karsies T, Dakhlallah A, Witwer KW, Sen CK, Eubank TD, Marsh CB. Circulating extracellular vesicle content reveals de novo DNA methyltransferase expression as a molecular method to predict septic shock. J Extracell Vesicles 2019;8:1669881. [20] Bird A, Taggart M, Frommer M, Miller OJ, Macleod D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. Cell 1985;40:91–9. [21] Saxonov S, Berg P, Brutlag DL. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci USA 2006;103:1412–7.

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[22] Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987;196: 261–82. [23] Willmer T, Johnson R, Louw J, Pheiffer C. Blood-based DNA methylation biomarkers for type 2 diabetes: potential for clinical applications. Front Endocrinol 2018;9:744. [24] Pradhan S, Bacolla A, Wells RD, Roberts RJ. Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation. J Biol Chem 1999;274:33002–10. [25] Hermann A, Goyal R, Jeltsch A. The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites. J Biol Chem 2004;279: 48350–9. [26] Ginder GD, Williams Jr. DC. Readers of DNA methylation, the MBD family as potential therapeutic targets. Pharmacol Ther 2018;184:98–111. [27] Mortusewicz O, Schermelleh L, Walter J, Cardoso MC, Leonhardt H. Recruitment of DNA methyltransferase I to DNA repair sites. Proc Natl Acad Sci USA 2005;102:8905–9. [28] Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247–57. [29] Xie S, Wang Z, Okano M, Nogami M, Li Y, He WW, Okumura K, Li E. Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 1999;236:87–95. [30] Rasmussen KD, Helin K. Role of TET enzymes in DNA methylation, development, and cancer. Genes Dev 2016;30:733–50. [31] Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009;324:930–5. [32] Lio CJ, Rao A. TET enzymes and 5hmC in adaptive and innate immune systems. Front Immunol 2019;10:210. [33] Wade PA. Methyl CpG binding proteins: coupling chromatin architecture to gene regulation. Oncogene 2001;20:3166–73. [34] Wade PA, Wolffe AP. ReCoGnizing methylated DNA. Nat Struct Biol 2001;8:575–7. [35] Hendrich B, Tweedie S. The methyl-CpG binding domain and the evolving role of DNA methylation in animals. Trends Genet 2003;19:269–77. [36] Fatemi M, Wade PA. MBD family proteins: reading the epigenetic code. J Cell Sci 2006;119:3033–7. [37] Baubec T, Ivanek R, Lienert F, Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell 2013;153:480–92. [38] Villeneuve LM, Natarajan R. Epigenetics of diabetic complications. Expert Rev Endocrinol Metab 2010;5:137–48. [39] Goll MG, Bestor TH. Histone modification and replacement in chromatin activation. Genes Dev 2002;16:1739–42. [40] Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. [41] Greer EL, Shi Y. Histone methylation: a dynamic mark in health, disease and inheritance. Nat Rev Genet 2012;13:343–57. [42] Wang Y, Hou C, Wisler J, Singh K, Wu C, Xie Z, Lu Q, Zhou Z. Elevated histone H3 acetylation is associated with genes involved in T lymphocyte activation and glutamate decarboxylase antibody production in patients with type 1 diabetes. J Diabetes Investig 2019;10:51–61. [43] Vakoc CR, Sachdeva MM, Wang H, Blobel GA. Profile of histone lysine methylation across transcribed mammalian chromatin. Mol Cell Biol 2006;26:9185–95. [44] Spallotta F, Cencioni C, Straino S, Sbardella G, Castellano S, Capogrossi MC, Martelli F, Gaetano C. Enhancement of lysine acetylation accelerates wound repair. Commun Integr Biol 2013;6:e25466. [45] Davegardh C, Garcia-Calzon S, Bacos K, Ling C. DNA methylation in the pathogenesis of type 2 diabetes in humans. Mol Metab 2018;14:12–25. [46] Hall E, Dekker Nitert M, Volkov P, Malmgren S, Mulder H, Bacos K, Ling C. The effects of high glucose exposure on global gene expression and DNA methylation in human pancreatic islets. Mol Cell Endocrinol 2018;472:57–67. [47] Al-Haddad R, Karnib N, Assaad RA, Bilen Y, Emmanuel N, Ghanem A, Younes J, Zibara V, Stephan JS, Sleiman SF. Epigenetic changes in diabetes. Neurosci Lett 2016;625:64–9.

Epigenetics of diabetic wound healing

[48] Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA 2000;97:12222–6. [49] Wang M, Smith K, Yu Q, Miller C, Singh K, Sen CK. Mitochondrial connexin 43 in sex-dependent myocardial responses and estrogen-mediated cardiac protection following acute ischemia/reperfusion injury. Basic Res Cardiol 2019;115:1. [50] El-Osta A, Brasacchio D, Yao D, Pocai A, Jones PL, Roeder RG, Cooper ME, Brownlee M. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J Exp Med 2008;205:2409–17. [51] Turner BM. Cellular memory and the histone code. Cell 2002;111:285–91. [52] Dhliwayo N, Sarras Jr. MP, Luczkowski E, Mason SM, Intine RV. Parp inhibition prevents ten-eleven translocase enzyme activation and hyperglycemia-induced DNA demethylation. Diabetes 2014;63:3069–76. [53] Palsamy P, Bidasee KR, Ayaki M, Augusteyn RC, Chan JY, Shinohara T. Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts. Free Radic Biol Med 2014;72:134–48. [54] Ezhkova E, Lien WH, Stokes N, Pasolli HA, Silva JM, Fuchs E. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev 2011;25:485–98. [55] Shaw T, Martin P. Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes. EMBO Rep 2009;10:881–6. [56] Rafehi H, El-Osta A, Karagiannis TC. Genetic and epigenetic events in diabetic wound healing. Int Wound J 2011;8:12–21. [57] Sinha M, Sen CK, Singh K, Das A, Ghatak S, Rhea B, Blackstone B, Powell HM, Khanna S, Roy S. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue. Nat Commun 2018;9:936. [58] Malissen B, Tamoutounour S, Henri S. The origins and functions of dendritic cells and macrophages in the skin. Nat Rev Immunol 2014;14:417–28. [59] Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij S, Peri F, Wilson SW, Ruhrberg C. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood 2010;116:829–40. [60] Ogle ME, Segar CE, Sridhar S, Botchwey EA. Monocytes and macrophages in tissue repair: implications for immunoregenerative biomaterial design. Exp Biol Med (Maywood) 2016;241:1084–97. [61] Yan J, Tie G, Wang S, Tutto A, DeMarco N, Khair L, Fazzio TG, Messina LM. Diabetes impairs wound healing by Dnmt1-dependent dysregulation of hematopoietic stem cells differentiation towards macrophages. Nat Commun 2018;9:33. [62] Gallagher KA, Joshi A, Carson WF, Schaller M, Allen R, Mukerjee S, Kittan N, Feldman EL, Henke PK, Hogaboam C, Burant CF, Kunkel SL. Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes. Diabetes 2015;64:1420–30. [63] Li B, Wang JH. Fibroblasts and myofibroblasts in wound healing: force generation and measurement. J Tissue Viability 2011;20:108–20. [64] Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am J Pathol 2003;162:303–12. [65] Gallego-Perez D, Pal D, Ghatak S, Malkoc V, Higuita-Castro N, Gnyawali S, Chang L, Liao WC, Shi J, Sinha M, Singh K, Steen E, Sunyecz A, Stewart R, Moore J, Ziebro T, Northcutt RG, Homsy M, Bertani P, Lu W, Roy S, Khanna S, Rink C, Sundaresan VB, Otero JJ, Lee LJ, Sen CK. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nat Nanotechnol 2017;12:974–9. [66] Park LK, Maione AG, Smith A, Gerami-Naini B, Iyer LK, Mooney DJ, Veves A, Garlick JA. Genomewide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts. Epigenetics 2014;9:1339–49.

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[67] Singh K. Cutaneous epithelial to mesenchymal transition activator ZEB1 regulates wound angiogenesis and closure in a glycemic status-dependent manner. Diabetes 2019;68(11):2175–90. https://doi.org/ 10.2337/db19-0202. [68] Tonnesen MG, Feng X, Clark RA. Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5:40–6. [69] Hadi HA, Suwaidi JA. Endothelial dysfunction in diabetes mellitus. Vasc Health Risk Manag 2007;3:853–76. [70] Khullar M, Cheema BS, Raut SK. Emerging evidence of epigenetic modifications in vascular complication of diabetes. Front Endocrinol 2017;8:237. [71] Rajasekar P, O’Neill CL, Eeles L, Stitt AW, Medina RJ. Epigenetic changes in endothelial progenitors as a possible cellular basis for glycemic memory in diabetic vascular complications. J Diabetes Res 2015;2015:436879. [72] Ti D, Li M, Fu X, Han W. Causes and consequences of epigenetic regulation in wound healing. Wound Repair Regen 2014;22:305–12. [73] Zhong Q, Kowluru RA. Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon. J Cell Biochem 2010;110:1306–13. [74] Rakyan VK, Down TA, Balding DJ, Beck S. Epigenome-wide association studies for common human diseases. Nat Rev Genet 2011;12:529–41. [75] Gomes FS, de Souza GF, Nascimento LF, Arantes EL, Pedro RM, Vitorino DC, Nunez CE, Melo Lima MH, Velloso LA, Araujo EP. Topical 5-azacytidine accelerates skin wound healing in rats. Wound Repair Regen 2014;22:640–6. [76] Kim SY, Hong SW, Kim MO, Kim HS, Jang JE, Leem J, Park IS, Lee KU, Koh EH. S-adenosyl methionine prevents endothelial dysfunction by inducing heme oxygenase-1 in vascular endothelial cells. Mol Cells 2013;36:376–84. [77] Spijkerman AM, Smulders YM, Kostense PJ, Henry RM, Becker A, Teerlink T, Jakobs C, Dekker JM, Nijpels G, Heine RJ, Bouter LM, Stehouwer CD. S-adenosylmethionine and 5-methyltetrahydrofolate are associated with endothelial function after controlling for confounding by homocysteine: the Hoorn study. Arterioscler Thromb Vasc Biol 2005;25:778–84. [78] Spallotta F, Cencioni C, Straino S, Nanni S, Rosati J, Artuso S, Manni I, Colussi C, Piaggio G, Martelli F, Valente S, Mai A, Capogrossi MC, Farsetti A, Gaetano C. A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair. J Biol Chem 2013;288:11004–12. [79] Saito A, Yamashita T, Mariko Y, Nosaka Y, Tsuchiya K, Ando T, Suzuki T, Tsuruo T, Nakanishi O. A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci USA 1999;96:4592–7.

CHAPTER 10

Role of lipid mediators in diabetic wound healing Dayanjan S. Wijesinghe

Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Richmond, VA, United States

1. Introduction Lipids are a highly heterogeneous group of biomolecules [1, 2]. They are amphipathic to hydrophobic in nature and as such dissolve very poorly in aqueous media. In aqueous environments, most lipids tend to self-assemble into either micelles or bilayer structures such as vesicles [2]. Lipids in these structures arrange themselves such that hydrophobic portions stack next to each other to minimize interactions with water [2]. This selfassembling nature has resulted in lipids being the predominant biomolecules to make up cellular and subcellular membranes as well as extracellular vesicles. Such membranes are also self-healing to a large extent thereby minimizing loss of intracellular content due to accidental damage [2]. Due to their heterogeneity, the grouping of lipids is challenging. However, based on structural similarity, lipids have been classified into eight different categories. Namely, fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, saccharolipids, polyketides, prenol lipids, with each category further divided into classes and subclasses [3]. Lipids were long thought to have only two functions, provide structure to membranes and to act as energy reserves. An ever-increasing body of evidence fueled by advances in lipidomic technology are now pointing to lipids with important roles in inter- and intracellular signaling [4, 5]. The fact that the lipids are also structural components of membranes have resulted in multiple modes by which lipids can undertake signaling functions. The composition of the lipidome will determine the three-dimensional structure of membranes [6]. As the behavior of proteins and enzymes is affected by this 3D structure, any alterations to it can directly result in changes to the activity of associated proteins and enzymes. A majority of signaling receptors are membrane associated. As such, changes in membrane lipid composition have the capacity to greatly affect cell signaling. A case in point are the lipid rafts, a relatively sturdy platform made of more hydrophobic lipids floating in a more loose arrangement of other lipids [7]. Those signaling proteins sequestered within a lipid raft will interact for longer lengths of time and thereby altering the length and magnitude of signaling response [7].

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00010-1

© 2020 Elsevier Inc. All rights reserved.

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While lipids have the capacity to cause signaling changes via compositional changes to membranes, certain lipids can also act as direct signaling molecules [5]. However, lipid signaling is functionally very different to most other small molecule signaling modalities. While most nonlipid signaling molecules can be presynthesized and stored until needed, this is not possible with signaling lipids. This is due to the fact lipids can freely diffuse through any lipid membrane within which they are contained. As such, lipid signaling molecules are synthesized on demand, often at or in proximity to the site of action. Often, the precursors of the bioactive lipid molecule are present as components of other membrane lipids close to their potential sites of action, ready to be liberated and processed into the active form when needed. Other signaling lipids are found tightly bound to carrier proteins ready to be liberated when needed. The amount of lipid signaling molecules present at any time is determined by the difference between their rate of synthesis and the rate of inactivation of the bioactive lipid molecule. Due to this more complicated nature of lipid signaling, the process is often more susceptible to disruption via external influences. The first reports of lipids as a source of signaling molecules were published in 1953 and 1958 by Lowell and Mabel Hokin who demonstrated that the secretion of amylase from pancreas slices in response acetylcholine also caused the incorporation of 32P into phosphoinositides [8]. Dubbed the “PI Effect” these seminal studies resulted in the exploration of other lipid species and lipid signaling events [8]. Many additional signaling lipids species have been discovered since then. Examples include ceramides and sphingosines and their phosphates, lysophosphatidic acid, eicosanoids, protectins, resolvins, maresins, platelet-activating factors, endocannabinoids, steroidal hormones, retinoic acids and their derivatives, etc. A majority of the signaling lipids act via G-protein-coupled receptors (GPCR), while several others act via nuclear receptors such as peroxisome proliferator-activating receptors (PPAR). Lipid-signaling events are involved in a multitude of biological functions and as such are also of diagnostic and prognostic relevance [9–14]. Several published studies demonstrate roles for lipids in multiple cellular and biological mechanisms that are relevant to the wound-healing process. For example, lipid autacoids such as the metabolites of di-homo gamma linolenic acid (20:3) arachidonic acid (20:4), eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) as well as ceramide-1-phosphate, sphingosine, sphingosine-1-phosphate (S1P), and sphinganine have proven biological roles in the modulation of biological processes relevant to wound healing. These include the onset of inflammation [15] and its resolution [16], neutrophil chemotaxis [17], and its inhibition [18], enhancement of monocyte chemotaxis [19], and enhancement of phagocytosis of apoptotic neutrophils by nonphlogistic monocytes [18]. Additionally, lipids are also found to have roles in the migration and proliferation of fibroblasts [20–23] and enhanced angiogenesis by endothelial cells [24]. As such, changes to bioactive lipid synthesis often result in alterations to the rate of healing of cutaneous wounds [25].

Role of lipid mediators in diabetic wound healing

Diabetes mellitus (DM) is often the most frequent endogenous cause of lipid metabolism-disorders [26–29]. As such, a significant component of the delayed wound healing observed among diabetics may be attributed to diabetes-associated alterations in lipid metabolism and signaling. Diabetic wounds are characterized by poor perfusion, substandard neutrophil response as well as poor fibroblast and keratinocyte migration. Lipids are known to play a role in each of these healing deficits. Summarized is how diabetes affects the natural metabolism of each lipid category and how that in turn may affect the healing of cutaneous wounds among patients with diabetes.

2. Fatty acyls Fatty acyls are a diverse group of molecules either synthesized in situ by a series of chain elongation and desaturation or acquired by diet and then modified as needed such as the Omega-3 and Omega-6 lipids. These lipids are characterized by a carboxyl group on one end of the acyl chain with additional modifications to contain other atoms such as oxygen, nitrogen, cyclic structures, etc. Common signaling mediators in this category include eicosanoids, protectins, resolvins, maresins, nitrolipids, etc. Among the eicosanoids, prostaglandins, leukotrienes, thromboxanes, hydroxyeicosatetraenoic (HETE) acids, epoxyeicosatrienoic (EET) acids, etc. are all involved in multiple cellular processes associated with wound healing. These include coagulation, inflammation, keratinocytes and fibroblast migration and proliferation, extracellular matrix production to name a few [30]. Protectins, resolvins, and maresins are a relatively new class of lipids identified that are oxygenated metabolites arising primarily from Omega-3 polyunsaturated lipids such as docosahexaenoic acids and eicosapentaenoic acids and have now been established as active endogenous resolvers of inflammation [31]. Many of these bioactive lipids have been reported to be present in human skin. A recent study by Kendall et al. identified 18 prostaglandins, 12 hydroxy fatty acids, 9 endocannabinoids, and N-acylethanolamides indicating potential roles for these bioactive lipids in the wound-healing process [32] (Fig. 1).

2.1 Prostaglandins Several published studies have now established dysregulated eicosanoid signaling to be associated with impaired wound healing among DM patients. Early studies demonstrated a marked loss in prostaglandin synthesis in diabetes-impaired wound tissue of ob/ob mice attributed to a decrease in cyclooxygenase-1 expression that is not compensated by a corresponding increase in cyclooxygenase-2 expression [33]. Specific eicosanoids affected included prostaglandin E2 (PGE2) prostaglandin D2 (PGD2) and also prostacyclin (prostaglandin I2, PGI2) [33]. Prostacyclin is associated with increased perfusion around the wound tissue due to vasodilation and subsequent increase in angiogenesis, both of which are negatively affected in diabetic wound healing. Additional evidence for the need for

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Fig. 1 Schematic outline of the production of bioactive fatty acyl of relevance to cutaneous wound healing. PLA2, phospholipase A2; DAG lipase, diacylglycerol lipase; PLD, phospholipase D; MAG lipase, monoacylglycerol lipase; FAAH, fatty acid amide hydrolase; PUFA, polyunsaturated fatty acid; LOX, lipoxygenase; CYP 450, cytochrome p450; COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid.

Role of lipid mediators in diabetic wound healing

prostacyclin in diabetic wound healing was established in a previous study where the application of a stable analog of prostacyclin to the wound tissue of diabetic mice demonstrated improved perfusion, increased angiogenesis, and subsequent faster healing of the wound [34]. The requirement for normal levels of prostaglandins in diabetic wound healing was also demonstrated when the inhibition of prostaglandin transporter led to enhanced vascularization and accelerated re-epithelialization of cutaneous wounds in a type 1 DM mouse model [35]. Downstream metabolites of PGD2 such as 15-deoxy-Delta (12, 14) prostaglandin J2 (15d-PGJ2) are known activators of peroxisome proliferator-activated receptor γ (PPARγ) [36]. The transition of macrophages from proinflammatory to healing-associated phenotypes is associated with the upregulation of PPARγ. However, this upregulation is hindered in diabetic wounds due to the presence of high levels of IL-1β. Additionally, decreased incidence of PGD2 reported in diabetic wounds also indicates a decreased level of 15d-PGJ2 indicating severely limited PPARγ signaling. Confirming this are new findings that demonstrate the exogenous addition of 15d-PGJ2 to decrease the proinflammatory macrophage phenotype in diabetic wounds leading to an increase in the expression or prohealing genes [37]. These findings indicate dysregulated prostaglandin signaling to be a significant cause to delayed healing in diabetic wounds.

2.2 Leukotrienes and cytochrome p450 metabolites Diabetic wounds are characterized by a high inflammatory status with excessive neutrophil and macrophage recruitment. A primary driver of recruitment of these inflammatory cells are the leukotrienes. Diabetic mice have been demonstrated to produce higher levels of leukotrienes in the skin correlating with larger nonhealing wounds, excessive neutrophil migration, and uncontrolled collagen deposition [38]. This unbalanced inflammatory response was observed to be overcome via the inhibition of B leukotriene receptor 1 [38]. Conversely, a decreased expression of cytochrome P450 epoxygenases is also observed in diabetic wounds [39]. Treatment of these wounds with the product of this enzyme, specifically 11,12 EET, resulted in decreased neutrophil and macrophage infiltration to the wound site with concomitant decreases in MMP-9 expression and increased collagen accumulation [39]. These lines of studies indicate the potential for modulating the unfavorable immune cell response and associated wound-healing complications via the modulation of associated lipid mediators.

2.3 Endocannabinoids Endocannabinoids are another important group of signaling lipids. Ongoing studies are elucidating new roles for endocannabinoids in the wound-healing process. The two major groups of endocannabinoids, namely, N-acylethanolamides (N-arachidonylethanolamide also called anandamide being the most studied) and

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2-arachidonyl glycerol, signal via the cannabinoid receptors 1 and 2 (CB1 and CB2). Both CB1 and CB2 are reported to be present in human skin. CB1 was identified to be present on keratinocytes, single epidermal nerve fibers, small unmyelinated subepidermal nerves, and large dermal unmyelinated nerves [40]. Presence of CB2 was identified in large myelinated nerve fiber bundles of the superficial and deep reticular dermis, at the dermal-epidermal junction and occasionally within the epidermis and small nerve fibers associated with hair follicles [40]. Additionally, CB2 was also identified in basal keratinocytes of the epidermis and resident skin macrophages and mast cells [40]. Considering the fact that 9 endocannabinoids and N-acylethanolamides have also been identified in the human skin [32], it is likely that a robust endocannabinoid signaling network is present in human skin with likely roles in wound healing [41, 42]. One of the identified functions of the cutaneous endocannabinoid system includes the regulation of cutaneous inflammation with the endocannabinoids acting as antiinflammatory agents [42]. Other functions include the proliferation and differentiation of epidermal keratinocytes. An additional receptor target of the endocannabinoid system, specifically anandamide, is the transient receptor potential vanilloid receptor 1 (TRPV1). TRPV1 is primarily involved in heat and paint sensations felt by the skin [43]. With respect to the involvement of the skin endocannabinoid, system and its role in diabetic wound healing, very few studies have so far been undertaken. However, insights can be gleaned from existing studies. Current evidence from multiple studies indicates that an overactive endocannabinoid system may contribute to the development of diabetes [44]. The elevated CB1 receptor signaling has been shown to enhance inflammatory and oxidative processes leading to both neuronal and microvessel damage and may contribute to some aspects of diabetic neuropathy [44]. Studies also demonstrate counts of TRPV1 containing nerve fibers to be significantly lower in both the epidermis and the subepidermis of the human skin [43]. Despite these findings, as of now, information with respect to the role of the endocannabinoid system in human skin wound healing in general and diabetic wound healing specifically is lacking and is a critical gap in our knowledge with respect to the role of lipid mediators in diabetic wound healing.

3. Glycerolipids Diacylglycerol (DAG) is a glycerolipid with two fatty acyl chains attached to the central glycerol back bone [45]. It is also a potent second messenger signaling lipid often produced from the phospholipid phosphatidylinositol 4,5-bisphosphate by the enzyme phospholipase-C [45]. However, there are additional biosynthetic pathways for formation of DAG such as via the hydrolysis of triacylglycerol lipids by neutral triglyceride lipase, as a product of formation of sphingomyelin by sphingomyelin synthase, etc. [45]. DAG is a potent activator of protein kinase C (PKC) via its recruitment from the cytosol to membranes [45]. Interestingly DAG is formed within a few seconds of

Role of lipid mediators in diabetic wound healing

wounding at a single-cell level and the biosynthetic process appears to be via the sphingomyelin synthase pathway [46]. Additionally, inhibition of this early DAG synthesis significantly affects the ability of wounds to heal at a cellular level indicating DAG signaling is a required process for wound healing [46]. Early production of DAG was also identified as being needed for the sealing of injured plasma membranes of individual cells [46]. It is worthwhile noting that dysregulated DAG synthesis leading to intracellular DAG accumulation and or ectopic DAG accumulation is closely associated with dysregulated cell signaling in response to IR [45]. Dysregulation of DAG mediated PKC signaling is now considered a major driver of impaired diabetic wound healing [47]. Synthesis of DAG analogs for exogenous application and activation of PKC signaling is an active area of research in diabetic wound healing [47]. However, more studies are needed to properly elucidate the role of DAGs in diabetic wound healing.

4. Glycerophospholipids 4.1 Phosphatidylinositol phosphates Phosphatidylinositol phosphates refers to a class of lipids with 1 (PIP), 2 (PIP2), or 3 (PIP3) phosphate groups attached to inositol portion of phosphatidylinositol lipids. Out of this broad class of lipids, PIP2 and PIP3 have recently been demonstrated to be very early responders to wounds at a cellular level with changes occurring within a minute of wounding [46]. Other PIPs are also involved in signaling associated with wound healing. For example, EGF is a well-reputed cytoprotective growth factor with roles in wound healing, and its activity is driven by the agonistic stimulation of the phosphatidylinositol 3-kinase (PI3K)-Akt axis by EGFR phosphorylation [48]. Similarly, some aspects of platelet-derived growth factor signaling are also mediated by PI3K and the resultant PI3P [49]. As such, PI3K/AKT signaling axis and, hence, phosphatidylinositol phosphate lipids are heavily involved in signaling during normal wound healing [50]. While the PI3K/AKT signaling axis is important for normal wound healing, the same signaling axis has been implicated in the development of diabetes and its complications [51]. Of note, PI3K/AKT signaling axis signaling is altered during DM resulting in increased cellular apoptosis, decreased proliferation and thus delayed wound healing [52]. PI3K activation is also one of the major downstream signaling pathways in response to activation of the insulin receptor by insulin. Insulin receptors are expressed in the skin, especially in keratinocytes and regulate their migration, proliferation, and differentiation [53]. The PI3K/AKT signaling axis appears to be blunted in diabetic wound healing and exogenous application of topical insulin appear to reverse this and improve the healing of diabetic wounds [53]. All in all, these established studies demonstrate the relevance of phosphatidylinositol phosphate lipids in diabetic and normal wound healing.

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4.2 Lysophosphatidic acid Lysophaphatidic acid (LPA) is a single acyl chain glycerophospholipid demonstrating bioactivity in cell signaling. While there are many different LPA species varying in the number of carbons of the acyl chain, double bonds and their position and the acyl chain position, the most potent and well-studied LPA species is 1-oleoyl-LPA (18:1 LPA). Multiple biosynthetic pathways regulate the synthesis and catabolism of LPA [54, 55]. LPA is implicated in the regulation of wound healing. Specifically, LPA has been implicated in the proliferation and migration of endothelial cells, smooth muscle cells, and fibroblasts [54]. LPA was also found to be present in blister wound fluids and to directly affect the migration of cultured keratinocytes [56]. Topical application of LPA has been demonstrated to improve wound healing in vivo [57]. LPA signals through LPA receptors 1–6, which are class A rhodopsin like GPCRs coupled to one or more of the four heterotrimeric Gα proteins, i.e., G12/13, Gq/11, Gi/o, and Gs [55]. Human mast cells have been demonstrated to undergo LPA signaling via LPAR-5 receptor to induce the production of the macrophage inflammatory protein MIP 1-β [58]. LPA was observed to signal via both LPAR-1 and LPAR-5 in human keratinocytes and to be involved in the expression of filaggrin, thus contributing to the formation of stratum corneum [59]. LPA receptor signaling has been implicated in obesity and insulin resistance [60] and specifically in diabetic nephropathy [61]. Blocking LPAR-1 signaling was observed to inhibit diabetic nephropathy in a mouse model of diabetes [62]. Elevated levels of LPA as well as one of its biosynthetic enzymes, namely autotaxin, are found to be elevated in the serum of older diabetic patients [63]. By inference, this indicates a higher-thannormal concentration of LPA at the very early stages of diabetic wound healing and consequently greater-than-average immune response by macrophages due to the elevated production of MIP 1-β by resident mast cells. While this relationship is inferred from existing literature, to this date no detailed studies have been undertaken investigating LPA signaling with respect to diabetic wound healing. This is again a critical gap in our knowledge with respect to the role of lipid mediators in wound healing.

5. Sphingolipids Sphingolipids are a category of lipids consisting of aliphatic amino alcohols and distinguished from other lipids by the presence of a primary or secondary amine group. First discovered in brain extracts and named after the mythological sphynx due to their enigmatic nature at the time of discovery, sphingolipids are now known to be important structural and signaling lipids with high relevance to wound healing. This lipid category consists of several of lipids, including sphingoid bases and their phosphates, ceramides and their phosphates, sphingomyelins, glycosphingolipids, etc. [64, 65] All cells in our body have the capacity to synthesize ceramides and most other sphingolipids via the de novo

Role of lipid mediators in diabetic wound healing

ceramide biosynthetic pathway [66]. Additionally, existing complex sphingolipids can also be recycled via the salvage pathway to regenerate ceramides [66]. With respect to cutaneous wound healing, sphingolipids are involved in multiple functions.

5.1 Ceramides and their phosphates A major portion of the structural barrier of the skin consists of specialized ceramide species [67]. In fact, there are 12 separate classes of ceramides, most of which are only found in the stratum corneum [67]. Any changes to the biosynthetic processes that alter the synthesis of these ceramides result in incomplete wound healing with an impaired barrier function. A recent study demonstrated that patients with type 2 DM have significantly lower skin ceramide content with an associated reduction in skin barrier function and antimicrobial defense [68]. Conversely, ceramides are known inducers of apoptosis in neutrophils [69], fibroblasts [70], and in keratinocytes [71]. Plasma ceramides are often found elevated among patients with DM [72]. As such, it is likely that the early wound environment of diabetic patients would experience the presence of higher-than-normal ceramide levels. Considering the impact of ceramide on cellular functions, this early exposure to high levels of ceramides may have a significant role with respect to delayed wound healing among DM patients. Many additional functions are associated with sphingolipids in relation to wound healing and is described in detail elsewhere [67, 71]. Following the formation of a wound, ceramide-1-phosphate has been demonstrated to act as a master regulator of eicosanoid synthesis and fibroblast migration [73].

5.2 Sphingosine-1-phosphate S1P is a highly bioactive lipid capable of inducing a wide range of biological responses. These include cell proliferation and differentiation, survival, motility, angiogenesis, to name a few [74]. As such, it is unsurprising that S1P is one of the bioactive lipids that is heavily involved in the wound-healing process [75–78]. S1P is also one of the very few lipids that are sufficiently hydrophilic to enable their storage for release as needed. As such, S1P is stored in platelets and are released upon platelet activation during the hemostasis stage of wound healing. S1P signaling is mediated via a family of five specific GPCRs named S1P receptors 1–5 (S1PR 1–5) all with low nM Kds for S1P binding [74]. S1P has been demonstrated to facilitate wound healing by increasing angiogenesis and more efficient inflammatory cell recruitment with the final healed wound demonstrating less scar formation [77]. Several studies demonstrate that S1P leads to the improvement of wound healing among diabetic rats [76] and in diabetic mice [75]. Significant enhancements were observed in the formation of granulation tissue and angiogenesis in wounded diabetic mice receiving injections of S1P at the wound site [75]. In addition to the direct benefits of S1P, this potent signaling lipid also has the ability to influence the signaling of other important lipid mediators. For example, S1P is known to upregulate the expression

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of COX-2 expression with a concomitant increase in the production of PGE2 [79]. As such, changes to the levels of S1P also have the capability to alter prostaglandin synthesis. As such, promotion of S1P signaling appears to improve the rate of diabetic wound healing.

5.3 Gangliosides Gangliosides are sialic acid containing glycosphingolipids [80]. GM3 (M referring to monosialic gangliosides) was observed to be the most prominent ganglioside in human skin followed by GM2, GM1, and GD3 (D refers to disialic gangliosides) [81]. In human skin, gangliosides are one of the lipids that mediate growth factor signaling [82]. A growing body of evidence suggests that GM3 mediates TNF-α and glucose-induced IR and type 2 DM [83]. GM3 was recently demonstrated to be a driver of impaired cutaneous wound healing by directly suppressing the cutaneous IGF-1/insulin signaling axis [83]. Depletion of GM3 was observed to reverse impaired wound healing in diabetic mice [84]. These studies demonstrate a significant role for the ganglioside GM3 in diabetic wound healing and identify it as target for drug development for the treatment of diabetic wounds.

6. Sterol lipids Sterol lipids constitute a large and diverse group of lipids, including cholesterol, cholesterol esters, steroidal hormones such as estrogens and their derivatives and androgens and their derivatives, bile acids, and many other lipid classes. Cholesterols and cholesterol esters are essential components of the permeability barrier of the stratum corneum, the proper formation of which is an essential part of full and complete wound healing [67]. Suppression of cholesterol synthesis is demonstrated to inhibit the growth of human fibroblasts [85]. Cholesterol accumulation is associated with an elevated inflammatory response [85]. The role of steroidal hormones with respect to wound healing has been well established [86]. The high incidence of chronic and nonhealing wounds among elderly males is associated with reduced levels of estrogen and the maintenance of high levels of androgen hormones [87]. Additionally, application of topical estrogen has been demonstrated to be beneficial in both males and females [87]. Bile acids are also known to have roles in wound healing. Specifically the bile acid deoxycholate and ursodeoxycholate has been demonstrated to regulate colonic epithelial wound healing [88]. However, little to no information is available with respect to bile acids in cutaneous wound healing. With respect to diabetic wound healing, the estrogen has been demonstrated to improve healing in diabetic mice and the beneficial effect of estrogen is attributed to the increasing function of multiple bone-marrow-derived progenitor cells [87]. Conversely, selective blockage of estrogen receptor beta was shown to improve diabetic

Role of lipid mediators in diabetic wound healing

wound healing [89] indicating the observed beneficial effects on healing in diabetic mice may be through an estrogen receptor independent pathway. Topical application of 17β-estradiol has been shown to improve diabetic wound healing by decreasing matrix metalloproteinase activity [90]. 17β-estradiol has also been demonstrated to be protective toward mesenchymal stem cells against high glucose-induced mitochondrial reactive oxygenase generation [91]. Conversely adequate testosterone levels are also required for normal wound healing [92], and a recently published study demonstrates enhanced wound healing in diabetic rats in response to 5α-dihydrotestosterone [93]. While a substantial body of literature has investigated the role of steroidal hormones in diabetic wound healing, there is currently very little information with respect to other sterol lipid mediators and any roles they may play during diabetic wound healing.

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[57] Balazs L, Okolicany J, Ferrebee M, Tolley B, Tigyi G. Topical application of the phospholipid growth factor lysophosphatidic acid promotes wound healing in vivo. Am J Physiol Regul Integr Comp Physiol 2001;280(2):R466–72. https://doi.org/10.1152/ajpregu.2001.280.2.R466. [58] Lundequist A, Boyce JA. LPA5 is abundantly expressed by human mast cells and important for lysophosphatidic acid induced MIP-1β release. PLoS One 2011;6(3). e18192. https://doi.org/10.1371/ journal.pone.0018192. [59] Sumitomo A, Siriwach R, Thumkeo D, et al. LPA induces keratinocyte differentiation and promotes skin barrier function through the LPAR1/LPAR5-RHO-ROCK-SRF axis. J Invest Dermatol 2019;139(5):1010–22. https://doi.org/10.1016/j.jid.2018.10.034. [60] D’Souza K, Paramel GV, Kienesberger PC. Lysophosphatidic acid signaling in obesity and insulin resistance. Nutrients 2018;10(4). https://doi.org/10.3390/nu10040399. [61] Lee JH, Kim D, Oh YS, Jun H-S. Lysophosphatidic acid signaling in diabetic nephropathy. Int J Mol Sci 2019;20(11):2850. https://doi.org/10.3390/ijms20112850. [62] Li HY, Oh YS, Choi J-W, Jung JY, Jun H-S. Blocking lysophosphatidic acid receptor 1 signaling inhibits diabetic nephropathy in db/db mice, Kidney Int 2017;91(6). https://www.sciencedirect. com/science/article/pii/S0085253816306810?via%3Dihub. (Accessed 30 November 2019). [63] Reeves VL, Trybula JS, Wills RC, Goodpaster BH, Dube JJ, Kienesberger PC, Kershaw EE. Serum autotaxin/ENPP2 correlates with insulin resistance in older humans with obesity, Obesity 2015; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700540/. (Accessed 30 November 2019). [64] Fahy E, Subramaniam S, Brown HA, et al. A comprehensive classification system for lipids. J Lipid Res 2005;46(5):839–62. https://doi.org/10.1194/jlr.E400004-JLR200. [65] Fahy E, Subramaniam S, Murphy RC, et al. Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res 2009;50(Suppl):S9–S14. https://doi.org/10.1194/jlr.R800095-JLR200. [66] Larsen PJ, Tennagels N. On ceramides, other sphingolipids and impaired glucose homeostasis. Mol Metab 2014;3(3):252–60. https://doi.org/10.1016/j.molmet.2014.01.011. [67] Wijesinghe DS, Warncke UO, Diegelmann RF. Human as the ultimate wound healing model: strategies for studies investigating the dermal lipidome. Curr Dermatol Rep 2016;5(4):244–51. https://doi. org/10.1007/s13671-016-0156-3. [68] Kim J-H, Yoon NY, Kim DH, et al. Impaired permeability and antimicrobial barriers in type 2 diabetes skin are linked to increased serum levels of advanced glycation end-product. Exp Dermatol 2018;27 (8):815–23. https://doi.org/10.1111/exd.13466. [69] Seumois G, Fillet M, Gillet L, et al. De novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis. J Leukoc Biol 2007;81(6):1477–86. https://doi.org/10.1189/ jlb.0806529. [70] Ishihara H, Yoshimoto H, Fujioka M, et al. Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor. J Invest Dermatol 2000;115(6):1065–71. https://doi.org/10.1046/j.1523-1747.2000.00180.x. [71] Uchida Y. Ceramide signaling in mammalian epidermis. Biochim Biophys Acta 2014;1841(3):453–62. https://doi.org/10.1016/j.bbalip.2013.09.003. [72] Haus JM, Kashyap SR, Kasumov T, et al. Plasma ceramides are elevated in obese subjects with type 2 diabetes and correlate with the severity of insulin resistance. Diabetes 2009;58(2):337–43. https://doi. org/10.2337/db08-1228. [73] Wijesinghe DS, Brentnall M, Mietla JA, et al. Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts. J Lipid Res 2014;55(7):1298–309. https://doi.org/10.1194/jlr.M048207. [74] Maceyka M, Harikumar KB, Milstien S, Spiegel S. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol 2012;22(1):50–60. https://doi.org/10.1016/j.tcb.2011.09.003. [75] Kawanabe T, Kawakami T, Yatomi Y, Shimada S, Soma Y. Sphingosine 1-phosphate accelerates wound healing in diabetic mice. J Dermatol Sci 2007;48(1):53–60. https://doi.org/10.1016/j. jdermsci.2007.06.002. [76] Yu H, Yuan L, Xu M, Zhang Z, Duan H. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury 2014;45(7):1054–8. https://doi.org/10.1016/j.injury.2014.03.003.

Role of lipid mediators in diabetic wound healing

[77] Aoki M, Aoki H, Mukhopadhyay P, et al. Sphingosine-1-phosphate facilitates skin wound healing by increasing angiogenesis and inflammatory cell recruitment with less scar formation. Int J Mol Sci 2019;20(14). https://doi.org/10.3390/ijms20143381. [78] Vogler R, Sauer B, Kim D-S, Sch€afer-Korting M, Kleuser B. Sphingosine-1-phosphate and its potentially paradoxical effects on critical parameters of cutaneous wound healing. J Invest Dermatol 2003;120 (4):693–700. https://doi.org/10.1046/j.1523-1747.2003.12096.x. [79] V€ olzke A, Koch A, Meyer Zu Heringdorf D, Huwiler A, Pfeilschifter J. Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells. Biochim Biophys Acta 2014;1841(1):11–21. https://doi.org/10.1016/j.bbalip.2013.09.009. [80] Kolter T. Ganglioside biochemistry. Int Sch Res Not 2012;https://doi.org/10.5402/2012/506160. [81] Jacques IP. Distribution of gangliosides in human epidermis, dermis and whole skin. J Clin Exp Dermatol Res 2015;06(03). https://doi.org/10.4172/2155-9554.1000282. [82] Wang X, Rahman Z, Sun P, et al. Ganglioside modulates ligand binding to the epidermal growth factor receptor. J Invest Dermatol 2001;116(1):69–76. https://doi.org/10.1046/j.1523-1747.2001.00222.x. [83] Dam DHM, Paller AS. Gangliosides in diabetic wound healing. Prog Mol Biol Transl Sci 2018;156:229–39. https://doi.org/10.1016/bs.pmbts.2017.12.006. [84] Wang X-Q, Lee S, Wilson H, et al. Ganglioside GM3 depletion reverses impaired wound healing in diabetic mice by activating IGF-1 and insulin receptors. J Invest Dermatol 2014;134(5):1446–55. https://doi.org/10.1038/jid.2013.532. [85] Tall AR, Yvan-Charvet L. Cholesterol, inflammation and innate immunity. Nat Rev Immunol 2015;15(2):104–16. https://doi.org/10.1038/nri3793. [86] Gilliver SC, Ashcroft GS. Sex steroids and cutaneous wound healing: the contrasting influences of estrogens and androgens. Climacteric J Int Menopause Soc 2007;10(4):276–88. https://doi.org/ 10.1080/13697130701456630. [87] Zhuge Y, Regueiro MM, Tian R, et al. The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells. J Vasc Surg 2018;68 (6, Suppl):127S–35S. https://doi.org/10.1016/j.jvs.2018.04.069. [88] Mroz MS, Lajczak NK, Goggins BJ, Keely S, Keely SJ. The bile acids, deoxycholic acid and ursodeoxycholic acid, regulate colonic epithelial wound healing. Am J Physiol Gastrointest Liver Physiol 2018;314(3):G378–87. https://doi.org/10.1152/ajpgi.00435.2016. [89] Sunkari VG, Botusan IR, Savu O, et al. Selective blockade of estrogen receptor beta improves wound healing in diabetes. Endocrine 2014;46(2):347–50. https://doi.org/10.1007/s12020-013-0144-3. [90] Pincus DJ, Kassira N, Gombosh M, et al. 17β-estradiol modifies diabetic wound healing by decreasing matrix metalloproteinase activity. Wounds Compend Clin Res Pract 2010;22(7):171–8. [91] Oh JY, Choi GE, Lee HJ, et al. 17β-Estradiol protects mesenchymal stem cells against high glucoseinduced mitochondrial oxidants production via Nrf2/Sirt3/MnSOD signaling. Free Radic Biol Med 2019;130:328–42. https://doi.org/10.1016/j.freeradbiomed.2018.11.003. [92] Demling RH. The role of anabolic hormones for wound healing in catabolic states, J Burns Wounds 2005;4: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1501119/. (Accessed 30 November 2019). [93] Gonc¸alves RV, Novaes RD, Sarandy MM, et al. 5α-Dihydrotestosterone enhances wound healing in diabetic rats. Life Sci 2016;152:67–75. https://doi.org/10.1016/j.lfs.2016.03.019.

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CHAPTER 11

Role of cytokines and chemokines in wound healing Harrison Stranga, Aditya Kaula, Umang Parikh, Leighanne Masri, Swetha Saravanan, Hui Li, Qi Miao, Swathi Balaji

Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of Surgery, Baylor College of Medicine and Texas Children’s Hospital, Houston, TX, United States

1. Introduction Diabetes mellitus (DM) is a significant health concern affecting millions of patients around the world [1]. It is a metabolic disorder characterized by hyperglycemia, and associated with complications, including neuropathy and impaired wound healing, which predispose to diabetic foot ulcers [2]. Despite existing treatment protocols and novel cellular, genetic and molecular therapies [3–14], successful treatment of diabetic ulcers remains limited. This often results in lower-limb amputations, a major threat with high morbidity and mortality in diabetic patients [15–17]. Successful wound healing is regulated by an interplay of multiple cell types, signaling molecules, growth factors, and extracellular matrix, with cytokines and chemokines intimately involved in the orchestration of wound-healing processes. In contrary, impaired healing observed in diabetic wounds is associated with complex changes in the wound microenvironment and simultaneous disruption of several pathways involved in the healing response. The diabetic wound environment is associated with inflammatory dysregulation [18, 19], intensified by increased proteolysis [20] and resulting in insufficient neovascularization and accumulation of granulation tissue. Along with a severely altered wound microenvironment, deficiencies are also seen on the cellular level in the diabetic condition [21]. The migration and function of these cells are controlled by the local cytokine and chemokine milieu in the wound microenvironment. Chemokines are a class of bioactive signaling molecules, first identified for their role in leukocyte migration, are now known to play key roles in all the phases of healing [22]. Along with regulating resident cell migration, chemokines also play an important role in the recruitment and localization of the circulating inflammatory and angiogenic cells to the wound. In particular, they contribute to the mechanisms governing differentiation of stem cells and macrophages, as well as the polarization of T-cells. Their role in the a

Contributed equally.

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00011-3

© 2020 Elsevier Inc. All rights reserved.

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Wound healing, tissue repair, and regeneration in diabetes

wound-healing process can be as agonists or antagonists depending on the G-proteincoupled receptors they bind and signal to [23]. Understanding how the cytokines and chemokines are locally produced and regulated during wound healing and how the chemokine milieu differs in diabetic wounds may help us identify ways in which we can target chemokine pathways to reduce chronic wound development and promote diabetic wound healing. Moreover, coadministration of chemokines (or chemokine pathway inhibitors) might complement the local application of stem cell and biomaterial-based therapies and growth factors to improve healing via promoting the migration of resident and inflammatory cells.

2. Chemokine classification and function Chemokines are a diverse family of chemotactic cytokines. They are small (8–10 kDa), positively charged proteins that facilitate cell-cell communication via both autocrine and paracrine mechanisms with the specific endpoint of cell trafficking. To date, approximately 50 human chemokines have been identified, which are classified into four subgroups according to the placement and number of cysteine residues at the N-terminal: C, CC, CXC, and CX3C [24, 25]. They share 20%–50% homology in amino acid sequence [22, 26], including the presence of cysteine residues in conserved locations that determine their shape [27, 28]. Members of the C chemokine group have only two cysteines, one at N-terminal and one downstream. CC chemokines have two adjacent cysteines near their N-terminal end. Members of the CXC group share two conserved cysteines separated by an amino acid [26]. The CXC family is further divided into two groups based on the presence or absence of glutamic acid-leucine-arginine (ELR) motif immediately before the first cysteine, either ELR
(angiostatic) or ELR+ (angiogenic). This ELR sequence is important for receptor selectivity and binding. The CX3C family contains two cysteines separated by three amino acids (Fig. 1). Within tissues and at the cell surface, chemokines are bound to extracellular matrix proteoglycans, particularly those containing the heparin sulfate glycosaminoglycan (HSGAGs) [29]. In the context of wound healing, chemokines are produced by a variety of cells, including endothelial cells, fibroblasts, keratinocytes, and leukocytes (Table 1). Chemokines can interact with 20 different receptors, which belong to the superfamily of rhodopsin-like, G protein-coupled seven-transmembrane receptors present in the lipid bilayer of the target cells [30, 31]. As with the chemokines themselves, the chemokine receptors are likewise divided into four families with distinct chemokine binding properties: XCR1 binds XC chemokines, CCR1–10 receptors bind CC chemokines, CXCR1–7 receptors bind CXC chemokines, and CX3CR1 binds CX3CL1. Although some chemokine-chemokine receptor interactions are selective, many chemokine receptors bind multiple chemokines, engendering both redundancy and plasticity in chemotactic responses [28]. Furthermore, atypical chemokine receptors bind chemokines,

Role of cytokines and chemokines in wound healing

Fig. 1 Chemokine family structure. Chemokines contain cysteines in conserved positions. The spacing between the first two cysteines determines the type of chemokine. The C subfamily contains only one of the proximal N-terminal cysteines. In the CC subfamily the first two cysteines are adjacent to each other, in the CXC family there is one amino acid between the first two cysteines, and they are further divided into ELR+ or ELR
. The CX3C family the two cysteines separated by three amino acids. The first cysteine (C) in the sequence forms a covalent bond with the third, the second and the fourth cysteines also form a disulfide bond to create the tertiary structure characteristic of chemokines.

but do not signal through G proteins. The resulting receptor/ligand interactions influence the function of the chemokines as well as regulate the composition of the local chemokine milieu, chemokine diffusion and clearance. The sequestration and diffusion of chemokines creates gradients within tissues that govern cell chemotaxis [29]. Even though bone marrow-derived cells are the primary targets of chemokines, chemokine receptors are also expressed in many other types of cells, including endothelia, smooth muscle cells, stromal cells, neurons, and epithelial cells [32]. On a functional basis, chemokines can be categorized as either being homeostatic, in which case they are constitutively expressed to maintain homeostatic leukocyte trafficking, or proinflammatory, in which case they are produced by activated cells to recruit leukocytes in the setting of an inflammatory response such as in wound healing. These distinctions are likely to be highly relevant to chemotaxis during wound-healing responses where an inflammatory influx occurs into previously homeostatic tissues [26]. However, in debilitating chronic wounds that are associated with poor wound-healing outcome, such as in diabetes, the distinctions between basal and inflammatory chemokine levels blur, with important consequences for immune regulation at these sites [33]. Along with these contextual associations, chemokines have particular tropisms in terms of the cell types they impact, reflecting the distribution of chemokine receptors (Table 1).

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Table 1 Chemokines and chemokine receptors in wound healing. Chemokines Systemic name

Human ligand

Mouse ligand

Chemokine receptors

Stage of wound Target cells in wound healing healing

CCL1 CCL2

I-309/TCA-3 MCP-1

TCA-3 MCP-1

CCR8 CCR2

Inflammation Inflammation

CCL3 CCL4 CCL5

MIP-1α ΜΙΡ-1β RANTES

MIP-1α ΜΙΡ-1β RANTES

CCL7

MCP-3

MARC

Neutrophils

Inflammation

CCL8

MCP-2

MCP-2

Eotaxin MCP-4

X NCC-1

CCL14 CCL15

HCC-1 HCC-2, Lkn-1, MIP-1d, MIP-5 HCC-4, LEC, LMC, LCC-1 TARC DC-CK1, PARC, AMAC-a, MIP-4 ΜΙΡ-3β, ELC, Exodus-3 MIP-3α, LARC, Exodus-1 6Ckı¨ne, SLC, Exodus-2 MDC MPIF-1

NCC-2 HCC-2, Lkn-1

CCR1 CCR1, CCR3

Mast cells, monocytes, T-lymphocytes Eosinophils Monocytes, eosinophils, T cells, basophils Monocytes T cells, monocytes

Inflammation

CCL11 CCL13

CCR4, CCR1, CCR1, CCR4, CCR1, CCR3, CCR1, CCR3, CCR3 CCR2,

Monocytes, macrophages Monocytes, T-lymphocytes, mast cells, keratinocytes, endothelial cells, fibroblasts Monocytes, macrophages Monocytes, macrophages Monocytes, macrophages

Proliferation Inflammation

NCC-4, MTN-1 CCR1

Monocytes, lymphocytes

Inflammation

ABCD-2 AMAC-1

CCR4 X

T cells Lymphocytes

Inflammation Inflammation

ΜΙΡ-3β, ELC

CCR7

T cells

Inflammation

MIP-3α, LARC

CCR6

T cells, lymphocytes

Inflammation

6Ckine, SLC

CCR7

Lymphocytes

Inflammation

ABCD-1 CCL6, C10

CCR4 CCR1

Inflammation Inflammation

MPIF-2, Eotaxin-2, CKb-6 TECK, MIP-4a

MPIF-2, CKb-6

CCR3

TECK

CCR9

T-lymphocytes Neutrophils, monocytes, T-lymphocytes Eosinophils, basophils, mast cells, Th2 T cells Macrophages

CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25

CCR5 CCR5 CCR3, CCR5 CCR2, CCR5 CCR2, CCR5 CCR3

Inflammation Inflammation Inflammation

Inflammation Inflammation

Inflammation Inflammation

CCL26

Eotaxin-3

TSC-1

CCR3

Eosinophils, basophils, T cells T-lymphocytes Neutrophils, keratinocyte, endothelial cells, fibroblasts

CCL27 CXCL1

ELR +

CTACK/ILC GROa/MGSAa

CTACK/ILC GRO/KC

CCR10 CXCR2

CXCL2

ELR +

GROb/MGSAb

GRO/KC

CXCR2

Endothelial cells

CXCL3 CXCL4 CXCL5

ELR + ELR
ELR +

GROg/MGSAg PF4 ENA-78

GRO/KC PF4 GCP-2/LIX

CXCR2 CXCR3b CXCR2

Endothelial cells Fibroblasts Neutrophils

CXCL6 CXCL7

ELR + ELR +

GCP-2 NAP-2

GCP-2/LIX NAP-2

CXCL8

ELR +

IL-8

MIP-2

CXCL9

ELR


MIG

MIG

CXCL10

ELR


IP-10

IP-10

CXCL11

ELR


l-TAC

l-TAC

CXCR1, CXCR2 Neutrophilic granulocytes CXCR2 Neutrophils, leucocytes, macrophages, keratinocytes, endothelial cells, fibroblasts CXCR1, CXCR2 Neutrophils, leucocytes, macrophages, keratinocytes, endothelial cells, fibroblasts CXCR3 T-lymphocytes, endothelial cells, fibroblasts CXCR3 T-lymphocytes, endothelial cells, fibroblasts CXCR3 T-lymphocytes

CXCL12

ELR


SDF-1a/b

SDF-1a/b

CXCR4

CXCL13 CXCL14 CX3CL1

ELR
ELR


BLC/BCA-1 BRAKFractalkine

BLC/BCA-1 X Fractalkine

CXCR5 X CX3CR1

Proliferation Inflammation Hemostasis, inflammation, proliferation Inflammation, proliferation Proliferation Hemostasis Hemostasis, inflammation, proliferation Proliferation Hemostasis, inflammation, proliferation Hemostasis, inflammation, proliferation Inflammation Proliferation, remodeling Proliferation, remodeling Hemostasis, inflammation, proliferation

T-lymphocytes, keratinocytes, endothelial cells, endothelial progenitor cells B cells, T-lymphocytes Inflammation Monocytes Inflammation Fibroblasts, NK cells, T cells, Inflammation endothelial cells, epithelial cells, macrophages, and vascular smooth muscle cells

202

Wound healing, tissue repair, and regeneration in diabetes

3. Cytokine and chemokine expression during acute vs diabetic wound healing Normal cutaneous wound healing occurs through a series of sequential, overlapping stages, namely hemostasis, inflammation, proliferation, and remodeling (Fig. 2). Coordinated actions executed by platelets, neutrophils, monocyte, macrophages, T cells, B cells, mast cells, stem and progenitor cells, keratinocytes, fibroblasts, and endothelial cells contribute to the healing in different stages. These cells are active producers and regulators of various cytokines, chemokines, and growth factors in the wound (Table 2), which further control the trafficking of specialized cell types to local sites of injury in a time- and context-dependent manner in the orchestration of multiple processes to promote healing. Normal Skin

Acute Wound Healing Inflammation

Proliferation

Remodeling

Keratinocytes

Fibroblast

Collagen Fiber

Tissue Resident Macrophage

Blood Vessel M1 Macrophage

T-Cell M2 Macrophage

Neutrophil

Fig. 2 Chemokines and their roles in the various phases of wound healing. Normal wound healing progresses through a series of sequential, overlapping stages. The initial stage of a wound response is hemostasis, occurring within minutes to hours of injury. Clot formation occurs, which sets stage for the inflammatory phase of healing, which begins with neutrophils coming in first, followed by macrophages. This phase is followed by proliferative and maturation phases. Reepithelialization and granulation tissue formation in which the keratinocytes migrate to cover the wound, and the wound tissue begins its repair by cell proliferation, ECM production, and blood vessel development. Finally, during the remodeling, much of the extracellular elements are removed by apoptosis, and the ECM is remodeled. During these stages, chemokines control the trafficking of specialized cell types to local sites of injury in a time- and context-dependent manner.

Role of cytokines and chemokines in wound healing

Table 2 Inflammatory cytokines involved in wound healing. Name

Synonym(s)

Receptor(s)

Hematopoietin-1 Catabolin IL-1 receptor antagonist lnterferon-γ inducing factor

CD121a, CDw121b CD121a, CDw121b CD121a IL-18Rα, β

Interleukins (IL1-like)

IL-1α IL-1β IL-1RA IL-18

Common G chain (CD132)

IL-2 IL-4 IL-7 IL-9 IL-13 IL-15

T cell growth factor BSF-1 T cell growth factor P40 P600

CD25, 122,132 CD124,213a13, 132 CD127, 132 IL-9R, CD132 CD213a1, 213a2, CD1243, 132 IL-15Ra, CD122, 132

Common B chain (CD131)

IL-3 IL-5

Multipotential CSF, MCGF BCDF-1

CD123, CDw131 CDw125, 131

CSF-2

CD116, CDw131

IFN-β2, BSF-2 AGIF

CD126, 130 IL-11Ra, CD130

CSF-3 NK cell stimulatory factor Leukemia inhibitory factor Oncostatin M

CD114 CD212 LIFR, CD130 OSMR, CD130

CSIF IL-10D

CDw210 IL-20Rα, β

HMW-BCGF LCF CTLA-8 IL-17E

IL-14R CD4 CDw217 IL17RB

Also related

GM-CSF IL6-like

IL-6 IL-11 Also related

G-CSF IL-12 LIF OSM IL10-like

IL-10 IL-20 Others

IL-14 IL-16 IL-17 IL-25 (Fort 2001)

Continued

203

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Table 2 Inflammatory cytokines involved in wound healing—cont’d Name

Synonym(s)

Receptor(s)

Interferons

IFN-α IFN-β IFN-γ

CD118 CD118 CDw119

TNF

CD154 LT-β TNF-α TNF-β 4-1BBL APRIL CD70 CD153 CD178 GITRL LIGHT OX40L TALL-1 TRAIL TWEAK TRANCE

CD40L, TRAP Cachectin LT-α TNFSF9, CD137L TALL-2 CD27L CD30L FasL TNFSF18, TL6 TNFSF14, LTg, CD258 TNFSF3, CD252 TNFSF13B, CD257 Apo2L Apo3L OPGL

CD40 LTβR CD120a, b CD120a, b CDw137(4-1BB) BCMA, TACI CD27 CD30 CD95 (Fas) GITR LTbR, HVEM OX40 BCMA, TACI TRAILR1–4 Apo3 RANK, OPG

TGF-β

TGF-β1 TGF-β2 TGF-β3

TGF-β

TGF-βR1 TGF-βR2 TGF-βR3

Miscellaneous hematopoietins

Epo Tpo Flt-3L SCF M-CSF MSP

Erythropoietin MGDF FLT3LG Stem cell factor, c-kit ligand CSF-1 Macrophage stimulating factor, MSI-1

EpoR TpoR Flt-3 CD117 CD115 CDw136

Bekeschus et al. [34] determined the concentrations of 37 different cytokines, chemokines, and growth factors in the superficial wound fluid from nonhealing wounds of either diabetic patients (>180 days postwound) or large acute wounds of stationary patients (13 days postwound). As reported, IL-6, IL-18, IL-1β, and IL-8/CXCL8 were significantly increased in the chronic diabetic wound sites, while many others were

Role of cytokines and chemokines in wound healing

strikingly decreased, including RANTES/CCL5, I-TAC/CXCL11, IP-10/CXCL10, IL-17A, IL-23, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), platelet-derived growth factor (PDGF)-AA, PDGF-BB, SCF, M-CSF, and EPO. However, different from preceding findings, the levels of the following factors were not altered much: activator protein (AP1), granulocyte-macrophage colony-stimulating factor (GM-CSF), MIG/CXCL9, G-CSF, TARC/CCL17, GROα/CXCL1, ENA-78/CXCL5, macrophage inflammatory protein (MIP-1α)/CCL3, IFN-α, IFN-γ, TNF-α, Eotaxin/CCL11, MIP-1β/ CCL4, MCP-1/CCL2, MIP-3α/CCL20, IL-10, IL-12, IL-33. Of note, the functions of some of the highly differentially expressed factors have not been extensively studied in the diabetic wounds (Fig. 3, Table 3). Here, we will review the fundamental biology of key cytokines and chemokines in wound healing, organized according to the stages of an acute wound response, and compare to diabetic conditions.

3.1 Insult before injury 3.1.1 The AGE of ruin Diabetes mellitus can be characterized by persistent hyperglycemia due to either insufficient levels of insulin (T1DM) or insensitivity to naturally produced insulin (T2DM). Due to persistent hyperglycemia, both groups accumulate greater quantities of advanced glycation end-products (AGEs) within both tissue matrices and within cells [21, 35, 36]. AGEs arise from nonenzymatic glycation events, which can generate multiple different intracellular cytotoxins and inappropriate crosslinks between tissue matrix proteins. Worryingly, reactive oxygen species (ROS) form as byproducts of AGE generation, and these ROS can accelerate subsequent AGE production in a pathological positive feedback loop [36]. AGE(s) influence chemokine production through their interactions with cell-surface advanced glycation end-product receptors (RAGE), which are upregulated in diabetic individuals [37]. These AGE-RAGE interactions generate proinflammatory cytokines, chemokines, and cell-adhesion molecules, leading to a sustained and aggressive inflammatory response. In addition to their interactions with AGE, RAGE can also bind certain members of the S100/calgranulin family of polypeptides [38]. These polypeptides are referred to as extracellular newly identified RAGE-binding proteins (EN-RAGEs). Leukocytes produce EN-RAGEs, and their interactions with RAGEs activate inflammatory cells, leading to the excessive synthesis of proinflammatory cytokines. In mononuclear phagocytes, ligation between RAGEs and either AGEs or EN-RAGEs induces these phagocytes to increase the production of IL-1, IL-6, and TNF-α. Additionally, the stimulation of mononuclear phagocytes by these RAGE-agonist enhances the production of O2 within the phagocytes [39]. Specifically, this helps to explain why mononuclear

205

Hemostasis

Remodeling

Inflammation

Proliferation

I-309 (CCL1) MCP-1 (CCL2) MIP-1α (CCL2) MIP-1β (CCL3) RANTES (CCL4)

MCP-1 (CCL2) RANTES (CCL4) CTACK (CCL27)

Gro-α (CXCL1) PF-4 (CXCL4) ENA-78 (CXCL5) GCP-2 (CXCL6) NAP-2 (CXCL7) IL-8 (CXCL8)

Gro-β (CXCL2) Gro-γ (CXCL3) IL-8 (CXCL8) IP-10 (CXCL10) I-TAC (CXCL11) SDF-1 (CXCL12)

MIG (CXCL9) IP-10 (CXCL10) I-TAC (CXCL11) SDF-1 (CXCL12)

Fractalkine (CX3CL1)

Preinjury

1 Day

2 Days

3 Days

4 Days

5 Days

6 Days

Hemostasis

7 Days

8 Days

9 Days

10 Days

Proliferation

Inflammation

MCP-1 IL-8 IL-17 TGF-β VEGF SDF-1

MCP-1 IL-8 IL-17

3–9 Months

Remodeling

MCP-1 IL-8 IL-17 TGF-β VEGF SDF-1

TGF-β VEGF Diabetes induces chronic inflammation

Preinjury

CRP IL-1β IL-6 LTB4 TNF-α 1 Day

2 Days

3 Days

4 Days

5 Days

IL-10 MIF PDGF RANTES SCF 6 Days

7 Days

8 Days

9 Days

10 Days

3–9 Months

Fig. 3 Chemokine and cytokine differences in acute vs diabetic wounds. Diabetic wounds are predisposed to a baseline increase in low-grade systemic inflammatory preinjury, which perturbs the wound-healing cascade. These wounds are stalled in inflammatory phase for prolonged periods making them refractory to timely resolution and repair.

Role of cytokines and chemokines in wound healing

Table 3 Characteristic differences in acute vs. chronic diabetic wounds. Notable differences between acute and diabetic wounds Variable

Acute skin wounds

Chronic skin wounds

Inflammatory phase

Robust induction and quick resolution Robust recruitment and clearance by apoptosis to allow macrophage recruitment Recruited after neutrophils Low Smaller and ovoid Present Good Yes

Extended

Delayed recruitment Very high Larger and polygonal Scarce Poor No

Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased Increased

Moderately increased Decreased Moderately increased Mildly increased Decreased Decreased Decreased Significantly increased Increased Significantly increased Significantly increased Little to none expressed Decreased Decreased Increased Increased Decreased Increased Decreased

Neutrophils

Macrophages MMP activity Fibroblast conformation Granulation tissue Angiogenesis Re-epithelialization Cytokine/chemokine/GF CCL2 CCL5 CXCL2 CXCL8 CXCL10 CXCL11 CXCL12 IL-1 IL-6 IL-8 IL-10 EGF bFGF GM-CSF IFN-γ MCP-1 PDGF TGF-β1 VEGF

Remain longer and reduced apoptosis

phagocytes from diabetic subjects tend to release more reactive oxygen species following an injury than mononuclear phagocytes from nondiabetics. More generally, the priming of mononuclear phagocytes due to an abundance of EN-RAGEs and RAGEs illustrates how the pathophysiology of diabetes can predispose diabetic individuals to overly aggressive wound-healing response well before an injury occurs. Importantly, concurrent hyperglycemia and AGE enrichment reduces macrophage efferocytosis of apoptotic neutrophils. Macrophage efferocytosis of apoptotic neutrophils drives the resolution of inflammation in acute wounds because it induces micro-RNA-21

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in macrophages [40, 41]. Micro-RNA-21 silences the proinflammatory activity of PDCD4 in favor of cJun-API activity, which increases the secretion of the antiinflammatory IL-10. Because AGEs inhibit macrophage-mediated efferocytosis, diabetic wounds must deal with the dual stresses of a high apoptotic cell load and absence of antiinflammatory M2 macrophages. Additionally, AGE-mediated reduction of IL-10 mitigates the body’s ability to rein in inflammation. The maladaptive effects of AGEs, RAGEs, and ENRAGEs extend well beyond promotion of inflammation. The deleterious effects of AGEs on keratinocytes, fibroblast, and endothelial cells suggest that AGEs hinder these cells’ ability to respond to wounds, and in particular, obstruct the normal chemotaxis of these cells by modifying the interstitial structure. 3.1.2 A spoonful of sugar does not help the medicine Notable damage also occurs to cells prominently involved in the inflammatory, proliferative, and remodeling stages of wound healing. Multiple studies have focused on effects of diabetes on wound cells, including fibroblasts, endothelial cells, keratinocytes, and inflammatory cells. 3.1.2.1 Impact of diabetes on fibroblasts Cutaneous fibroblasts incubated in hyperglycemic conditions show marked decline in synthetic, secretory, and proliferative capabilities. Eventually, they become refractory to growth factors like insulin-like growth factor (IGF-1) and EGF, which constitute the initial wound cues for these cellular trafficking [42–44]. Fibroblasts from diabetes concurrently exhibit impairments in IGF-1, VEGF, nitric oxide (NO), and collagen production, while secretion of matrix metalloprotease type 2 (MMP2), MMP3, MMP8, and MMP9 is enhanced. Additionally, the migratory speed of hyperglycemiaincubated fibroblasts is astoundingly reduced by almost 40% [45]. Studies suggest that these negative attributes arise from oxidative stress generated within the fibroblasts. High intracellular glucose levels increase electron transport chain (ETC) activity in fibroblast mitochondria, and ROS accumulate within the fibroblasts as byproducts of this increased ETC activity. Additional sources of oxidative stress that act upon diabetic fibroblasts include glucose auto-oxidation, the polyol pathway depleting antioxidant reserves, and the formation of advance-glycation end products (AGEs) [46]. Relatedly, AGEs reduce the regenerative capacities of fibroblasts. Further, it was demonstrated that AGE precursor 3-deoxyglucosone (3DG)-rich collagen hindered fibroblast migration, proliferation, and collagen expression by activating p38 mitogen-activated protein kinase (MAPK), which downregulated extracellular regulated kinase 1/2 (ERK1/2) and Akt. In addition, Loughlin and Artlett discovered that 3DG-enriched collagen-induced oxidative stress, endoplasmic reticulum stress, and in some cases caspase-3-mediated apoptosis within fibroblasts [47–49]. Indeed, when fibroblasts were stimulated with the AGE N

Role of cytokines and chemokines in wound healing

(epsilon)-(carboxymethyl)lysine (CML)-collagen, intermediate triggers (ROS, NOS, and ceramides) enhanced caspace-3 activity in these fibroblast via JNK and p38 pathways, and the concurrent upregulation of the transcription factor FOXO1 made these fibroblast far more likely to undergo apoptosis [50]. 3.1.2.2 Impact of diabetes on endothelial and endothelial progenitor cells Hyperglycemia-related microvascular pathology is yet another major contributor to impaired diabetic healing and is associated with deficiencies in both endothelial cells [21, 51] and endothelial progenitor cells (EPC) [52, 53]. In particular, hyperglycemia can affect endothelial function and impair cell activity via several major mechanisms, including toxic effects on endothelial cells via NO, oxidative stress and inflammation [54–56], as well as accumulation of AGEs that results in altered extracellular matrix and disrupted angiogenic growth factor signaling [18, 57–59]. Endothelial cells from diabetics exhibit disturbed cell cycles, with increased DNA damage, delayed replication, and extensive cell death [60]. Studies in vitro have utilized high-glucose culture conditions that demonstrated increased cytochrome-c-mediated caspase-induced endothelial cell apoptosis [61, 62], impaired proliferation, adhesion and tube formation [63–65], and changes in cytokine production [66]. With regards to chemokines, hyperglycemic conditions stimulate the uncontrolled release of proinflammatory chemokines, including tumor necrosis alpha (TNF-α) secretion, the death receptors TNF-R1 expression, Fas, and Bax protein levels [62, 67], interleukin-1β (IL-1β), and IL-6. Additionally, insulin resistance disrupts NO-mediated angiogenic signaling of VEGF, fibroblast growth factor (FGF), and transforming growth factor beta (TGF-β) [68, 69]. Further experimental evidence suggests that oxidative stress results in sustained activation of antiangiogenic, proinflammatory pathways in endothelial cells even after glycemia is normalized; in other words, cell phenotype becomes altered permanently under the “hyperglycemic memory” (reviewed in Ref. [46]). The important implication of these findings is that there is a need for novel therapies that can reverse hyperglycemic memory of endothelium. Neovascularization in diabetes also receives much less contribution from endothelial progenitor cells (EPCs), because the tandem stimulation of excessive glucose and runaway ROS shifts EPCs toward the production of proinflammatory cytokines and inducible nitric oxide synthase (i-NOS) at the expense of a key component to angiogenesis, endothelial NOS (e-NOS) [70]. Excessive glucose and RAGE-agonist can trigger a proinflammatory profile in endothelial cells, transforming these cells in a cytokine and ROS factories. Previous research has shown that the accumulative pressure of hyperglycemia and AGE overproduction disrupts endothelial cell proliferation and migration by interfering in pathways that respond to angiogenic growth factor, such as VEGF. Further, both hyperglycemia and AGEs interfere in the release and recruitment of endothelial progenitor cells (EPCs) from bone marrow [71]. EPCs exposed to AGES suffered a p38 MAPK

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(ERK/JNK)-dependent downregulation of both endothelial nitric oxide synthase (eNOS) and the apoptosis regulator Bcl-2 [72], while simultaneously upregulating cyclooxygenase-2, Bax, nuclear-factor kappa B (NF-κB), and caspase-3, which helps to explain why EPCs from diabetics show limited replicative and migratory potential relative to EPCs from nondiabetics. 3.1.2.3 Impact of diabetes on keratinocytes Chronic diabetic wounds will often fail to re-epithelialize. Instead, neodermis facing creases of mitotically active keratinocytes border the wound’s edges. AGE modification of type-I collagen and other ECM components impairs the integrin-mediated adhesion of keratinocytes to the basement membrane, thus hindering keratinocyte migration [73]. In the absence of proper integrin-mediated adhesion, proliferating keratinocytes will have no option other than forming these multilayered borders around the wound. Additionally, keratinocytes incubated in AGE-rich bovine serum albumin show simultaneous upregulation of MMP9 and downregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) [73]. Long-term incubation of keratinocytes with AGEs significantly reduces their proliferative ability. Taken together, AGEs can inhibit wound-induced keratinocyte proliferation and migration well before the actual injury occurs. Pairing this with hyperglycemia-mediated cytotoxicity and a decline in insulin-mediated proliferation and VEGF release, and it becomes clear that diabetes handicaps keratinocyte function prior to the creation of a wound. 3.1.2.4 Impact of diabetes on immune cells and cytokines To better understand the hyperinflammatory cytokine and chemokine profiles seen in chronic diabetic wounds, one must first understand how diabetes-induced alterations to normal physiology could predispose diabetic skin to excessive baseline inflammation well before an injury occurs. Diabetic patients are characterized by systemic chronic lowgrade inflammation and high levels of the proinflammatory cytokines TNF-α, IL-1, IL-6, and regulated on activation, normal T-cell expressed and secreted (RANTES) [74]. IL-1β has been implicated in insulin resistance and aberrant healing in diabetes [75]. IL-1β is mainly produced by blood monocytes and tissue resident macrophages and activated via caspase-I by NALP3 inflammasome, which allows IL-1β to further amplify its own secretion via activation of this inflammasome. Sustained release of IL-1β from adipose tissue in obese patients, which is a common comorbidity in T2D, could have broad effects on wound healing. In fact visceral fat is also a significant source of IL-6. Diabetic insulin resistance and β-cell inflammation are associated with increased IL-6 levels. Diabetic patients with foot ulcers displayed significantly higher circulating levels of IL-6 than those without foot ulcers, with wound chronicity strongly correlated with the expression of IL-6 levels in the ulcer.

Role of cytokines and chemokines in wound healing

TNF-α is a proinflammatory cytokine, which can activate intracellular transduction cascades interfering with insulin signaling through the inhibition of insulin receptor substrate 1 (IRS-1) [76]. TNF-α has harmful effects on diabetic wound healing, through the inhibition of fibroblasts and keratinocytes proliferation and migration [77], the induction of apoptosis in endothelial cells and pericytes, and the increase in the expression of the FOXO1 transcription factors. TNF-α is also produced by visceral fat, with elevated serum levels of TNF-α observed in obese patients. Acute hyperglycemia triggers a much stronger upregulation in diabetic patients than in normal patients. Together with IL-1β and IL-6, TNF-α acts as potent chemoattractant for neutrophils and stimulates M1 macrophage activation, while stimulating apoptosis of fibroblasts, keratinocytes, and endothelial cells. The systematic proinflammatory environment extends to various tissues, including skin [77]. In diabetic wounds, the immune system is unable to mount the effective immune response required for pathogen control and tissue regeneration [78]. Interestingly, diabetes is accompanied by high levels of systemic LTB4 further contributing to the systemic inflammation [79]. During skin infection, diabetic mice produce higher levels of LTB4 in the skin, which is associated with larger nonhealing lesion areas, dysregulated cytokine and chemokine production, excessive neutrophil chemotaxis, insufficient bacterial clearance, and uncontrolled collagen deposition [79]. Noticeably, the neutrophils can secrete proteolytic enzymes. These proteases degrade key wound-healing factors, including TGF-β1, PDGF-BB, and VEGF [80, 81], as well as the ECM. Also, the neutrophil overloading can lead to sustained release of NO, which suppresses the expression of RANTES/CCL5. Neutrophils are also recruited to the adipose tissue in diabetic individuals, where they differentiate to the proinflammatory N1 phenotype and contribute to chronic inflammation [82]. In diabetes, macrophage polarization favors the M1 proinflammatory phenotype [83] that promotes low-grade chronic inflammation and insulin resistance through IL-6 and TNF-α secretion. Hyperglycemia significantly elevates IL-6 production in a dose-dependent manner in normal macrophages, which explains why macrophages isolated from either streptozotocin-injected (T1D) or db/db (T2D) mice have increased IL-6 expression levels. In diabetic patients, the systemic proinflammatory environment is also primarily maintained by T cells [84]. Depending on cytokine environment during antigenic challenge, CD4 T cells can be polarized into a diversity of subsets with specific effector functions. These subsets can be identified based on the expression of specific transcription factors and cytokines. Proinflammatory T helper (Th) subsets include Th1, Th17, and Th22 cells that produce proinflammatory cytokines. Th1 cells produce IFN-γ and TNF-α. Th17 cells produce IL-17A, IL-17F, IL-21, and IL-22, are positively regulated by IL-6, IL-23, and IL-1β. Th22 also produce IL-22 in the absence of IL-17. Antiinflammatory T cells include Th2 cells (producing IL-4 and IL-13), and regulatory T cells (Tregs, producing the antiinflammatory cytokines IL-10 and TGF-β). It has been documented extensively in the literature that ligands for the chemokine receptors CCR1,

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CCR2, CCR5, and CXCR3 are ubiquitous in chronic inflammation, whereas in acute inflammation, CXCR1 and CXCR2 dominate. Activated neutrophils and T lymphocytes have high expression of CXCR1 and CXCR2, and the Th1 lymphocytes also express CXCR3. Monocytes, eosinophils, and basophils express CCR1 and CCR2, and monocytes also express CCR5. The differentiation and functions of T cells are influenced by diabetes, furthered with the loss of T-cell homeostasis (reviewed by Touch et al. [85]). In the blood of patients with T2D, the proinflammatory Th cells, Th1, Th17, and Th22 are notably increased [86–88]. High levels of Th22 are positively correlated with HOMA-IR (the homeostasis model of assessment for insulin resistance index), and likely serve as a phenotypic distinction between metabolically healthy persons and T2D patients. Thus, the nearly constant hyperglycemia seen in both forms of diabetes creates an unfavorable environment to major cells types in both survive and respond to injury. Understanding these preinjury factors will help explain why wounds in diabetic patients tend toward chronic inflammation more often than similar wounds in nondiabetic individuals.

3.2 Hemostasis Hemostasis is the first stage in the process of wound healing and begins at the onset of wounding. During hemostasis, degranulation of platelet α-granules releases a variety of cytokines and chemokines that initiate coagulation, inhibit premature blood vessel formation, and traffic variety of cells to the wound. CXCL4 is the predominant chemokine released by platelet α-granules, along with the release of CXCL1, CXCL 5, CXCL 7, CXCL 8, CXCL 12, and CCL2, CCL3, CCL5 to lesser extent. CXCL4 (platelet factor 4) inhibits hematopoiesis and collagenase activity, and also inhibits angiogenesis by binding to VEGF and bFGF through its receptor CXCR3. Interestingly, CXCL4 binding to CXCR3 disrupts CCL5-directed chemotaxis of monocytes on the endothelium, whereas CXCL4 is poorly chemotactic to T cells. Additionally, platelet degranulation activates many tissue resident cells, including macrophages, keratinocytes, fibroblast, and mast cells [89]. These activated cells in turn produce inflammatory mediators such as the proinflammatory chemokines CXCL8 (IL-8) and CCL2 (monocyte chemoattractant protein-1 (MCP-1)), which promote the directional migration of inflammatory cells and endothelial cells to the wounded area, further regulating the inflammation and angiogenesis at the tissue repair site (reviewed in Ref. [89]). Little is known of how T2DM-induced alterations to hemostatic chemokines impacts wound healing, since many studies into this matter have focused on the prolonged inflammation stage of chronic diabetic wounds. However, studies in T2DM instigated cardiovascular diseases do offer some tantalizing insights. Research has shown that dysregulated circulating cytokines contribute directly to both chronic inflammation and

Role of cytokines and chemokines in wound healing

hypercoagulation in T2DM. Diabetic patients have also shown significant upregulation in serum TNF-α during high blood glucose events compared with a relatively little change observed in normal patients [90]. Human diabetic foot ulcers have increased levels of IL-1β, inhibiting healing progression [91].Concurrent elevation of both TNF-α and IL-1β common in T2DM increases ICAM-1 levels in monocytes and leukocytes, which increases these cells affinity for activated endothelium [92]. Additionally, elevated levels of C-reactive protein found in both Type 1 and Type 2 diabetes upregulate the expression of cell adhesion molecules on endothelial cells and stimulates these cells to release proinflammatory chemokines like TNF-α, IL-1β, and IL-6 [93]. These proinflammatory chemokines can both stimulate the release of several different procoagulant factors, such as von Willebrand factor (vWF), plasminogen activator inhibitor 1 (PAI-1), and inhibit the release of many different anticoagulants. When the proinflammatory chemokines IL-1β and IL-6 bind to platelets, they stimulate various inflammatory pathways. Blocking IL-1β effects with either IL-1 receptor antagonist (IL1Ra) or anti IL-1β antibody treatments have correlated with improved beta islet cell functionality in T2D patients, as well as decreased IL-6, TNF-α and M1/M2 ratio, with improved wound healing [94–96]. Anti-TNF-α neutralizing antibody administration to ob/ob mice has similarly shown a significant decrease in inflammation and improvement in healing. TNF receptor fusion proteins have also been shown to reduce TNF-α activity and decrease fibroblast apoptosis and increase matrix formation in db/db wounds, suggesting a potential for such interventions in improvement of diabetic wound healing. Given the significant proinflammatory pressure exerted by these chemokines during hemostasis, it is not unreasonable to believe these pressures could hinder proper wound healing in diabetics. The combination of TNF-α, IL-1β, and IL-6 is known to stimulate an acute-phase response, attract neutrophils through chemotaxis, and classically activate macrophages (reviewed in [97]). High levels of these chemokines during hemostasis may prime the body with an excessive number of inflammatory cells that will congregate at wound site.

3.3 Inflammatory stage of wound healing: Damnation by perpetuation The inflammation phase of wound healing generally occurs within the first few days of wounding. It is characterized by an influx of inflammatory cells that remove the dead cells, debris, and potential pathogens to decontaminate the wounds to facilitate the migration, proliferation, and differentiation of endothelial cells, fibroblasts, and keratinocytes to repair the wound. 3.3.1 The neutrophil influx As platelets degranulate, CXCL1 (growth-regulated oncogene alpha, abbreviated GRO-a), CXCL2, CXCL5, and CXCL8 (IL-8) released from α-granules shepherd

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polymorphonuclear neutrophils (PMN) along chemotactic gradients toward the wound, such that within 24 h of wounding, 50% of cells in the wound are PMNs [98]. Neutrophils begin the phagocytosis of debris in the wound and further release chemokines, including CCL2, CCL3, CCL5, CCL7, CCL8, and CCL13, which promote the recruitment of monocytes to the wound. CCL2, also referred to as MCP-1, is predominant chemokine in the monocyte recruitment. Within the wound, PMNs also secrete additional IL-8, as well as TNF-α and IL-1β. IL-8 further attracts additional PMNs to the wound via CXC chemoreceptor 2, inhibits PMN apoptosis, and facilitates the eventual monocyte infiltration by increasing the permeability of the endothelium. The secreted IL-1β and TNF-α activate the NF-κB pathway in PMNs, which leads to the secretion of multiple different MMPs. Accumulating evidence reveals that multiple proinflammatory cytokines provoke leukocyte recruitment at wound site in response to damage cues. The TGF-β1 and platelet-derived growth factors (PDGFs) secreted by platelets can activate mesenchymal cells, and recruit and activate neutrophils and macrophages. Endothelial cells and keratinocytes at wound site also secrete numerous chemokine ligands, including C-X-C chemokine ligand (CXCL)-1, -5, and -8 [99]. Mediated by these factors and their cell surface receptors like C-X-C motif chemokine receptor 2 (CXCR2), neutrophils are the first, though transient, inflammatory cells recruited to the wound site. During acute wound repair, neutrophils are short-lived and apoptose to help prepare the wound bed for the upcoming wave of monocytes, which differentiate to macrophages and takeover the process by targeting dying cells and apoptotic neutrophils and foreign bodies. However, the dysregulated levels in multiple cytokines and chemokines in chronic diabetic wounds set these PMNs upon a proinflammatory path that negatively alters their role in wound healing and the roles of all subsequent cellular actors. In acute wounds, IL-8 concentrations are lower than they are in chronic wounds, and these lower levels seem to promote keratinocyte migration and proliferation as well as drive leukocytes to secrete MMPs (reviewed by Barrientos et al. [100]). However, IL-8 accumulates in many chronic wounds, including chronic diabetic wounds. High levels of IL-8 decrease keratinocyte migration and proliferation, as well as reduce collagen lattice contraction by fibroblasts. The elevated levels of IL-8 also result in the aggressive and persistent homing of PMNs to wound bed, as well as protect PMNs from physiological apoptosis. Neutrophils from chronic diabetic mouse wounds have been shown to undergo reduced apoptosis during staphylococcal infection, which lead to less neutrophil clearance and sustained secretion of proinflammatory cytokines, including TNF-α [101]. These PMNs will further contribute to the proinflammatory environment within the wound through the positive-feedback loop production of both TNF-α and IL-1β [102, 103]. TNF-α stimulates its own secretion and the secretion of IL-1β in PMNs, while simultaneously affecting TGF-β function by inhibiting SMAD phosphorylation via the JNK pathway. This latter effect reduces TGF-β expression in PMNs, which means PMNs cannot generate enough TGF-β to counteract their own inflammatory

Role of cytokines and chemokines in wound healing

stimuli. Consequentially, these PMNs overproduce MMPs, elastase, ROS, and reactive nitrogen species (RNS). Due to the diabetes-induced reduction of antiinflammatory chemokines like IL-10, these PMNs will drench the wound in inflammatory signal and have fewer, if none, means of redressing this issue. Thus, these PMNs will become locked in a hyperinflammatory and apoptosis-refractory phenotype in chronic wounds. Experiments have also implied that the dysregulated overexpression of both MIP-2 and macrophage chemoattractant protein-1 (MCP-1) in diabetic wounds directly contributes to the intense and protracted infiltration of both PMNs and macrophages [104]. The excessive neutrophil activity due to continued recruitment and/or reduced apoptosis and clearance by macrophages can exacerbate the proinflammatory microenvironment and contribute to the nonhealing state of chronic wounds. 3.3.2 When 2 does not follow 1 (macrophages) Within 2–4 days of an acute wound forming, macrophages supersede PMNs as the dominant inflammatory cell type in wounds. These macrophages arise from circulating naı¨ve monocytes following multiple CC chemokines released by PMNs, keratinocytes, endothelial cells, and tissue resident macrophages into the early inflammatory wound microenvironment. Monocytes express chemokine receptor CCR2 that plays an important role in the initial MCP-1-mediated monocyte recruitment. These cells subsequently decrease the expression of CCR2 and increase CCR1/CCR5 that contributes to guided tissue localization. Intact db/db wounds have been shown to have lower expression of CCL2 than controls within 24 h after wounding, but a higher expression of the chemokine after 13 days, which is not conducive to healing as it results in elevated levels if neutrophils and macrophages and increased inflammation during later stages of healing (Fig. 4). Db/db macrophages also show a significant decrease in chemotaxis to CCL2 compared to controls, despite similar expression of the receptor CCR2, suggesting diabetic wounds may contain macrophages that are less responsive to the wound signals. Within the wound, these naı¨ve monocytes are classically activated into inflammatory macrophages (M1). M1 macrophages secrete a variety of cytotoxic and proinflammatory cytokines, including IL-1, TNF-α, IL-1β, and IL-6 and promote microbicidal effects. M1 polarization also promotes type-I immune responses via interferon (IFN-γ) responsive chemokines, including CX3CL1, CXCL9–11, CXCL16, and CCL5. The growth factor secretion, including FGF-2, PDGF, and VEGF, by M1 macrophages also promotes proliferation of endothelial and fibroblasts. In acute wounds, the efferocytosis of apoptotic PMNs catalyzes the transformation of these M1s into antiinflammatory and proregenerative phenotype (M2). These M2 macrophages will secrete TGF-β, IL-10, placental growth factor, and a panoply of other cytokines and growth factors that enable resolution of inflammation and promote the proliferative and remodeling phases of wound healing [105]. The wound microenvironment strongly influences the polarization and functional heterogeneity of the macrophages (reviewed in Rees et al. [106]) (Fig. 5).

215

Fig. 4 Inflammation in acute vs diabetic wounds. In comparison to acute normal wounds, diabetic wounds are characterized by lower levels of chemokines that are necessary to mount a robust inflammatory response to injury at the onset of the wound. However, expression is sustained throughout for an extended period, which correlate with elevated levels of neutrophils, macrophages, and inflammatory cytokines and chemokines at later stages of healing, which is not conducive for successful healing.

Role of cytokines and chemokines in wound healing

Fig. 5 Wound macrophages. Macrophages can rapidly change behavior in response to wound microenvironmental stimuli to promote inflammation (M1), tissue deposition (M2a), or remodeling (M2c), and function as major regulators of healing.

However, comparing histological samples from chronic diabetic wounds and comparable acute nondiabetic wounds reveals that the diabetic wounds contain a higher number of PMNs and M1s, and a greater ratio of M1s to M2s [107]. At the chronic wound margin, approximately 80% of cells are proinflammatory M1s, with these cells playing a major role in the pathogenesis and the ongoing chronicity of wounds [108, 109].The imbalance of M1 and M2 results in increased production of proinflammatory cytokines that prevents subsequent tissue repair [105]. These chronic diabetic wound macrophages secrete an exorbitant amounts of IL-1β, TNF-α, MCP-1, MMP-9, FGF-2, IL-17, ROS, and inducible nitric oxide synthase (iNOS) [96, 110, 111]. In contrast, hyperglycemic conditions perturb macrophages from secreting stromal cell-derived factor 1 (SDF-1, otherwise known as CXCL12) [111]. Additionally, the loss of the M2s leads to a reduction in growth factor levels that regulate the proliferation and neovascularization stages of repair, such as TGFβ1, IGF-1, and VEGF. It is suspected that the sustained hyperglycemia seen in diabetic patients inhibits autophagy, induces ROS, and activates the Nod-like receptor protein (NLRP3) inflammasome in their macrophages, which pushes these M1s to secrete excessive amounts of proinflammatory cytokines [112]. Additionally, genetic analysis of macrophages from diabetic patients and murine models show multiple proinflammatory aberrations when compared to nondiabetic counterparts. Db/db mice macrophages have decreased expression of the M2-related genes Ym1 and Arg-1 [113]. Chromatin immunoprecipitation analysis of macrophages from diet-induced TD2 model mice revealed increased histone-3lysine-4 methylation and decreased histone-3 lysine-27 methylation along the IL1β promoter [113], which indicates that diabetes upregulates IL-1β expression

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in macrophages at an epigenetic level. Moreover, hyperglycemia induces FOXO1 to bind directly to the IL1β promoter, which further amplifies IL-1β production [114]. In total, the preponderance of macrophages stalled at the M1 phase perpetuates the inflammation phase in chronic diabetic wounds. The inherent and prevalent stressors of diabetes perturbs most antiinflammatory and proregenerative properties of macrophages. Couple this with the multitude of ways diabetes predisposes macrophages toward hyperaggressive inflammation, and the net result is the perpetual generation of inflammatory chemokines without a counterbalancing response. 3.3.3 Mast cells and lymphocytes Mast cells (MCs) are recruited early to wounds, and are one of the initial cell types to respond to trauma with the release of inflammatory mediators. There is growing evidence to demonstrate that chemokines and their receptors regulate mast cell tissue localization and function. Human mast cells of different origin express several chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4, and CCR5). Chemokines CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5, and CCL11 have been shown to act on some of these receptors and to induce mast cell migration [115]. The chemokine MCP-1 has been shown to be a potent attractant for mast cells, which are increasingly considered to be important mediators in wound healing [116]. Mast cells degranulate after injury releasing multiple proinflammatory mediators and vasoactive amines [117]. MCs also release chemokines such as IL-8, MIP-1α, and MIP-1β independent of degranulation, when stimulated via the activation of the CD30 pathway [118]. Mast cells synthesize IL-4 and stimulate fibroblast proliferation [116]. Mast cells can have both stimulatory and inhibitory functions in skin inflammation that are regulated by chemokines (reviewed by Harvima and Nilsson [119]). Studies by Tellechea et al. [120] show that the number of degranulated mast cells is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice. Conversely, postwounding, mast cell degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate and topical application of indole-carboxamide type stabilizer rescue diabetes-associated wound-healing impairment in mice and shifted macrophages to the regenerative M2 phenotype [121]. Nevertheless, nondiabetic and diabetic mice deficient in mast cells have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human mast cells is reduced in hyperglycemic conditions. T-lymphocytes are present in the wounds from as early as day 1 until resolution of the wound [122], where they serve as immunological effector cells and produce additional chemokines and cytokines, contributing to the modeling of the wound milieu. CCL3,

Role of cytokines and chemokines in wound healing

CCL4, and CCL5 and major lymphocyte chemoattractants in the wound. B-lymphocytes produce antibody responses to antigens present in the wound and T-lymphocytes produce cytokines and promote cytolytic activity. Different types of T cells play crucial roles in inflammation resolution and tissue remodeling. In the inflammatory phase, T cells are attracted to wound site by IFN-γ released by macrophages, which also promote Th1 polarization [123]. Tissue resident γδT cells regulate proliferation and differentiation of keratinocytes through releasing growth factors such as FGF-7, FGF-10, and IGF-1, which are also activated during tissue damage and participate in wound repair [124]. Importantly, these cells stay unresponsive in chronic wounds. Regulatory T cells (Tregs facilitate antiinflammatory macrophage polarization and suppress the inflammatory responses via releasing antiinflammatory cytokines (including IL-10 and TGF-β1) [125]. In the final stages of acute wound repair (matrix formation and remodeling), antiinflammatory macrophages and T cells (Tregs and Th2) are involved in repair and fibrosis outcomes [126, 127]. Th2 and Tregs release TGF-β, IL-4, IL-5, IL-10, IL-13, and IL-21. These factors support ECM formation by stimulating myofibroblast differentiation (TGF-β), and triggering fibroblasts and myofibroblasts to produce new ECM (TGF-β, PDGF, and IL-4). These cytokines play distinct roles in fibrogenesis, favoring antiinflammatory and antifibrotic macrophage polarization and suppressing other types of inflammatory cells. Moura et al. [74] analyzed T-cell receptor (TCR) repertoire diversity in TCR-αβ + T cells, and the distribution and phenotype of T cells from the blood of healthy controls and diabetic individuals with or without DFU. TCR-β gene diversity is diminished in diabetic patients, especially in those with active foot ulcerations, which persists after the DFU has healed. Correspondingly, in diabetic individuals, the number of naive CD4 + T cells (CD27+ CD28+CD45RO
) significantly decreases, while there is a significant increase in the pool size of activated/memory (CD27+CD28+CD45RO+) and effector (CD27
CD28
) T cells. Notably, these T-cell populations produce inflammatory cytokines at different levels, while IL-2 is primarily generated by naive and activated/memory T cells, IFN-γ and TNF-α are only detected on the activated/memory and effector T cells. Even though no difference was found in the effector T-cell percentages between diabetic patients with chronic DFU and both the diabetic patients without DFU and control groups, expression of cytokine receptors is altered. In the effector T cells from diabetic patients, expression of CCR5, CXCR3, and CXCR1 is strikingly reduced. These studies reveal that T-cell differentiation is impaired in DFU. In sum, compared with acute wounds, chronic wounds have (1) excessive proinflammatory cytokines (IL-6, IL-8, IL-1, and TNF-α), and deficit of antiinflammatory and healing-associated cytokines (like TGF-β and IL-10); (2) decreased production of growth factors, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin growth factor (IGF), nervederived growth factor (NGF), and platelet-derived growth factor (PDGF); and (3) much

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higher levels of protease activities, which shifts their equilibrium with their inhibitors, the TIMPs, in favor of the proteases, with detrimental effects on chemokine gradients by release of chemokines bound to cell surface or ECM, inactivation of chemokines and generation of more powerful chemotactic or antagonistic derivatives. These findings suggest that chemokines may be the major regulators of the association between inflammation and remodeling stages during wound-healing processes.

3.4 Proliferation and remodeling stages of wound healing The hallmark events of the proliferation stage (fibroplasia and granulation tissue formation, neovascularization, and re-epithelialization) and the remodeling stage (cell regression, ECM turnover, and collagen deposition and maturation) chronologically overlap within their respective stages, and at certain times even between stages. This is partly due to the shared cellular ensembles of these two stages performing multiple roles simultaneously. Chronic diabetic wounds show remarkable absences or reductions in all of these events and lack a clear temporal delimitation for when these impairments “begin.” Experiments have revealed how the chemokine milieu of chronic diabetic wounds negatively affects the cells that are paramount to these stages. 3.4.1 Granulation tissue deposition ECM protein synthesis and deposition is mainly carried out by fibroblasts during this phase of wound healing, which is also highly regulated by chemokines and cytokines. Fibroblasts, alongside keratinocytes, proliferate around the wound’s edge and migrate toward the center, which closes the wound. Fibroblasts are also potent effector cells that manipulate the chemokine expression in the wound milieu. MCP-1 can induce the expression of TGF-β and collagen synthesis by rat fibroblasts [128]. MCP-1 has also been shown to enhance MMP-1 and TIMP-1 gene expression in primary human dermal fibroblasts [129]. CXCL11 is important in dermal–epidermal interactions and in maturation of the healing tissue. Feugate et al. [130, 131] demonstrated that chicken chemotactic and angiogenic factor (cCAF), a CXC chemokine orthologue of human IL-8, stimulates fibroblasts to produce early granulation tissue and ECM components, including tenacin, fibronectin, and collagen, as well as stimulates the differentiation of fibroblasts to myofibroblasts. Chemokines are also important in the control of absolute fibroblast number and the extent of fibroplasia. For example, IL-8 stimulates differentiation of fibroblasts into myofibroblasts. Fibroblasts and myofibroblasts will coordinate to synthesize mature connective tissue and degrade nascent matrix tissue using MMPs. Noticeably, MMPs have been shown to inactivate specific chemokines and generate antagonistic derivatives that influence healing. Key such factors is the inactivation of chemokine CXCL12 (SDF-1), which is inactivated by MMP1–3, MMP9, MMP13, and MMP14. MMP8 also inactivates CXCL1, which promotes bone-marrow-derived

Role of cytokines and chemokines in wound healing

stem cell recruitment and their subsequent differentiation into granulation-tissueproducing cells: endothelial cells and fibroblast (reviewed in Ridiandries et al. [23]). In vitro analysis of diabetic dermal fibroblasts demonstrated reduced TGF-β RII signaling and fibroblast migration. Diabetic wounds in both STZ-induced and db/db models demonstrated reduced levels of CXCL12, and overexpression of CXCL12 resulted in improved granulation tissue formation and wound healing. Although it would be reasonable to believe the elevation of MCP-1 in chronic diabetic wounds would actually assist fibroblast migration and function, this cannot overcome the negative impacts of excessive TNF-α, which impedes fibroblast proliferation, hinders migration, degrades their ability to secrete matrix components, and ultimately destines these cells to apoptosis. The chronic supraoptimal of TNF-α in diabetic wounds permanently impairs fibroblast function. What fibroblasts remain likely acquire an antagonistic phenotype, as already noted. 3.4.2 Neovascularization The onset of the proliferation stage is also characterized by the formation of many neovessels that form to support the metabolic demands of the wound with rapid cell infiltration and matrix deposition. Early vessel formation depends upon tissue resident cells (macrophages, keratinocytes, fibroblast, etc.) secreting proangiogenic factors like VEGF and proteolytic enzymes to facilitate endothelial cell proliferation and migration. Many of the proangiogenic chemokines that mediate neovascularization include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, which stimulate CXCR1 and/or CXCR2 molecules that dot the surface of an endothelial cells. Early wound angiogenesis is associated with high expression of XCL1 and CXCL8. CXCL12 is another important chemokine expressed by endothelial cells, which stimulated endothelial migration in a VEGF-dependent manner. Its primary receptor, CXCR4, is expressed by lymphocytes and monocytes. CXCR7 had been recently reported to be a receptor for CXCL1, which is mainly found on macrophages in pathological conditions as opposed to no expression on blood leukocytes, suggesting a role for the receptor/ligand signaling in the orchestration of inflammation. The CXCL12CXCR4 axis also plays a role in endothelial progenitor cells mobilization and homing, and the decrease in expression of CXCL12 in diabetic wounds may contribute to impaired neovascularization in these wounds. The other major chemokines and respective receptors that regulate EPC activation and homing are IL-8 and CXCR2, growth-regulated oncogene-a and CXCR1, CCL5, CCR5, and C-C chemokine and chemokine (C-C motif ) receptors 2 and 5 [132]. Upon interaction with tissue-specific chemokines, EPCs become activated and initiate integrin-mediated adhesion to endothelial vascular cells and, consequently, trans-endothelial migration into sites of vascular and tissue remodeling. CXCL8 stimulates vascular permeability, which is important for both angiogenesis and vasculogenesis. EPC invasion into the vascular injury site

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depends on the breakdown and remodeling of the vessel basement membrane and the interstitial wound space mediated by extracellular proteases. As discussed previously, chemokines play a major role in the regulation of the ECM remodeling, and may thereby influence wound neovascularization mediated via both angiogenesis and stem-cell-dependent vasculogenesis mechanisms. When the wound defect is filled, angiogenesis has to cease. A temporal change in the balance between proangiogenic and angiostatic factors regulated by chemokines plays an important role in the orchestration of this cessation. Endothelial cells differentially express several chemokines in response to the wound microenvironmental stimuli [133]. These include MCP-1 and RANTES as well as IL-8, GRO-α, GRO-β (CXCL2), GRO-γ (CXCL3). CTAP-III, β thromboglobulin, and NAP-2 have also been described to induce endothelial cell proliferation in vitro and angiogenesis in vivo. ELR-positive chemokines IL-8 and GRO-α are significantly induced at the neovascularization sites in the wound granulation tissue immediately after wounding and then markedly decline after day 4 [98]. CXCR2 chemokine receptor, which binds to all ELR-positive chemokines, is also believed to play an important role in the mediation of neovascularization (reviewed by Gillitzer et al. [134]). Furthermore chemokines indirectly influence neovascularization by facilitating the recruitment of support cells that are essential for tissue revascularization. DiPietro et al. [135] reported that depletion of MIP-1α significantly reduced angiogenic activity of murine wounds. It was suggested that MIP-1α promotes recruitment of macrophages to wound sites, which in turn act as a source of angiogenic cytokines. Several ELR-negative CXC chemokines, including IFN-γ-inducible cytokines IP-10, CXCL-9 (Mig), CXCL4, 9, 10, and 11 or I-TAC exhibit growth-inhibitory functions and inhibition of angiogenesis. IP-10 and Mig have also been shown to be highly expressed after day 4 during the later stages of normal wound healing, with an inhibitory action on fibroblast motility and recruitment. Arguably, disturbance in the chemokine and cytokines in diabetes contributes to impaired neovascularization by affecting both angiogenesis and vasculogenic pathways. 3.4.3 Re-epithelialization During re-epithelialization, factors, including GRO-α, IL-8, FGF, and keratinocyte growth factor (KGF, otherwise known as FGF7), direct wound-edge keratinocytes to proliferate and migrate. Both GRO-α and IL-8 mediate this activity through CXCR2 receptors on keratinocytes. These stimulated keratinocytes will receive reinforcements in the form of undifferentiated keratinocytes migrating toward the wound due to basal keratinocytes secreting CXCL11 [136] and from bone-marrow-derived keratinocyte stem whose CCR10 receptors were stimulated with CCL27 [137]. In acute wounds, TGF-β secreted from M2s will stimulate keratinocyte proliferation, endothelial cell proliferation, keratinocyte migration, endothelial cell migration, collagen formation, ECM remodeling, and initiate the formation of granulation tissue. CXCL12 elevated around

Role of cytokines and chemokines in wound healing

the edges of the wound may also enhance keratinocyte proliferation, and thus contribute to re-epithelialization. 3.4.4 Remodeling This is perhaps the longest phase for spanning several months after closure of the wound. During wound maturation and remodeling, ECM turnover occurs with disorganized collagen fibers being rearranged and crosslinked, along with a decrease in cellularity and regression of the neovasculature. Fibroblasts and myofibroblasts synthesize mature connective tissue, as well as produce metalloproteinases that remove the nascent matrix and facilitate matrix maturation. In postnatal tissues this results in scar formation, but in diabetic wounds may produce unsatisfactory healing that predisposes them to repeat injury. CCL2 was found to promote expression of MMP-1 and TIMP-1 in fibroblasts, indicating its role in regulation of matrix production and remodeling. Chemokines CXCL9, CXCL10, and CXCL11 have been shown to play role in the process of epidermal and dermal maturation and regression of neovasculature. The wound repair and regeneration is thought to be further influenced by stem cell recruitment and mechanical forces that impact the wound bed, both of which are governed in part by chemokines.

4. Diabetic challenges and potential therapeutic opportunities 4.1 Biofilms Diabetic foot ulcers especially have been shown to have higher levels of bacterial biofilm than nondiabetic wounds. Biofilms are complex structures composed of extracellular polymeric substances secreted by the microbes residing within them. They form an extracellular matrix that is important for structural stability and is a potential reason for the antibiotic therapy resistance as well as the persistence of chronic nonhealing wounds [138]. Recent studies have begun to characterize the composition of the microbiome, which changes over time with wound acuity and severity. The most common components of the biofilm are Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium striatum, and Alcaligenes faecalis. When correlated to wound outcomes, S. aureus is associated with longer wound duration and impaired healing. Commensal organisms such as C. striatum and A. faecalis also impact healing, with increased concentrations in wounds greater than 12 weeks old [139]. The pathogenesis of the biofilm is mediated through cytokines and bacteriophages. Specifically, in P. aeruginosa diabetic wound infections, bacteria produce a filamentous phage, which affect host immunity. They are first engulfed by leukocytes, where phage RNA is recognized by TLR3- and TRIF-dependent PRRs. This interaction increases type 1 IFN expression, inhibiting TNF production and thus suppressing

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host-cell-mediated phagocytosis. These phages are found in between 60% and 68% of chronic human diabetic wounds, and correlate with the persistence of wounds, increased pathogenesis and virulence, as well as increased morbidity and mortality [140, 141]. Future studies should further explore the role of the biofilm and microbiome in influencing cytokine expression, as well as the interactions between hosts and bacteriophages.

4.2 Cytokines/chemokines that may alleviate diabetic wounds Cytokines of IL-10 family, including IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 [142], have been implicated in the pathogenesis of chronic inflammatory diseases. IL-22 can be produced by αβ T-cell classes (Th1, Th17, and Th22), γδ T cells, NKT cells, innate lymphoid cells (ILCs), neutrophils, and macrophages [143]. In T2DM subjects, the release of IL-17 and IL-22 by adipose tissue CD4+ T cells can be enhanced by IL-1β, the main macrophage-derived cytokine. Reciprocally, IL-22 upregulated IL-1β expression by adipose tissue macrophages, hence amplifying IL-1β–driven inflammation in human adipose tissue [144]. In contrast, IL-22 was also reported to play diverse beneficial roles in diabetes, improving insulin sensitivity, suppressing inflammatory responses, and alleviating tissue injury [145]. Also, IL-22, the most potent one among IL-20 subfamily cytokines that bind to the common receptor subunit IL-22R, can promote wound repair in D2T mouse model [146]. Presumably, the proinflammatory versus tissue-protective functions of IL-22 are regulated by other factors such as the coexpressed cytokine IL-17A [147]. CXCL6 appears an independent predictor of wound healing in DFU individuals. It has been implicated in fibrosis and vasculopathy in systemic sclerosis, colon cancer, and human lumbar disc degeneration [148]. It is present at much lower concentrations in the wound exudates from DFU patients. GM-CSF may act as both a proinflammatory and an antiinflammatory or regulatory cytokine, and several other growth factors (PDGF, VEGF, and bFGF) had been shown with promises in enhancing the healing of chronic wounds (reviewed in Barrientos et al. [149]).

4.3 Mesenchymal stromal cells Chemokines and chemoattractant signals also regulate the mobilization and homing of bone-marrow-derived as well as circulating stem cell populations to the tissue repair sites, including mesenchymal stem cells (MSCs). Under the guidance of chemokine signaling, MSCs can selectively migrate to injured sites, including skin wound healing [137, 150, 151], bone fractures [152, 153], myocardial infarctions [154, 155], and ischemic cerebral injuries [156], where they engraft and contribute to tissue recovery [156]. Although MSCs have been shown to exhibit low levels of long-term incorporation into healing wounds, a growing body of research suggests that their therapeutic benefit is attributed to their release of trophic mediators, rather than a direct structural contribution. Through the release of VEGF, stromal-cell-derived factor-1, epidermal growth factor,

Role of cytokines and chemokines in wound healing

keratinocyte growth factor, insulin-like growth factor, and matrix metalloproteinase-9, MSCs promote new vessel formation, recruit endogenous progenitor cells, and direct cell differentiation, proliferation, and extracellular matrix formation during wound repair. MSCs also exhibit key immunomodulatory properties though the secretion of interferon-λ, tumor necrosis factor-α, interleukin-1α, and interleukin-1β, as well as through the activation of inducible nitric oxide synthase. MSC secretion of prostaglandin E2 further regulates fibrosis and inflammation, promoting tissue healing with reduced scarring. Finally, MSCs display bactericidal properties through the secretion of antimicrobial factors and by upregulating bacterial killing and phagocytosis by immune cells. MSCs and their functions are impaired under diabetic conditions. MSCs have been administered both systemically and locally for the treatment of cutaneous wounds. Bonemarrow-derived MSCs are normally harvested from humans by aspiration from the iliac crest, followed by in vitro selection based on their ability to adhere to a plastic culture dish. Cells can then be expanded in culture and applied topically to wounds to promote tissue regeneration. Clinical trials using alternate delivery methods have further confirmed the potential therapeutic efficacy of BM-MSCs in human cutaneous regeneration. A study of human patients with chronic leg ulcers resistant to conventional treatment for at least 1 year showed that application of autologous BM-MSCs reduced wound size, increased vascularity, and increased dermal thickness [157, 158]. Exosomes are a type of extracellular vesicles secreted by the cells, defined by their size (50–150 nM) and cell surface markers. Recent studies suggest that exosomes secreted from MSC serve as key paracrine regulators for tissue repair. Exosomes contain a variety of cargo, including mRNAs, microRNAs (miRNA) proteins and lipids that could be transferred to target cells inducing genetic and epigenetic changes in target cells. Exosomes are not immunoreactive, can pass through biological barriers, and pose no risk of maldifferentiation [159]. As a result of these properties, exosomes have a great potential in the field of wound healing. MSC-derived exosomes could serve as novel treatment options for patients with complications such as diabetic nephropathy and CNS damage. Full-thickness wounds in diabetic murine models treated with exosomes isolated from MSCs have shown improved cutaneous wound healing [160]. MSC exosome cargo regulating glucose metabolism in diabetes is gaining traction. Transfer of miR-99b from adipose-derived MSCs to hepatic cells modulated the fibroblast growth factor 21 expression and is involved in glucose metabolism. MSC exosome therapy improved glucose uptake by inducing GLUT4 translocation to the cell membrane in the rat skeletal muscle. They also inhibited apoptosis and increased insulin secretion and islet regeneration [161]. Clinical trials using MSC-derived microvesicles for diabetes type I are currently ongoing (https://clinicaltrials.gov/ct2/show/NCT02138331?term¼MSC+ exosomes&draw¼1&rank¼1). MSC exosomes enhanced diabetic wound healing by resolving inflammation via M1–M2 macrophage polarization. Exosomes overexpressing certain miRNA (miR-126) were shown to promote proliferation of human dermal

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fibroblasts and microvascular endothelial cells in a dose-dependent manner and contribute to cutaneous wound healing [162]. Exosome therapy in diabetic rat models also promoted formation of collagen. Delivering these exosomes via bioactive hydrogels/ polysaccharide-based dressings to the wound site further improved the healing and is characterized by faster wound closure rates, collagen deposition and re-epithelialization, and less scar formation [163,164]. Preconditioning of exosomes with chelating agents (deferoxamine) increased angiogenesis by miRNA-mediated activation of certain growth-signaling pathways (PI3K). These studies show promise that diabetic-induced wound impairments can be overcome by stem and progenitor-cell-based therapeutics, which will be a huge boom for patients suffering from nonhealing ulcers.

4.4 Biomaterials Biomaterials are defined as any nondrug material used to enhance or replace the function of a native organ or tissue in an organism. Critically, biomaterials are engineered to elicit minimal to no adverse reaction from the recipient immune system [165]. As they relate to wound healing, biomaterials fall into three main categories: tissue-derived biomaterials, hydrogel-based biomaterials, and biomaterials that enable controlled release of signaling molecules. Tissue-derived biomaterials are typically found as scaffolds, three-dimensional structures impregnated with cells, which serve as a base for an organism’s own cells to adhere to and begin the process of native tissue formation. These scaffolds closely mimic the extracellular matrix, or ECM, of the local microenvironment. Hydrogel-based biomaterials also serve to closely mimic the ECM of the wound environment; however, their properties are more easily engineered per the requirements of the specific wound. Finally, biomaterials that enable controlled release of signaling molecules are necessary due to the highly proteolytic environment of wounds, which quickly degrade growth factors, cytokines and chemokines critical to the normal wound-healing process. For the purposes of this chapter, we will focus on the role biomaterials play on cytokines and chemokines in the wound-healing process and specifically their role in diabetic wound healing. Chronic diabetic wounds are characterized by the stalling of the normal wound healing process in the early inflammatory stage, thus preventing the later stages of proliferation and remodeling from taking place. Studies have shown that an imbalance of proinflammatory M1 macrophages and antiinflammatory M2 macrophages disrupts the wound-healing process and causes its arrest in the inflammatory phase. This imbalance affects the secretion of cytokines and chemokines necessary for downstream processes to occur. Macrophages have been noted to more readily bind to hydrophobic surfaces, although the macrophages that bind to neutral or hydrophilic surfaces are more likely to release cytokines. Furthermore, monocyte-derived macrophages have been

Role of cytokines and chemokines in wound healing

noted to modify their surface protein expressed dependent upon the surface chemistry they bind to. As a result, scientists have been able to engineer biomaterials, which take advantage of these binding characteristics and encourage the polarization needed to promote proper wound healing. It has also been shown that IL-22, a proinflammatory cytokine, actually enables wound healing in diabetic murine wounds by promoting keratinocyte proliferation. This seems to show that proper ratios of pro- and antiinflammatory cytokines in temporally spaced doses are more effective in controlling diabetic wound healing rather than antiinflammatory cytokines alone. Biomaterials that leverage this biphasic approach to delivering cytokines to the wound bed are encouraging as similar techniques have been used in bone repair with IFN-γ and IL-4 [111]. The importance of biomaterials that enable controlled release of signaling molecules becomes apparent when studying the various endogenous growth factors, cytokines, and chemokines implicated in the wound-healing process. Chronic or nonhealing wounds are often the result of degradation of these cytokines or the very absence of the cells that produce these molecules. As a result, delivery of exogenous cytokines to the wound site using various biomaterials has become a prominent area of interest in the field of regenerative tissue repair. This rather straightforward solution is complicated by the fact that each individual molecule must be delivered to the wound site in its respective functional state and in the proper concentration. In vivo, heparan sulfate binds and protects various growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in addition to other cytokines involved in the inflammatory phase. In order to recapitulate these interactions, heparan has been incorporated into woundhealing therapeutics for different growth factors, including VEGF and transforming growth factor beta (TGFβ). Other methods of incorporation include recombinant expression of growth factor fusion proteins, which can then be incorporated into the biomaterial scaffolds mentioned earlier in this chapter. Furthermore, biomaterial scaffolds inclusive of exogenous growth factors or cytokines have been shown to promote expression of endogenous growth factors providing some compensation for the proteolytic environment [166]. Useful growth-factor-binding domains can be isolated from various ECM molecules given the fact that the ECM naturally binds growth factors. For example, several growth factors possess specific interactions with the heparan sulfate proteoglycans of the ECM. As such, a number of biomaterial matrices have been modified with heparan or heparan sulfate-mimetic molecules to sequester heparan-binding growth factors and control their release. For example, synthetic hydrogel films crosslinked with heparan have been used to successfully control the delivery of FGF-2 in a full-thickness excisional wound model in db/db diabetic mice and showed acceleration of dermis formation and vascularization. Given the fact that heparan-binding growth factors can extend their half-life by being protected in the matrix, bioengineers have modified nonheparan-binding growth factors to increase their affinity to endogenous heparan sulfate in vivo. This concept has not yet

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been studied in wound-healing therapies, but it has been applied in cartilage tissue engineering. As such, the engineering of a heparan-binding IGF-1 (HB-IGF-1) variant has shown an improved retention in proteoglycan-rich environments and sustained bioactivity. In dermal wound healing, IGF-1 is also a key factor that promotes type I collagen synthesis, and fibroblasts, and keratinocytes proliferation. Its topical application on nonhealing diabetic skin has been correlated with a faster re-epithelization and enhanced scarring in rat model [167].

5. Conclusion The studies reviewed here show that normal wound healing requires the sequential stimulation and resolution of multiple phases, which are influenced by regulatory molecules such as the chemokines. Chemokines play multiple roles in injury repair, not confined to their well-characterized roles in leukocyte chemotaxis and angiogenesis. It is becoming clear that chemokines also play an integral part in re-epithelialization, granulation tissue formation, and remodeling of the healing wounds. Alteration in chemokine expression, longevity, and localization can result in pathological healing states. Chemokines may also play a critical role in the pathogenesis of chronic wounds, not only by altering the inflammatory and angiogenesis pathways, but also via their role in combating with biofilmforming multicellular organisms. In addition, chemokine overexpression has been demonstrated to be associated with fibrosis in multiple organs. In this context, understanding the differences in the chemokine profile in normal acute and chronic diabetic wounds can take us one step closer to improving diabetic-healing outcomes. Despite the great promise of chemokine-targeted therapy, much work still needs to be done to determine whether control of this signaling system will prove to be fruitful from bench to bedside.

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CHAPTER 12

The wound microbiome Aayushi Uberoi, Amy Campbell, Elizabeth A. Grice

Departments of Dermatology and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

1. Introduction Diabetes mellitus currently affects about 285 million adults worldwide, and this figure is expected to rise to over 400 million adults by 2030 [1]. Diabetic patients are at high risk for complications related to nonhealing wounds and up to one in four persons with diabetes will develop a diabetic foot ulcer (hereafter referred to as “DFU”) [2]. Complications from DFUs account for 2/3 of all nontraumatic lower-extremity amputations performed in the United States [2, 3] and 5-year mortality rates surpass those of prostate and breast cancer, among others [4, 5]. Lower-limb related problems constitute up to 1/3 of the cost of diabetes in the United States [6], a staggering figure when one considers that the estimated global cost of diabetes was $1.3 trillion in 2015 [7]. Very few interventions have demonstrated improvement in DFU healing: median time to heal is >2 months [8], and 2/3 of DFU will eventually require surgery [9]. Microbial bioburden is believed to play a significant role in impaired healing in DFU and other chronic wounds, and the development of infection-related complications, such as wound deterioration, osteomyelitis, and amputation [10]. Although all DFUs are colonized with microbes, their importance in the absence of clinical infection is unclear [9]. Furthermore, several dimensions of microbial bioburden may be important, including microbial load/quantity, microbial diversity, and the presence of pathogenic microorganisms [10]. While culture-based methods have been the gold standard for characterizing wound bioburden, they are now understood to be biased in their assessments. Fastidious, difficult-to-grow microorganisms are recalcitrant to isolation in culture, as are microorganisms that rely on social interactions to survive in the wound environment (e.g., biofilm) [11]. The introduction of culture-independent, DNA sequencing-based methods has greatly enhanced the ability to characterize wound microbes at the community-wide level, providing more precise surveys of what constitutes the wound microbiota [12]. Application of such methods has provided novel insights into the relationship between wound microbiota, impaired healing, and the progression to infection-related outcomes.

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00012-5

© 2020 Elsevier Inc. All rights reserved.

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Here, we will summarize the current understanding of the wound microbiome in DFU. Specifically, we will provide current knowledge about the components of the DFU microbiome, the cutting-edge techniques used to elucidate the composition and activities of the microbiota, and the future research directions that will be required for mechanistic understanding and clinical translation of the DFU microbiome.

2. From contamination to infection: The wound infection continuum The skin is inhabited by a diversity of microbiota, which generally exhibit commensal or mutualistic behavior under steady-state conditions [13]. However, breach of the skin barrier when wounding occurs exposes subcutaneous tissue and nutrient sources, which provide a favorable environment for microbial proliferation and colonization [14, 15]. Normal wound repair due to acute trauma follows a precisely orchestrated sequence of events (coagulation, hemostasis, inflammation, cell proliferation, cell migration, and tissue remodeling) and wound closure is achieved within 3–14 days [16]. All wounds are minimally contaminated with microbiota from the surrounding tissues and environment. However, not all contamination or colonization results in clinical consequences, since not all wounds progress to infection or result in complication [17]. On the other hand, chronic wounds such as DFUs fail to proceed through this series of events, become stalled in a chronic inflammatory state, and often result in infection or complication (Fig. 1). What constitutes problematic bioburden in a DFU, and its relationship to clinical signs and symptoms, is an ongoing debate. The first phase of the wound infection continuum is contamination, as all wounds acquire microorganisms from surrounding tissue and the environment, but these microbes do not necessarily survive or replicate [18]. Contamination may progress to colonization provided a suitable environment for replication, but not in sufficient numbers or virulence to provoke an immune response [19]. Early local wound infection occurs when colonizing microbes invade viable tissue but infection is localized to the wound. At this stage subtle signs of infection may be absent and remain difficult to diagnose clinically, especially in those with diabetes

Fig. 1 Wound infection continuum. Upon breach of the skin barrier, microbes from the environment and the skin contaminate the open wound. If the microbes replicate in wound tissue, they are considered colonizing. Colonization may progress to local infection, which can spread and eventually become a systemic infection.

The wound microbiome

[10, 20]. Later, local infection may exhibit overt clinical symptoms such as erythema and purulent discharge, while delayed wound closure is observed [21]. A local infection may spread with microbial invasion beyond the bounds of the wound to deeper tissues, and eventually systemically [22]. Microbial communities are hypothesized to form biofilms in wound tissue, which presents a number of challenges for the clinical management and treatment of wounds [23]. Biofilms may be simple or complex communities of aggregated microbes embedded in a self-secreted extracellular polysaccharide matrix (EPS). Bacterial adhesion to collagen, fibrinogen, and fibronectin, readily available in the wound, is a key step of biofilm formation [24]. Bacteria living within a biofilm behave differently than those in a planktonic, nonbiofilm state, slowing their metabolism and altering virulence factor production to evade host immune responses. These factors, in addition to the diffusion barrier produced by the layer of EPS, most likely contribute to the dramatic resistance of biofilms to conventional antibiotic and antimicrobial treatments [25]. Bacteria grown as biofilms invoke different wound responses compared to their planktonic forms both in vitro and in vivo [26, 27]. Early local infection and biofilm formation may precede clinically apparent infection and may represent a point of intervention for preventing further infection of DFU by appropriate cleansing and debridement methods [28]. Nonetheless, accurate and rapid detection of biofilm in situ is not currently possible, and bacteria within a biofilm are recalcitrant to culture-based identification [29]. Therefore, cutting-edge -omic techniques, such as those described hereafter, offer promise for assessing biofilm status in a patient wound while providing insight into pathogenicity behaviors of wound microbes.

3. Culture-based insights into wound microbes Historically, culture-based methods have been the gold standard for isolation and identification of microbes within wounds [30]. The majority of these studies have focused on bacterial isolation, where Gram-positive bacteria, especially cocci, are the most frequently isolated microbes from DFUs [31–34]. Among these, Staphylococcus aureus is consistently the most prevalent aerobic species, followed by coagulase-negative Staphylococci spp. and Streptococcus spp. (reviewed in [28]). Some studies have also isolated Corynebacterium species from DFUs, but these are usually regarded as contaminants from the skin [35, 36]. Common Gram-negative bacteria isolated from wounds include Pseudomonas aeruginosa, Enterobacteriaceae species, Klebsiella pneumonia, and Stenotrophomonas maltophilia. Several studies have provided the evidence that besides aerobic Gram-positive and Gram-negative communities, DFUs are also inhabited by anaerobic bacteria. However, the prevalence and abundance anaerobes have been greatly underestimated, due to the limitations of conventional culture methods in identifying them from samples and the overall time-consuming nature of anaerobe isolation. Consequently, their roles in wound

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healing outcomes are understudied (reviewed in [37]). In recent years, researchers have made efforts to optimize specimen collection and isolation of diverse species on larger scales. Citron et al. [33] identified the Gram-positive Finegoldia magna as the most abundant anaerobe in DFU, and other anaerobes identified included Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Anaerococcus spp., Prevotella spp., Porphyromonas spp., and Bacteroides fragilis. In vitro biofilms have discrete niches of oxygen depletion potentially explaining presence of anaerobes in polymicrobial biofilms [38]. Recent advances in ability to model anaerobes in wound infections will elucidate the role of anaerobes alone and as part of polymicrobial biofilms in delayed healing and aberrant host responses [39, 40]. Besides bacteria, fungal infections in immunocompromised patients are of major concern worldwide, and diabetic patients are also susceptible to fungal infections [41, 42]. However, contribution of fungi in DFUs has remained largely elusive. In a large-scale study comprising 518 patients, Candida parapsilosis, Candida tropicalis, Trichosporon asahii, Candida albicans, and Aspergillus species were isolated from DFU [43]. While several culture-based studies have reported Candida spp. as the most abundant fungi recovered, one study reported Aspergillus niger as the most abundant [44, 45]. Culture-based methods have been invaluable in shedding light on the microbial inhabitants of DFU and other chronic wounds. However, these techniques select for microorganisms that thrive under the typical nutritional and physiological conditions employed by diagnostic laboratories over fastidious microorganisms that may be key modulators of wound-healing responses. Further, strains of a single species are divergent across their pangenome, resulting in distinct strain phenotypes and consequently differing effects on pathogenesis [46]. For example, strain heterogeneity of Staphylococcus aureus contributes to differential disease severity in other dermatological conditions such as atopic dermatitis, where strains from more severe patients elicit stronger immune responses and skin inflammation [47]. Toxigenic Staphylococcus aureus strains are rarely isolated from DFUs but are often present in infections with a more severe grade and systemic impact, whereas nontoxigenic strains seem to remain localized in deep structures and bone with diabetic foot osteomyelitis [48]. Others have shown that different strains of Staphylococcus aureus and Streptococcus pyogenes induce variable adaptive immune responses [49]. These observations suggest that the virulence of specific strains of pathogens in a wound may be more meaningful than the totality of all pathogenic species. Strain variability can impact the structure of microbial communities or lead to differential effects on the host wound responses depending on strain virulence. Additionally, complete characterization of the DFU microbiota also requires analyzing viruses and protists that have eluded wound microbiologists [50]. With advent of next-generation sequencing technologies we now have the opportunity to characterize the composition of DFU microbiota at higher resolution and identify biomarkers of infection [28].

The wound microbiome

4. Culture-independent profiling of the DFU microbiome Culture-independent strategies using next-generation sequencing (NGS) have shed novel light on the diverse microbial communities that constitute the human microbiome. Advances in NGS platforms and analysis tools enable comprehensive whole-genome sequencing (WGS) and metagenomic profiling experiments that were not previously possible (reviewed in [51]). These methods circumvent the need to isolate microbes in culture prior to sequence-based identification, a step which limits the range of observable organisms by selecting for those which grow easily in a laboratory environment. Amplicon-based sequencing is the most common strategy used to construct community profiles of skin microbiota. This method has been extensively used to characterize prokaryotic communities by targeting the highly conserved 16S ribosomal RNA (rRNA) gene, which contains hypervariable regions that are widely divergent among different bacterial taxa [52] (Fig. 2). The similar approach can be applied to characterize fungal communities by targeting regions within the fungal rRNA gene operon [53]. Early 16S rRNA gene-based characterizations showed that culture-dependent methods had indeed underestimated bacterial load and diversity in DFUs and other types of chronic wounds [54–56]. More recently, metagenomic shotgun sequencing approaches have been used for profiling microbial communities of the skin. Here, extracted DNA is sequenced directly without being filtered to a specific gene, eliminating potential biases introduced by PCR amplification-based strategies and affording higher resolution of taxonomic data (Fig. 2). The utility, considerations, and challenges while conducting and analyzing data from high-throughput sequencing using either of these strategies are discussed in depth in Section 5 of this chapter. In the next section we will focus on the insights gained from these methodologies to characterize the DFU microbiome.

4.1 Who are the members of the DFU microbiome? Initial studies to catalog the DFU microbiome focused on bacterial communities as surveyed by 16S rRNA gene sequencing (reviewed in [57]). The first culture-independent study was published in 2008 [58]. Unlike culture-based studies, Corynebacterium spp. were identified as the most prevalent taxa (n ¼ 40 DFUs) in this study. Other common genera identified were Bacteroides, Peptoniphilus, Finegoldia, Anaerococcus, Streptococcus, and Serratia spp. In contrast, Corynebacterium were absent from the top 7 reported taxa found to colonize the DFU debridement material, reported by the same group [59]. Others have reported that Streptococcus was more prevalent in the wounds of persons with diabetes (n ¼ 12) than those free of diabetes. In a study of mixed wound types (n ¼ 32), one of the most prevalent types of bacteria, present in 25 of 32 wounds analyzed, was Clostridiales Family XI, a family of fastidious anaerobic bacteria [60]. In a study by Han et al.,

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Fig. 2 Strategies to characterize the wound microbiome. Overview of amplicon-based sequencing approaches (right) versus whole shotgun metagenomic sequencing approaches (left) to characterizing diabetic wound microbiota. Each method utilizes a specimen (e.g., a swab) taken from the wound, from which DNA is then extracted and subjected to either or each protocol.

microbiota colonizing 11 chronic wounds was analyzed and Staphylococcus was present in 10/11 wounds, but in highly variable amounts [61]. These early studies suggested that wound-sampling technique and well-defined patient cohorts were critical factors to obtaining meaningful data. To address the relationship between ulcer and patient-level factors and the microbiome, Gardner et al. analyzed 52 neuropathic, uninfected DFU sampled by Levine’s technique, which samples the deep tissue fluid. In this cohort, Staphylococcus, primarily of the species Staphylococcus aureus, was the most abundant taxa recovered (found in n ¼ 49/52 DFUs). Additionally, the relative abundance of Staphylococcus aureus was negatively correlated with relative abundance of anaerobic bacteria, such as Porphyromonas,

The wound microbiome

Anaerococcus, Finegoldia, Peptoniphilus, Prevotella, and Incertae Sedis XI [54]. Deeper ulcers and those of a longer duration contained a greater microbial diversity and a higher relative abundance of anaerobic bacteria and Gram-negative Proteobacteria. Shallow ulcers and those of a shorter duration were more likely to contain a greater abundance of Staphylococcus, in most cases, the pathogenic Staphylococcus aureus. Furthermore, poor control of blood glucose was associated with a higher relative abundant colonization by Staphylococcus and Streptococcus spp. These findings demonstrated that clinical characteristics of the DFU were associated with distinct microbial communities. Together these and other studies demonstrate that DFU microbiota are heterogeneous and have greater diversity than reported by traditional cultures. Subsequent 16S rRNA gene profiling-based studies of DFUs reached similar conclusions regarding the major taxa associated with DFUs [62–64]. To date, most studies using 16S rRNA gene sequencing and analysis have focused on cataloging the microbiota within the wound tissue. A recent study analyzed the differences in microbiota between normal foot skin and DFUs from the same subject [65]. Microbial diversity was depleted in wound tissue compared to intact skin, although dominant members of microbiota were similar at both sites. Higher abundances of Actinobacteria, Staphylococcus, Corynebacterium, and Propionibacterium were observed in skin specimens compared to wound tissue itself. This finding is consistent with previous reports and is likely because of the stark contrast in nutrient availability [66]. As described earlier, fungi may also colonize wounds (reviewed in [67]). The first study to employ culture-independent analysis of fungi in chronic wounds of mixed etiology found that 40.8% of DFUs harbored fungi and in many specimens, the predicted fungal:bacterial ratio was predicted to be greater than 50%. Candida was the most dominant and most ubiquitous genera with Candida parapsilosis, Candida albicans, Candida tropicalis, and Trichophyton mentagrophytes as the most dominating species in DFUs [68]. Kalan et al. employed PCR-based amplicon sequencing of the fungal ITS1 region to precisely define the prevalence and structure of fungal communities residing in DFUs (n ¼ 100) over time (6 months), in an attempt to link these polymicrobial, interkingdom microbiomes to clinical outcomes [69]. Of the specimens that were culture-positive for fungi, Candida spp. was the most commonly isolated. While Candida albicans was detected in 22% of subjects, contrary to prior studies [68] Cladosporium herbarum, a ubiquitous and saprophytic fungus, was the most abundant in this cohort. Additionally, a clear distribution of wounds containing either a high proportion of pathogenic or allergenic fungi was observed. As an important agent linked to allergic rhinitis and respiratory disease, Cladosporium spp. along with other allergic fungi such as Aspergillus spp., Penicillium spp., Alternaria spp., Pleospora spp., and Fusarium spp. was detected with high frequency in DFUs. Pathogenic and opportunistic fungi, such as Candida spp., Trichosporon asahii, and Rhodotorula spp., were associated with poor outcomes, such as stalled open wounds after 6 months’ treatment, or wounds resulting in an amputation.

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Viruses have been recognized as an important component of the human skin microbiome, but have not been studied extensively in the context of wound healing [70]. Notably, bacteriophage, the viruses that infect bacteria, may play key roles in altering the virulence of their hosts. For example, the filamentous Pf bacteriophage (Pf phage) are produced in abundance by Pseudomonas aeruginosa in wounds and promote Pseudomonas aeruginosa biofilm formation [71, 72]. Recently, Sweere et al. observed that vaccination against Pf phage protects against Pseudomonas aeruginosa wound infections, by preventing host immune responses triggered by Pf that prevented bacterial clearance [73]. Whole metagenomics shotgun sequencing strategies may help us further identify components of the DFU virome that contribute to pathogenicity of their hosts while suggesting potential targets for infection prevention and treatment.

4.2 Temporal stability of the DFU microbiome Most studies that have used culture-independent methods to examine DFUs and other chronic wound microbiomes, have employed cross-sectional designs, which only provide a picture of the microbiome at a single time point. Longitudinal sampling and analysis of the DFU microbiome and variability over time with respect to clinical outcomes allows the investigator to make hypotheses regarding causal mechanisms. However, these studies can be challenging in DFU, where sampling is required to be minimally invasive yet provide an accurate picture of the microbial burden. In one of the first prospective studies to assess the impact of DFU microbiomes and their wound healing outcomes, subjects with DFUs (n ¼ 100) were sampled every 2 weeks until healed, amputated, or study conclusion after 26 weeks of follow up [74]. Using mathematical modeling of diversity and community dynamics within a single wound niche, Loesche et al. showed that microbial community stability was significantly associated with poor clinical outcomes. Specifically, DFU with more dynamic microbiota healed faster than those with less dynamic microbiota [74]. An overview of the mathematical approaches to assess community dynamics is described in Section 5.2. Such methods to model microbial community succession in the wound have confirmed the highly dynamic nature of wound microbial communities in additional cohorts [75]. A longitudinal study design enables the examination of the effects of therapeutic intervention, including systemic antibiotic therapy and debridement, on the DFU microbiome. Kalan et al. observed that debridement elicited a decrease in bacterial diversity, driven by a reduction in the abundance of anaerobic bacteria in the overall community, in the subset of wounds that achieved complete closure within 12 weeks. These findings suggested that several species of anaerobic bacteria are abundant across DFUs in association with mixed aerobes, and successful debridement may disrupt anaerobic networks. In another study where microbiome analyses were performed on 23 DFU patients undergoing foot salvage therapy (FST), wound samples were collected

The wound microbiome

at 0, 4, and 8 weeks following wound debridement and antibiotics treatment. Healing DFUs had a higher abundance of Actinomycetales and Staphylococcaceae while nonhealing DFUs had a higher abundance of Bacteroidales and Streptococcaceae. Additionally, FST elicited marked increases in Actinomycetales in DFU, which was significantly greater in patients that healed [76]. These studies raise the intriguing possibility that the microbiome can serve as a prognostic marker of healing trajectory at the time of debridement. Longitudinal study designs also allow one to glean the effects of antibiotic therapy on the wound microbiome. Kalan et al. found evidence of widespread resistance genes in DFU microbiomes and in some cases, conferring resistance to 10 different classes of antibiotics. Of antibiotic resistance classes detected, the most widespread were genes associated with resistance to beta-lactams, aminoglycosides, and macrolide antibiotics. Examination of the effects of antibiotic administration at the community level suggested little change in overall diversity in healed or nonhealed wounds, suggesting little perturbation to the microbiome within the wound [77]. In contrast, characterization of the fungal mycobiome in the same wound specimens found that fungal diversity increased with antibiotic administration and onset of a clinical complication [69].

5. A focus on methodology: High-throughput sequencing approaches to characterize the diabetic wound microbiota As we will discuss here, there are tradeoffs to each high-throughput sequencing method for microbial community profiling in DFU, and different experimental design decisions enable different types of inference of the wound microbiota [78]. While so-called amplicon-based methods work by sequencing marker genes, which have undergone targeted PCR amplification using gene-specific primers, shotgun metagenomics methods work by directly sequencing fragments of DNA, which have been extracted from wound samples without being filtered to a specific gene. In both cases, the quality of the results depends in part on sequencing depth, which measures how many times each nucleotide is estimated to have been sequenced in the NGS step [79]. In this methodology-focused section, we discuss the contributions of each of these methods toward a more meaningful characterization of the diabetic wound microbiota in aggregate, as well as their experimental and computational limitations.

5.1 Amplicon-based sequencing approaches Sequencing of a single marker gene shared by all organisms of interest (e.g., bacteria or fungi) allows for efficient and scalable taxonomic profiling of diabetic wound specimens. In studies of bacteria, this is usually accomplished by amplifying and sequencing all or select parts of the 16S rRNA gene (Fig. 2). Both fungal and bacterial amplicon sequencing approaches rely on ribosomal genes, which are largely conserved over evolutionary time, but which have taxonomically specific, well-defined “hypervariable” regions

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(Fig. 2). Since these regions can differ in sequence conservation and diversity between phylogenetic clades, the choice of regions to prime for amplification often depends on experimental context. For example, despite the V3-V4 region of the 16S rRNA locus’s demonstrated sequence diversity [80, 81], the V1-V3 region is commonly amplified for wound bacterial profiling in particular because of its improved ability to distinguish between Staphylococcus species [10, 82, 83]. The fungal analog to bacteria’s 16S rRNA gene is the eukaryotic 18S rRNA gene, but fungal community profiling much more frequently targets the internal transcribed spacer regions (ITS-1 and ITS-2) due to their increased hypervariability [67]. Community-level studies of the wound microbiota commonly report taxonomic diversity metrics. Richness is the simplest summary of diversity, describing the raw number of unique taxa present in the sample at a given taxonomic level (e.g., species, or genus). Alpha diversity, which can be calculated several ways, describes richness but usually also accounts for how evenly distributed the abundances of each taxonomic group are in a given sample. Shannon Index and Simpson’s Index are two such measures of alpha diversity. Rather than summarizing single samples, beta diversity describes the “distance” in taxa presence and distribution between two groups of samples [84, 85]. Despite being a vast improvement upon prior methods of wound community profiling such as colony-forming unit (CFU) counts, characterizing microbial communities using 16S rRNA gene sequencing poses several computational problems and carries substantial limitations. First, it relies on a single, well-characterized genetic marker, which is conserved enough across taxa to amplify with a common set of primers, but which also contains enough variable regions to partition the pooled PCR product into different taxonomic groups. This requirement drastically reduces the feasibility of viral metagenomic profiling using even a combination of several PCR-amplified markers [86]. Even the canonical 16S and ITS phylogenetic markers are often indecipherable between species either because of low sequence variability or insufficient reference sequences in the database used for taxonomic assignment. The bioinformatic task of assigning marker gene sequences to their taxonomic origins is nontrivial even with a comprehensive reference database. The task of taxonomic assignment involves first binning reads into sequence-similarity-based groups termed operational taxonomic units (OTUs), and then assigning each OTU a taxonomic classification. Open source tools for binning and classifying 16S rRNA gene-sequencing reads include MOTHUR, QIIME, and HmmUFOtu [87–90]. All three of these amplicon-based profiling pipelines rely on a strong reference set of labeled example genes. This requirement, especially in the case of less well-studied bacterial clades and eukaryotic microbes (e.g., fungi), necessarily limits these methods’ ability to identify microbial community members’ lower-level taxonomic relationships [91].

The wound microbiome

5.1.1 Experimental bias, statistical corrections, and normalization Even in cases where most or all raw marker gene reads are accurately grouped into OTUs, and OTUs are accurately assigned taxonomic origin, the resulting tables of OTU assignments and counts can be difficult to interpret. First, these so-called raw counts of OTUs are inherently biased. Sources of error include differential PCR amplification efficiency (e.g., GC-content in 16S rRNA gene), sparsity associated with taxa of lower abundance in the wound, and inflated sequencing errors at the ends of reads during high-throughput sequencing [86]. The latter source of bias can be mitigated by pairedend sequencing, in which marker genes are sequenced from both the 50 and 30 end [92]. Reducing the sparsity resulting from rare but present taxa’s being “missed” during sequencing would require a dramatic increase in sequencing depth to the point of saturation, which means enough reads are sequenced to represent all variations of the marker gene in a wound sample [93]. In practice, this is technically infeasible in microbial communities as complex as those inhabiting chronic wounds, particularly since increasing sequence depth-by-sample imposes practical limits on the number of samples that can be processed. The observation that more rare species tend to be observed in samples sequenced at a greater depth further complicates the comparison of different samples’ microbial compositions [93]. There are several approaches to correcting these biases following OTU assignment. One common correction method, termed rarefaction, involves randomly drawing an equal number of reads from every sample’s total OTU-assigned reads to simulate a common sequencing depth across samples. As McMurdie and Holmes [94] point out, this approach can easily erase the presence of lower abundance taxa entirely or, alternatively, artificially inflate their abundances due to random error. They instead recommend normalizing by Variance Stabilization, a transformation borrowed from methods developed for normalizing RNA-seq output, which models the noise in counts of OTUs across samples as a negative binomial distribution, logarithmically transforming the data to enforce a shared variance between samples. 5.1.2 Current utility and future prospects for amplicon-based microbiome studies of diabetic wound Despite its technical challenges, limited resolution, and bacteria or fungi-specific nature, amplicon sequencing underlies most recent studies of the diabetic wound microbiota. About a fifth the cost of metagenomic shotgun sequencing by some estimates [95], 16S rRNA gene and ITS sequencing can provide a general, if not completely detailed, summary of taxa present in a wound. Because these methods are scalable and relatively inexpensive, they enable the profiling of diabetic wound samples from large clinical cohorts. Wolcott et al. [62], for example, obtained 16S rRNA gene-based bacterial

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profiles for 2963 chronic wounds, including 910 diabetic foot wounds, and reported abundant bacterial taxa in these wounds at a genus level. Larger sample sizes also enable computational analyses of microbial wound dynamics that require more biological replicates. Along these lines, Loesche et al. [74] characterized changes in wound microbial composition over time in terms of taxonomic clusters termed “community types” rather than specific bacterial taxa or traditional metrics such as species richness or diversity. Adopting Dirichlet multinomial mixture models from studies of gut microbial “enterotypes,” 16S rRNA gene profiles of 349 longitudinally collected wound samples were divided into four robust community types. Frequencies with which wounds changed from one type to another over the course of healing and infection were analyzed. Defining wounds by their community-wide characteristics showed that wound complications and chronic infections corresponded to bacterial communities that remained static over time, and which were dominated by a few bacterial taxa. Kalan et al. [69] similarly applied amplicon-based sequencing approaches to characterize the fungal contents of the DFU microbiome by amplifying and sequencing the eukaryotic ITS1 locus in longitudinal wound samples from 100 diabetic patients. Given the general lack of knowledge about the fungal role of diabetic wound healing, and given the lack of fungal examples in reference databases, they identified relationships between fungal community composition and clinical outcomes in the absence of reference-based OTU assignments. Where possible, fungal OTUs with taxonomic identifications were grouped into “pathogen” and “allergen” categories based on prior annotations of assigned taxa, rather than focusing on species or genera-specific relationships. Comparing the relative abundances of these two functional designations demonstrated that wounds that deteriorated due to necrosis tended to have higher proportions of fungi labeled as pathogenic. Amplicon-based microbiota sequencing allows for high-throughput, temporally specific, and culture-free profiling of many microbiome samples at once. Accordingly, longitudinal, marker gene-based studies of wound healing in large diabetic cohorts have provided more comprehensive insights into these microbial communities than the culture-based studies preceding them. Still, the fact that this approach relies on a single ribosomal gene marker introduces a world of bias, from PCR-bias that complicates its quantitative interpretation, to its frequent failure to characterize species and strain-level composition of wounds and its inability to account for viruses and bacteriophage. Whole metagenomic shotgun sequencing, though expensive and computationally challenging, addresses many of these limitations.

5.2 Whole metagenomic shotgun sequencing Unlike amplicon-based microbial profiling, whole metagenome shotgun sequencing does not require sample enrichment for a single, well-characterized target gene

The wound microbiome

(see Fig. 2). Instead, DNA is purified and fragmented directly from each metagenomic sample. The resulting fragments are then sequenced in bulk and filtered to remove host DNA (e.g., reads that map to the human genome). These filtered sequences can be used to construct detailed taxonomic and functional summaries of the microbial communities they represent. Whole metagenomic shotgun sequencing (WMS) can therefore capture far more complexity in wound microbial composition than the amplicon-based approaches which preceded it. In particular, it circumvents the amplification bias associated with the PCR step of amplicon-based methods, captures viral genomic content, and can distinguish between individuals of the same bacterial species with strain-level specificity. Given that specific strains of common skin microbes such as Staphylococcus epidermidis and Staphylococcus aureus can provoke adverse host immune responses in contexts where these species typically act as commensals, the strain-level resolution of WMS makes it a uniquely informative tool for finding clinically correlated abundance patterns in diabetic wounds. This was evident in the study of Kalan et al. [77], where Staphylococcus aureus was present in almost every DFU sample regardless of clinical outcome. Deconstruction of Staphylococcus aureus at the strain-level revealed that nonhealing DFU contained different strains than those which healed. At least one of these “nonhealing” strains contained virulence genes that may be the result of bacteriophage insertion. These results illustrate the importance of gene and strain-level microbial composition of diabetic wounds, and motivate additional WMS investigations into wound healing. Though this study is the first to investigate microbial communities in wounds using WMS approaches, multiple groups have demonstrated the strength of this method to identify clinically relevant dynamics in other skin microbial communities. Oh et al. [96] demonstrated the diversity of Propionibacterium acnes and Staphylococcus aureus strains in healthy skin, and even estimated the abundance of skin taxa lacking reference genomes by clustering them according to gene content similarity. Byrd et al. [47] identified Staphylococcus aureus and Staphylococcus epidermidis strains that are differentially associated with skin inflammation in atopic dermatitis. In a study highlighting the tremendous taxonomic and functional diversity of viruses on the skin, Hannigan et al. [70] used co-occurrence networks to investigate the interactions between bacterial species and the viral entities that infect them. Though not directly related to diabetic wound healing, these studies describe functional relationships that could prove to be important in identifying the mechanistic contributions of skin microbiota to wound-healing outcomes. 5.2.1 Experimental limitations and computational challenges WMS profiles can provide a wealth of information about the microbial contents of diabetic wounds, but the methods required to construct these profiles are labor-intensive, costly, and computationally arduous. First, each sample has to be sequenced with enough depth to capture the maximum proportion of genomes in the sample. Kalan et al. [77], for example, found that the positive relationship between the number of reads sequenced

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and the median sequencing depth only leveled off when they had sequenced 1  108 reads per sample. These stringent sequencing requirements impose a tradeoff between sample resolution and the number of samples sequenced. Achieving adequate depth is further complicated by the need to remove human sequence reads, since the bulk sequencing approach captures all available DNA rather than just an enriched bacterial or fungal marker gene. Even if libraries are constructed with adequate depth and coverage, and host/control reads are properly filtered out, processing WMS reads is still a computationally intensive task. First, summarizing the metagenome in terms of relative abundances of different genera, species, and strains necessitates the taxonomic assignment of the sequenced reads, and some way of estimating relative counts of reads from different taxa. The process of assembling full microbial genomes from these fragments is further complicated by the fact that the metagenome contains species of differing genome lengths at different abundances [97], as well as the lack of available reference assemblies in NCBI particularly for nonbacterial taxa (see Fig. 3). While some publicly available methods for this process attempt to patch together overlapping reads into contigs before mapping them against a database of reference genomes, others map reads directly to a database of small marker sequences associated with known taxa. Based on these read assignments, most methods estimate the relative abundance of each taxonomic group in the sample [97–99]. MetaPhlan2 (2015) is one freely available marker gene approach, and its derivative method StrainPhlan (2017) expands this previous marker database to estimate the abundance of different bacterial strains in addition to species [100, 101]. These publicly available, well-documented tools make shotgun metagenomic profile construction more feasible than ever before. Truong et al. [100] measured the performance of MetaPhlan2 specifically in the context of healthy skin microbiota, making a strong case for its utility in studying cutaneous microbial communities including those found in DFUs. 5.2.2 Functional characterization of DFU microbiota using gene-level metagenomic profiles The information contained in sequenced shotgun reads is not limited to taxonomic composition. Because these pooled reads, in theory, represent the raw genomic contents of the microbial community they sample, defining them in terms of their gene contents without attention to taxonomic assignments can elucidate wounds’ functional capabilities for antibiotic resistance, virulence, and responses to host tissue-healing processes. One way to do this is to compare the pool of sequenced reads to SEED, which aggregates functional annotations of microbial genes into an online database [102]. Kalan et al. [77] searched the sequencing reads from their DFU samples against SEED, and found that wounds with impaired healing or chronic nonhealing outcomes contained higher abundances of biofilm-annotated genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) can also serve as a reference database for annotating metagenomic

The wound microbiome

Fig. 3 Number of available reference genome assemblies (y-axis) deposited in NCBI for archaea, fungi, viruses, and bacteria that are complete (dark pink) or incomplete (light pink). NCBI was accessed on 03/24/19 through https://www.ncbi.nlm.nih.gov/genome/browse#!/overview/.

shotgun profiles by gene content, and the publicly available software MGS-Fast automates this process, enabling efficient analysis for functionally important pathways [103–105]. Though these methods are not useful for inferring which genes are expressed by which microbes in the wound, identifying the functional gene annotations enriched in diabetic wounds that show better or worse clinical outcomes might still suggest mechanisms by which the microbes in DFU affect their healing.

6. Future directions 6.1 Microbial lifestyles: Impact of biofilms As discussed in Section 2, it is widely believed that formation of polymicrobial biofilms is a part of the wound infection continuum. While there is consensus that biofilm impedes

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wound healing, detection and diagnosis of biofilm in wound tissue is difficult. In vitro studies have shed light on the life cycle of biofilm-forming organisms, but microbial behavior in complex tissues such as a chronic wound is less certain. This is partly because biofilms remain difficult to be diagnosed clinically and factors that drive polymicrobial biofilms remain unresolved. Currently microscopy-based techniques such as scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) remain the most powerful tool to diagnose wound biofilms [106]. However, these approaches are limited in dissecting the polymicrobial composition of biofilms. There has been progress in developing FISH methods (e.g., Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH)) that involves labeling microbes of interest with combinations of probes coupled with spectral imaging to allow distinguishing different taxa of microbes [107]. Use of CLASI-FISH in oral biofilm models suggests that spatial distribution of bacteria in communities is potentially an important factor in understanding the effects of microbial communities [108]. In these studies, the authors showed that in dental plaques filamentous Corynebacterium provided the scaffold for remaining bacteria. Interactions between some clinically important bacterial and fungal species have also been described. Candida spp. have been reported to interact with diverse bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cenocepacia, Streptococcus spp, Acinetobacter baumannii, E. faecalis, and E. coli [109]. Kalan et al. observed that Candida albicans and Citrobacter freundii that were coisolated from the same DFU formed three-dimensional biofilms in vitro and Candida albicans provided the scaffold for Citrobacter freundii to intimately attach and proliferate [69]. This suggests that fungi themselves are critical to the construction and establishment of the biofilm. These data suggest that the biogeography of the microbes is critical in understanding the physiology and ecology of bacterial communities as well [110]. A combination of advanced imaging approaches coupled with high-throughput sequencing could help dissecting spatial roles of microbes in polymicrobial communities.

6.2 From microbial census to gene expression Annotations of isolated whole-genome sequences or individual metagenomic shotgun reads can provide some insight into the functional potential of the microbes inhabiting diabetic wounds. Inferring the true functional activity of these microbes, however, requires measures of the mRNA transcripts expressed by the microbial genomes present. Quantitative real-time PCR (qPCR) is one way to do this; given a set of known genes of interest with known sequences, it is possible to extract the RNA transcripts present in a wound sample, reverse transcribe these transcripts into labeled cDNA labeled with genetargeted fluorescent probes, and infer the relative expression levels of the genes tested based on the strength of the fluorescent signal [111]. In practice, many more microbial genes are likely to be important in DFU infection and healing than can be analyzed feasibly by qPCR platforms, and it is difficult to identify a set of likely candidates to test.

The wound microbiome

One promising approach for characterizing microbial gene expression in DFU is RNA-seq, wherein all RNA in a wound specimen are sequenced in high throughput to yield a comprehensive picture of the genes expressed in a given sample at a given time. Because many orthologous genes are nearly identical between bacterial species, RNA-seq studies of wound microbial communities must currently be limited to a single species, and are thus far restricted to ex vivo bacterial cultures from wound environments. One such study, done in burn wounds and soft-tissue infections, performed RNA-seq directly from a polymicrobial wound sample, by filtering out most of the resulting reads (from host and other colonizing microbiota) to get a robust gene expression profile for Pseudomonas aeruginosa [112]. In another study, Pseudomonas aeruginosa PA01, a lab reference strain, was grown on human burn wound exudate and RNA expression profiles were compared to expression profiles of pure planktonic cultures [113]. These studies both made important discoveries about how the wound environment shifts Pseudomonas aeruginosa gene expression and adaptation to the wound environment. However, it remains a challenge to captured polymicrobial interactions and expression in vivo, and to simultaneously profile the host gene expression responses. If developed to better capture polymicrobial, in vivo bacterial and host expression, RNA-seq methods could infer causal mechanisms of microbial-host interactions in DFUs.

6.3 Harnessing the therapeutic potential of the microbiome Microorganisms produce bioactive secondary metabolites, or “natural products,” with unique chemical structure and potent cytotoxic, antitumor, antimicrobial, and antiinflammatory activity [114]. These metabolites often mediate interactions with other microbes and with the host tissue environment. For example, some commensal microbes produce short-chain fatty acids (SCFAs), and when injected or applied topically, SCFAs such as acetic, butyric, and propionic acid suppress cutaneous inflammation by promoting skin T-regulatory cells (Tregs) [115]. Human skin and nasal commensal bacteria, such as such as Staphylococcus epidermidis, Staphylococcus lugdunensis, and Staphylococcus hominis, produce antibiotic molecules that can impair colonization and infection of Staphylococcus aureus [116–118]. Some studies have suggested that using probiotic microbes can alter the skin microbiome and improve cutaneous healing [119]. In burn wounds Lactobacillus plantarum-containing probiotics inhibited biofilm formation by Pseudomonas, improving tissue repair in murine and rabbit skin and decreasing mortality in porcine models [120–122]. Together these suggest that the microbiome is still a largely unexplored area with tremendous potential to build cost-effective novel therapies.

7. Summary Culture-independent, next-generation sequencing-based approaches for characterizing wound bioburden have driven novel and significant insights into the role of the microbiome in tuning wound-healing responses and clinical outcomes, including

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infection-related complications. While amplicon-based sequencing methods (e.g., bacterial 16S rRNA gene sequencing, fungal ITS sequencing) provided substantial strides in uncovering the previously underappreciated microbial diversity in diabetic wounds, more sophisticated approaches such as shotgun metagenomic sequencing have provided clinically relevant insights into implications of strain-level diversity, mechanisms of virulence, and response to therapeutics. These methods will continue to evolve, with increased emphasis on functional implications of microbial inhabitants and their interactions with each other and the host. Furthermore, the microbiome is an accessible and modifiable therapeutic target; fundamental understanding of its components will reveal novel targets for the management and treatment of diabetic wounds.

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[46] Read TD, Massey RC. Characterizing the genetic basis of bacterial phenotypes using genome-wide association studies: a new direction for bacteriology. Genome Med 2014;6(11):109. [47] Byrd AL, et al. Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis. Sci Transl Med 2017;9(397):1–10. [48] Dunyach-Remy C, et al. Staphylococcus aureus toxins and diabetic foot ulcers: role in pathogenesis and interest in diagnosis. Toxins (Basel) 2016;8(7):1–20. [49] Sela U, et al. Strains of bacterial species induce a greatly varied acute adaptive immune response: the contribution of the accessory genome. PLoS Pathog 2018;14(1):e1006726. [50] Johnson TR, et al. The cutaneous microbiome and wounds: new molecular targets to promote wound healing. Int J Mol Sci 2018;19(9):1–19. [51] Hodkinson BP, Grice EA. Next-generation sequencing: a review of technologies and tools for wound microbiome research. Adv Wound Care (New Rochelle) 2015;4(1):50–8. [52] Grogan MD, et al. Research techniques made simple: profiling the skin microbiota. J Invest Dermatol 2019;139(4):747–52. e1. [53] Schoch CL, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A 2012;109(16):6241–6. [54] Gardner SE, et al. The neuropathic diabetic foot ulcer microbiome is associated with clinical factors. Diabetes 2013;62(3):923–30. [55] Thomsen TR, et al. The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods. Wound Repair Regen 2010;18(1):38–49. [56] Yarza P, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 2014;12(9):635–45. [57] Misic AM, Gardner SE, Grice EA. The wound microbiome: modern approaches to examining the role of microorganisms in impaired chronic wound healing. Adv Wound Care (New Rochelle) 2014;3(7):502–10. [58] Dowd SE, et al. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP). PLoS One 2008;3(10): e3326. [59] Dowd SE, et al. Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol 2008;8:43. [60] Price LB, et al. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota. PLoS One 2009;4(7):e6462. [61] Han A, et al. The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds. Wound Repair Regen 2011;19(5):532–41. [62] Wolcott RD, et al. Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing. Wound Repair Regen 2016;24(1):163–74. [63] Gontcharova V, et al. A comparison of bacterial composition in diabetic ulcers and contralateral intact skin. Open Microbiol J 2010;4:8–19. [64] Oates A, et al. Molecular and culture-based assessment of the microbial diversity of diabetic chronic foot wounds and contralateral skin sites. J Clin Microbiol 2012;50(7):2263–71. [65] Park JU, et al. Influence of microbiota on diabetic foot wound in comparison with adjacent normal skin based on the clinical features. Biomed Res Int 2019;2019:7459236. [66] Gardiner M, et al. A longitudinal study of the diabetic skin and wound microbiome. Peer J 2017;5: e3543. [67] Kalan L, Grice EA. Fungi in the wound microbiome. Adv Wound Care (New Rochelle) 2018;7 (7):247–55. [68] Dowd SE, et al. Survey of fungi and yeast in polymicrobial infections in chronic wounds. J Wound Care 2011;20(1):40–7. [69] Kalan L, et al. Redefining the chronic-wound microbiome: fungal communities are prevalent, dynamic, and associated with delayed healing. MBio 2016;7(5):1–12. [70] Hannigan GD, et al. The human skin double-stranded DNA virome: topographical and temporal diversity, genetic enrichment, and dynamic associations with the host microbiome. MBio 2015;6 (5). e01578-15.

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CHAPTER 13

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing Sayantan Maitraa,∗, Dibyendu Duttab,∗ a

Government of West Bengal, Department of Health and Family Welfare, Institute of Pharmacy, Jalpaiguri, India Bengal College of Pharmaceutical Sciences and Research, Durgapur, India

b

1. Introduction Disruption of the integrity of skin, mucosal surfaces, or organ tissue causes the formation of a wound. Wounds can be associated with a disease process or have an accidental or intentional etiology. At the time of damage to a tissue, several intracellular as well as extracellular pathways are activated in a coordinated manner in the aim of restoring tissue integrity [1]. Wound healing is a physiologic process involving a series of sequential yet overlapping stages. Classically this process of wound healing is divided into four distinct stages: hemostasis, inflammation, proliferation, and tissue remodeling [2]. The first stage, hemostasis, occurs with the onset of injury and is usually completed within hours. The second stage, inflammation, occurs immediate to hemostasis and usually is completed within 24–72 h of the injury. Proliferation, the third stage, usually aims to repair the injury and takes place after 1–3 weeks of injury. The fourth and final stage, remodeling, initiates generally after 3 weeks of injury and restoration to the normal physiologic condition of the injured tissue may take up to several months to years [2, 3]. The physiological process of wound healing is described as follows.

1.1 Hemostasis The onset of skin injury leads to bleeding, which in turn helps to flush the microorganisms and antigens out of the wound, as well as triggering the hemostasis. Blood loss at this stage is prevented by clot formation, which is achieved by three key mechanisms: intrinsic pathway (endothelial damage causes activation of factor XII, which further causes proteolytic cleavage cascade and activates factor X that converts prothrombin to thrombin and consequently fibrinogen to fibrin, which forms the clot); extrinsic pathway (endothelial damage leads to exposure of tissue factor to the blood and that activates factor VII, ∗

These authors contributed equally to this work.

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00013-7

© 2020 Elsevier Inc. All rights reserved.

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which in turn causes activation of thrombin); and platelet activation (activation of thrombin, thromboxane, adenosine diphosphate (ADP) results in morphological alteration of platelets [4]. The activated platelets aggregate and clump at the site of exposed collagens to form platelet plugs and transiently hinder the bleeding) [1].

1.2 Inflammation The major aim of this stage of wound healing is to prevent infection. Regardless of the etiology of the wound, the frontline defense system, i.e., the mechanical barrier, gets ruptured. Within minutes of injury, neutrophils, which are highly motile, infiltrate the wound and migrate at sustained levels for 48 h. The infiltration of neutrophils to the site of injury depends upon the chemical gradient (chemotactic movement), mediated by various chemical signaling including interleukin activation and transforming growth factor-β (TGF-β) signaling. In addition, other white blood cells (WBCs) including monocytes, lymphocytes and plasma cells migrate to the injury site. Among them, neutrophils are the most predominant for the first few days after injury and disappear when there is no more infection. In the absence of infection, the monocytes differentiate into macrophages and become major phagocytic cells at the site of injury. Along with surviving microorganisms, dead neutrophils, cell debris, and fibrins are ingested by macrophages. Macrophages also synthesize cytokines along with various growth factors involved in proliferation, organization of new connective tissue, and vascular beds within the wound [2, 5].

1.3 Proliferation The key aim of this stage is to cover the wound skin with new skin (reepithelialization), restoring vascular integrity to the site of injury (neovascularization), and repairing the altered tissue structure by recruiting new connective tissues (granulation). Shortly after the injury, hypoxia and acidosis develop within the vascular bed that in turn promote angiogenesis and synthesis of new collagen. As the process of angiogenesis proceeds, new vascular structure develops within the wound, aiming to restore the vascular integrity. Reepithelialization of a wound occurs when keratinocytes completely cover the surface of the wound skin. An embryological process known as epithelial to mesenchymal transition (EMT) allows the motility of epithelial cells to reach into the site of injury. Once the migration of keratinocytes is completed, they begin to stabilize themselves by establishing firm attachments with each other. When the skin is covered with epidermal cells, the wound is considered closed [2].

1.4 Remodeling This stage maximally prolongs up to 2 years and results in the development of normal epithelium and maturation of scar tissue. This involves a perfect equilibrium between

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing

the synthesis and degradation, as the collagen and other associated proteins accumulated in the wound become increasingly well organized. Ultimately, they restore a structure similar to that seen in the unwounded tissue [1]. The healing of a wound is a physiological process that can take up to a maximum of 2–3 years. There are several factors that can disrupt this physiological process of healing, such as increased stress, age, ischemia, alcoholism, smoking, improper nutrition, etc., which are the behavioral factors. Several disease conditions can also impede the normal wound healing process, such as fibrosis, jaundice, obesity, diabetes, cancer and acquired immune deficiency syndrome (AIDS) [6]. Among these, diabetes is the most prevalent cause of impaired wound healing. The incidence of diabetes mellitus is growing at an alarming rate and worldwide it is reaching epidemic proportions. The diabetic foot ulcer (DFU) is very common in diabetic patients and it has been estimated that the lifetime risk of DFU incidence is 25% [6]. The hyperglycemic milieu of the diabetic condition has deleterious effects on wound healing, as the advance glycation end products (AGEs) induce the production of inflammatory cytokines (TNF-α, IL-1) and hinder collagen synthesis [7]. High blood glucose level causes the alteration of cellular morphology, decreased rate of cellular proliferation, and abnormal differentiation of keratinocytes, which in turn are associated with delayed wound healing [8]. Moreover, the diabetic condition affects the immune response and also decreases the resistance to infection. Thus diabetic wounds impair healing through several mechanisms (Fig. 1) [9].

2. Influence of diabetes on functions of neutrophils It is widely accepted that diabetic patients, irrespective of the type of their disease, are more prone to infections and this is attributed to metabolic derangements in the diabetic milieu [10]. Polymorphonuclear neutrophils (PMNs) are the main defensive weapon in the innate immune system. They should attach to the endothelium, surrounding the inflammatory focus (adhesion) and migrate toward the chemotactic factor (chemotaxis). They must contact and ingest the invading organism (phagocytosis) and kill the microbe (bactericidal capacity) [11].

2.1 Hindrance in energy supply In diabetes, the functions of PMNs are altered and the normal physiological processes in wound healing are hindered. The phagocytic activity of PMNs impairs due to lack of intracellular adenosine triphosphate (ATP) resulting from disturbances in carbohydrate metabolism [12]. In diabetes, the excess blood glucose is converted into sorbitol by the polyol pathway. The calorific value of glucose is 3.9 kcal/g whereas in the case of sorbitol it is 2.4 kcal/g. The difference in energy produced by glucose and sorbitol metabolism clearly supports the altered activity of PMNs. The chemotaxis of PMNs to the site

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Fig. 1 Events that cumulatively impair wound healing in diabetes: increased expression and activities of aldose reductase and sorbitol dehydrogenase that decrease the activity of myoinositol, resulting in impaired cell signaling and altered growth factor production; the increased production of reactive oxygen species (ROS) that leads to impaired angiogenesis and oxidative killing; and decreased activities of hexokinase and phosphofructokinase result in a shortage of ATP synthesis, which is required for several functions of neutrophils. All these events cumulatively complicate the wound healing in diabetes.

of injury is completely diminished at a blood glucose level of 300 mg/dL due to intracellular energetic disturbances [11].

2.2 Involvement of polyol pathway Neutrophil dysfunction is also associated with the polyol pathway (Fig. 2). Under normal physiological conditions, glucose is converted to glucose-6-phosphate by hexokinase. But in a hyperglycemic milieu the hexokinase pathway becomes saturated, resulting in conversion of excess glucose into sorbitol by aldose reductase, followed by conversion of sorbitol to fructose by sorbitol dehydrogenase and then to fructose-3-phosphate by

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing

Fig. 2 Polyol pathway in impaired energy metabolism: In normal physiological conditions, glucose is converted into glucose 6-phosphate (G-6-P) by the action of hexokinase and one molecule of ATP is converted into ADP. This is followed by conversion of glucose 6-phosphate to fructose 6-phosphate (F-6-P), which is a key intermediate to produce energy in cells (glycolytic pathway). Glucose is also converted into sorbitol by aldose reductase and furthermore to fructose by sorbitol dehydrogenase in a very lower degree. Fructose then is converted to fructose 6-phosphate by hexokinase (polyol pathway). In diabetic conditions, the rate of sorbitol synthesis is elevated and the function of the glycolytic enzyme hexokinase is decreased (depicted by #), which is further associated with altered cellular energetics.

3-phosphokinase. Once sorbitol is produced, it does not diffuse easily across the cell membrane and this intracellular deposition of sorbitol gives rise to diabetic complications. Sorbitol in the diabetic condition gives rise to intracellular osmolarity, decreases the availability of enzyme cofactor nicotinamide adenine dinucleotide phosphate (NADPH), leading to alteration of endothelial cell functions, that changes the course of leukocyte-endothelial cell interaction [13, 14].

2.3 Disrupted bactericidal activity The bactericidal activity of neutrophils is partially mediated by oxidative killing. The oxidative burst required to kill pathogens is highly reliant upon biochemical events, and those are susceptible to impaired glucose metabolism. The elevation of the polyol pathway in high glucose concentration decreases the depletion of superoxide anions on the membrane of neutrophils, resulting in dysfunction of the oxidative killing [14, 15].

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2.4 Inhibition of chemotaxis Polymorphonuclear neutrophils (PMNs) are the first line of defense against invading pathogens. PMNs possess specialized receptors, namely formyl peptide receptors (FPRs), that recognize the formylated peptides that shade the bacteria, such as N-formyl-Met-Leul-Phe (FMLP). Stimulation of FPR induces intracellular release of ATP. Release of ATP in extracellular space through pannexin-1 (panx1) channels facilitates ATP to be released at specific membrane regions to promote gradient sensing, pseudopod protrusion, and coordinated cell migration during PMN chemotaxis. When released into extracellular space, ATP acts as an intercellular messenger and activates purinergic receptors via paracrine or autocrine mechanisms. Purinergic receptors are broadly classified into two families: P1 adenosine receptors and P2 nucleotide receptors. P2 receptors are further divided into the P2X and P2Y receptor subfamilies. The seven-membered P2X receptor subfamily functions as ATP-gated ion channels, and the P2Y receptor subfamily consists of eight members that are G-protein coupled receptors (GPCRs) and recognize ATP, uridine triphosphate, and related molecules. Extracellular ATP regulates chemotaxis of PMNs via P2Y2 receptors. PMNs contain very few mitochondria and mainly rely upon the glycolysis to produce ATP that triggers the purinergic signaling cascade to activate PMNs for carrying out regulatory functions [16, 17]. Production of ATP depends upon the conversion of glucose into lactate via glycolysis. In diabetic conditions, the activity of the key glycolytic enzyme, i.e., hexokinase, is downregulated [18, 19], which in turn suppresses the production of intracellular ATP as well as extracellular available ATP required for activation of PMNs and leads to impaired wound healing (Fig. 3).

3. Influence of diabetes on functions of keratinocytes In the proliferative stage of wound healing, keratinocytes migrate and proliferate in wound edges or skin adnexal structures with the aim of reepithelialization. The migration of keratinocytes is induced by fibroblast growth factor (FGF), endothelial growth factor (EGF), and keratinocyte growth factor (KGF). The proteolysis of matrix proteins enables migration of epithelial cells into wounds. Matrix metalloproteinases (MMPs), belonging to the family of zinc-dependent endoproteinases, regulate the degradation of extracellular matrix (ECM) proteins, such as laminin, fibronectin, and collagen. MMPs play a pivotal role in many physiological processes including wound healing [20].

3.1 Impaired migration Many factors are associated with the locomotion of keratinocytes, including proteolysis of ECM proteins by MMPs. Another important factor that monitors the migration and spreading of keratinocytes is focal adhesion kinase (p125FAK), a nonreceptor

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing

Fig. 3 Inhibition of chemotactic movement of PMNs: Bacterial FMLP stimulates FPRs that trigger a first phase of Ca2+ mobilization, mitochondrial activation, and ATP production, followed by rapid ATP release in extracellular space via Panx1 channels. The release of ATP initiates a second round of Ca2+ signaling via activation of P2Y2 receptor. ATP generation via the glycolytic pathway and its release leads to activation of P2X receptors that contribute to a third phase of Ca2+ signaling due to influx from the extracellular space. The second phase of ATP signaling maintains intracellular Ca2+ levels and regulates functional PMN responses. But the high glucose environment in the diabetic condition downregulates the activity of hexokinase that leads to a cut down to the ATP supply for a second phase of ATP signaling, followed by inactivation and impaired chemotaxis of PMNs.

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protein-tyrosine-kinase. Increased p125FAK and its phosphorylation is essential to modulate the cytoskeletal proteins, which in turn regulates the motility of cells [21, 22]. In diabetic patients, the high blood glucose is associated with the decreased expression of MMPs and p125FAK, which are the key factors for migration of keratinocytes to the inflamed tissue. This downregulation of essential factors hinders the reepithelialization process and thereby impairs wound healing [20, 23].

3.2 Impaired angiogenesis Angiogenesis is essential for reconstructing the damaged vascular integrity. The downregulation of antiangiogenic factors and upregulation of angiogenic factors stimulates angiogenesis. Thrombospondin-1 (TSP-1), a high molecular weight glycoprotein, is a potent physiological inhibitor of angiogenesis, which is continuously synthesized in normal human cells [24]. Keratinocyte-derived TSP-1 is a key regulator of angiogenesis during wound healing. The high glucose environment upregulates the expression of this angiogenesis suppressor via elevated oxidative stress induced DNA hypomethylation at the TSP-1 promoter region in keratinocytes. Thus the hyperglycemic condition triggers a plethora of defects in normal function of the physiology of keratinocytes, leading to impaired wound healing in diabetes (Fig. 4) [20, 25].

4. Influence of diabetes on apoptosis The excess blood glucose present in the diabetic condition leads to formation of advanced glycation end products (AGEs). The carbonyl group of glucose or glucose 6-phosphate interacts with free amino acid of circulatory protein and forms unstable Schiff bases. After a few days, structural rearrangements of Schiff bases lead to generation of keto-amines or Amadori products. The Amadori products then undergo further structural rearrangement and modifications via an oxidative or nonoxidative pathway or through polymerization in order to form more stable, irreversible AGEs [26, 27]. An elevation in AGEs has been linked to many complications associated with several diseases, such as delayed wound healing and vascular complications seen in patients with diabetes and cardiovascular disease [28–31].

4.1 Contribution of AGEs An interaction between AGEs and receptor for AGE (RAGE) activates a transcription factor, namely nuclear factor-κβ (NF-κβ), which regulates synthesis of several genes essential for the immune system; its activation has been associated with the expression of numerous inflammatory cytokines, including IL-6, IL-1a, TNF-α, and various growth factors. This eventually leads to a sustained inflammatory phase in wound healing.

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing

Fig. 4 Effect of AGEs on diabetic wound healing: Elevation of increased matrix metalloproteinase (MMP) activity in the diabetic condition leads to an aberrant collagen breakdown and the decreased release of fibroblast growth factors (FGFs) results in minimal collagen synthesis; these events aggravate faulty keratinocyte function and impair the repairing of the wound. Advanced glycation end products (AGEs) are formed in hyperglycemia, which raises the expression of proapoptotic transcription factor FOXO1 and also elevates oxidative stress, leading to an increased apoptosis of fibroblasts followed by impaired healing of the wound.

In order for wounds to heal effectively, the inflammatory phase has to cease so that later stages of wound healing may proceed [32–34]. Fibroblasts and FGFs are essential for collagen synthesis and reduced collagen synthesis leads to impaired dermal wound healing. AGEs stimulate apoptosis and decrease proliferation of fibroblasts and also diminish the activity of FGF. AGEs induce apoptosis of fibroblasts by activation of caspase 3 and caspase 8, thereby signaling apoptosis via a cytosolic pathway, and caspase 9, thereby signaling apoptosis through a mitochondrial pathway [35, 36].

4.2 Apoptosis of fibroblasts N€-(carboxymethyl) lysine (CML) is a prevalent AGE structure and is generally produced by oxidative cleavage of Amadori intermediates. AGE, CML-collagen induce fibroblast apoptosis via reacting to RAGE, which in turn stimulates cytosolic and mitochondrial apoptosis [37]. CML-collagen increases the generation of the intracellular ROS, NO,

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ceramide, and mitogen activated protein kinase (MAPK) cascade, which eventually activates different transcription factors including NF-κβ [38]. Stimulation of the transcription factor enhances the expression of inflammatory cytokines such as IL-1, TNF-α that in turn activate JNK and p38 MAPK followed by the activation of proapoptotic transcription factor FOXO1; this ultimately leads to fibroblast apoptosis [39, 40]. The role of AGEs in delayed wound healing is articulated in Fig. 4.

5. Conclusion Wound healing in diabetes is greatly disrupted by a plethora of events that are associated with each other. In the current scenario diabetes is one of the most prevalent diseases and is growing at an alarming rate. Adequate information is needed regarding the impeded healing of wounds in the diabetic condition in order to cut down the rate of morbidity. High blood glucose concentrations lead to increased oxidative stress, altered glucose metabolism, deficiency in ATP synthesis as well as its utilization, increased apoptosis and decreased proliferation of fibroblasts, and increased expression of MMPs thereby decreasing the function of collagen; all these events cumulatively aggravate the healing of wounds. Proper blood glucose monitoring is warranted in such conditions along with the conventional treatment approaches. New targets such as FOXO1, p125FAK and TSP-1 can be modulated to improve the conditions associated with diabetic wound healing.

References [1] Singh S, Young A, McNaught C-E. The physiology of wound healing. Surgery (Oxford) 2017; 35(9):473–7. [2] Strodtbeck F. Physiology of wound healing. Newborn Infant Nurs Rev 2001;1(1):43–52. [3] Qing C. The molecular biology in wound healing & non-healing wound. Chin J Traumatol 2017; 20(4):189–93. [4] Zarbock A, Polanowska-Grabowska RK, Ley K. Platelet-neutrophil-interactions: linking hemostasis and inflammation. Blood Rev 2007;21(2):99–111. [5] Turabelidze A, Dipietro LA. Inflammation and wound healing: inflammation and wound healing. Endod Top 2011;24(1):26–38. [6] Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res 2010;89(3):219–29. [7] Boulton AJM. The pathway to foot ulceration in diabetes. Med Clin North Am 2013;97(5):775–90. [8] Hennessey PJ, Ford EG, Black CT, Andrassy RJ. Wound collagenase activity correlates directly with collagen glycosylation in diabetic rats. J Pediatr Surg 1990;25(1):75–8. [9] Tsourdi E, Barthel A, Rietzsch H, Reichel A, Bornstein SR. Current aspects in the pathophysiology and treatment of chronic wounds in diabetes mellitus. Biomed Res Int 2013;2013:1–6. [10] Dinh TL, Veves A. A review of the mechanisms implicated in the pathogenesis of the diabetic foot. Int J Low Extrem Wounds 2005;4(3):154–9. [11] Rayfield EJ, Ault MJ, Keusch GT, Brothers MJ, Nechemias C, Smith H. Infection and diabetes: the case for glucose control. Am J Med 1982;72(3):439–50.

Downregulation of hexose sugar metabolism in diabetes decreases the rate of wound healing

[12] Wierusz-Wysocka B, Wysocki H, Wykretowicz A, Klimas R. The influence of increasing glucose concentrations on selected functions of polymorphonuclear neutrophils. Acta Diabetol Lat 1988; 25(4):283–8. [13] Esmann V. The polymorphonuclear leukocyte in diabetes mellitus. J Clin Chem 1983;21(9):561–7. [14] Alba-Loureiro TC, Munhoz CD, Martins JO, Cerchiaro GA, Scavone C, Curi R, et al. Neutrophil function and metabolism in individuals with diabetes mellitus. Braz J Med Biol Res 2007; 40(8):1037–44. [15] Hotta N. New approaches for treatment in diabetes: aldose reductase inhibitors. Biomed Pharmacother 1995;49(5):232–43. [16] Boland OM, Blackwell CC, Clarke BF, Ewing DJ. Effects of ponalrestat, an aldose reductase inhibitor, on neutrophil killing of Escherichia coli and autonomic function in patients with diabetes mellitus. Diabetes 1993;42(2):336–40. [17] Bao Y, Ledderose C, Seier T, Graf AF, Brix B, Chong E, et al. Mitochondria regulate neutrophil activation by generating ATP for autocrine purinergic signaling. J Biol Chem 2014;289(39):26794–803. [18] Chen Y, Yao Y, Sumi Y, Li A, To UK, Elkhal A, et al. Purinergic signaling: a fundamental mechanism in neutrophil activation. Sci Signal 2010;3(125):ra45. [19] Gupta A, Raghubir R. Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing. Mol Cell Biochem 2005;270(1–2):71–7. [20] Da Silva D, Ausina P, Alencar EM, Coelho WS, Zancan P, Sola-Penna M. Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice. IUBMB Life 2012;64(9):766–74. [21] Lan C-CE, Liu I-H, Fang A-H, Wen C-H, Wu C-S. Hyperglycaemic conditions decrease cultured keratinocyte mobility: implications for impaired wound healing in patients with diabetes. Br J Dermatol 2008;159(5):1103–15. [22] Bockholt SM, Burridge K. Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin. J Biol Chem 1993;268(20):14565–7. [23] Golubovskaya VM. Focal adhesion kinase as a cancer therapy target. Anti Cancer Agents Med Chem 2010;10(10):735–41. [24] Hu SC-S, Lan C-CE. High-glucose environment disturbs the physiologic functions of keratinocytes: focusing on diabetic wound healing. J Dermatol Sci 2016;84(2):121–7. [25] Lawler J. Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 2002;6(1):1–12. [26] Lan C-CE, Huang S-M, Wu C-S, Wu C-H, Chen G-S. High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation. Transl Res J Lab Clin Med 2016;169:91–101.e1-3. [27] Ashraf JM, Ahmad S, Choi I, Ahmad N, Farhan M, Tatyana G, et al. Recent advances in detection of AGEs: immunochemical, bioanalytical and biochemical approaches. IUBMB Life 2015; 67(12):897–913. [28] Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol 2014;18(1):1–14. [29] Yamagishi S-I, Fukami K, Matsui T. Evaluation of tissue accumulation levels of advanced glycation end products by skin autofluorescence: a novel marker of vascular complications in high-risk patients for cardiovascular disease. Int J Cardiol 2015;185:263–8. [30] Peppa M, Uribarri J, Vlassara H. Glucose, advanced glycation end products, and diabetes complications: what is new and what works. Clin Diabetes 2003;21(4):186–7. [31] Sherif EM, Abdelmaksoud AA, Issa HM, Mohamed SA. Soluble receptor for advanced glycation end products (sRAGE) and carotid intima-media thickness (CIMT) in type 1 diabetes mellitus: possible association with diabetic vascular complications. Egypt J Med Hum Genet 2014;15(4):361–7. [32] Yamagishi S-I, Nakamura N, Suematsu M, Kaseda K, Matsui T. Advanced glycation end products: a molecular target for vascular complications in diabetes. Mol Med 2015;21(Suppl. 1):S32–40. [33] Byun K, Yoo Y, Son M, Lee J, Jeong G-B, Park YM, et al. Advanced glycation end-products produced systemically and by macrophages: a common contributor to inflammation and degenerative diseases. Pharmacol Ther 2017;177:44–55.

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[34] Mahali S, Raviprakash N, Raghavendra PB, Manna SK. Advanced glycation end products (AGEs) induce apoptosis via a novel pathway: involvement of Ca2 + mediated by interleukin-8 protein. J Biol Chem 2011;286(40):34903–13. [35] Shaikh-Kader A, Houreld NN, Rajendran NK, Abrahamse H. The link between advanced glycation end products and apoptosis in delayed wound healing. Cell Biochem Funct 2019;37(6):432–42. [36] Duraisamy Y, Slevin M, Smith N, Bailey J, Zweit J, Smith C, et al. Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: possible relevance to wound healing in diabetes. Angiogenesis 2001;4(4):277–88. [37] Alikhani M, Alikhani Z, Boyd C, MacLellan CM, Raptis M, Liu R, et al. Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways. Bone 2007;40(2):345–53. [38] Alikhani Z, Alikhani M, Boyd CM, Nagao K, Trackman PC, Graves DT. Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Biol Chem 2005;280(13):12087–95. [39] Denis U, Lecomte M, Paget C, Ruggiero D, Wiernsperger N, Lagarde M. Advanced glycation endproducts induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction. Free Radic Biol Med 2002;33(2):236–47. [40] Alikhani M, Maclellan CM, Raptis M, Vora S, Trackman PC, Graves DT. Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor. Am J Physiol Cell Physiol 2007;292(2):C850–6.

Further reading Wang Y, Zhou Y, Graves DT. FOXO transcription factors: their clinical significance and regulation. Biomed Res Int 2014;2014:925350.

CHAPTER 14

Biomaterials for diabetic wound-healing therapies Nava P. Rijal, Daria A. Narmoneva

Department of Biomedical Engineering, College of Engineering and Applied Sciences, University of Cincinnati, Cincinnati, OH, United States

1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder that negatively impacts health and results in significant economic burden for patients and healthcare around the globe [1–3]. Diabetic ulcer is a severe complication associated with DM; as many as one in four diabetic patients will develop an ulcer in their lifetime [4]. In the United States, more than 22.3 million patients are affected with diabetes annually, and the cost for diabetes was $245 billion in 2012 [5] and continues to grow. The annual care for diabetic lowerextremity ulcers alone ranges from $9 billion to $13 billion, in addition to the cost for management of DM alone [3]. Chronic nonhealing ulcers are responsible for 50%–85% of all diabetic lower-extremity amputations in this population and 27% of $116 billion in diabetic health care costs in the United States and are the primary cause of for hospitalization among people with diabetes [2,6–11]. Generally, ulcers can be classified into several grades: grade 1, partial-thickness wound involving epidermis and dermis layer; grade 2, full-thickness wound extending to subcutaneous tissue; grade 3, wound with exposed tendons, ligaments, and joints; grade 4, wound with partial gangrene; grade 5, wound with full gangrene [12]. Partial-thickness wounds heal naturally through normal healing cycle, whereas full-thickness wounds are more difficult to heal naturally due to disruptions in wound-healing mechanisms. The grades can be further classified using several scoring systems, including PEDIS (perfusion, extent, depth, infection, sensation), SINBAD (site, ischemia, neuropathy, bacterial infection, depth), and University of Texas (UT) [13]. Diabetic ulcers are commonly classified using PEDIS system. Normal wound healing consists of three phases: inflammatory response (that includes hemostasis, i.e., blood clot formation), proliferation, and maturation. See Fig. 1 for overview of the wound-healing process. During the hemostasis part of inflammatory phase, leakage of blood causes platelet aggregation, activation and formation of the fibrin blood clot, which acts as a primary scaffold for infiltrating cells. Platelets also secrete growth factors such as platelet-derived growth factor (PDGF), which is one of the first factors Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00014-9

© 2020 Elsevier Inc. All rights reserved.

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Fig. 1 Overview of normal and diabetic wound-healing processes.

that initiate the subsequent healing cascade. Neutrophils are recruited next and secrete proteases, reactive oxygen species (ROS), and proinflammatory cytokines [14–16]. These inflammatory proteins amplify the inflammatory response and stimulate angiogenic factors, such as vascular endothelial growth factor (VEGF), CC-chemokines ligand (CCL2), and CXC-chemokine ligand (CXCL1, CXCL8) for an adequate repair response [17,18]. The inflammatory phase lasts for several days, usually subsiding by day 7–10 postwounding. At the end of inflammatory phase and beginning of proliferation phase (starting at day 3–4) macrophages differentiate from monocytes and enter the wound. Macrophages play a crucial role in the removal of apoptotic cells, providing the first line of defense against infection, and in production of a variety of proteins to support cell proliferation, synthesis of the extracellular matrix (ECM, mostly collagens I and III and glycosaminoglycans) and formation of granulation tissue. They also express a variety of growth factors for tissue repair such as transforming growth factor-beta (TGFβ), VEGF, fibroblast growth factor (FGF), PDGF, interleukin-1 (IL-1), tumor necrosis factor (TNF), insulin-like growth factor (IGF), interferon (IFN), hepatocyte growth factor (HGF) [19–21]. These factors are essential for blood vessel formation (vasculogenesis and angiogenesis), granulation tissue formation, ECM deposition, wound contraction, and re-epithelization [14-16,22,23]. During remodeling, the final phase of wound healing that usually starts at about 3 weeks postwounding, granulation tissue matures into repair tissue/scar, where collagen type III is replaced by collagen I. Collagen remodeling involves matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs). Change in collagen type increases the mechanical strength of tissue to up to 80% of uninjured skin [14,24].

Biomaterials for diabetic wound-healing therapies

In contrast to the normal wound-healing process described, chronic diabetic wounds appear to be stalled in the inflammatory phase for an extended period (more than 12 weeks) and do not progress to proliferative and remodeling phases (Fig. 1), which results in dysregulated molecular and cellular wound microenvironment [25]. Diabetic wounds exhibit abnormal collagen metabolism, high oxidative stress, inflammatory dysregulation, and increased proteolysis, as well as alterations in cellular phenotype, and altered neovascularization [8–10]. Diabetic wounds are also prone to infection due to the failure in functions of MMPs and impaired immune responses such as defective phagocytosis [10,26,27]. A large number of inflammatory cells recruited into the diabetic wound produce high amounts of ROS, causing damage to structural elements of the extracellular matrix (ECM) [18,28]. Biomaterials such as films, foams, hydrogels, antimicrobials, and different types of dressings have been used in a variety of therapeutic applications for treatment of chronic wounds. However, despite many successes, there is still no “ideal” solution that is able to directly address the complexity of problems associated with impaired healing of diabetic ulcers. This chapter focuses on the recent advances and remaining challenges in the development of biomaterials for the management of diabetic wounds, including a review of the major classes of biomaterials (i.e. synthetic, natural, and hybrid) used in wound dressings and skin substitutes, their physiochemical properties, critical aspects of biomaterial processing, as well as emerging therapies for diabetic wound treatment that incorporate biomaterials as a key component.

2. Successes and challenges in the use of biomaterials for the management of diabetic wounds Current treatment options for chronic ulcers include a variety of wound dressings, hyperbaric oxygen treatment, revascularization surgery, autografts, allografts, and bioengineered skin substitutes [29]. While partially successful, these options are often focused on wound closure rather than addressing the underlying pathophysiological conditions of the diabetic wounds, which often leads to ineffective healing, wound reoccurrence, and extended healing times. However, improved scientific understanding of woundhealing mechanisms in the last decade resulted in several recent breakthroughs in biotechnology for designing and fabricating biomaterials-based devices or scaffolds for wound treatment. Fig. 2 shows exponential growth for the number of scientific publications in the field of biomaterials related to diabetic wounds. The primary advantage for the use of natural and synthetic biomaterials-based scaffold for diabetic wound healing is their proven biocompatibility and their resorbable biodegradation products. The ultimate goal is to develop highly effective scaffolds able to direct the healing process of impaired tissues through functional restoration and maintenance, consistent with the basic principles of tissue engineering and regenerative medicine.

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Fig. 2 Exponential growth in the number of scientific publications related to the use of biomaterials for diabetic wound-healing applications. Results are presented as the number of hits following the search using the keywords “biomaterials,” “diabetic,” and “wound healing” in the Science Direct database.

Biomaterials play a crucial role in wound healing by enabling interactions between cells, ECM, and various growth factors. ECM is primarily composed of fibronectin, collagens, laminin, proteoglycans, and glycosaminoglycans, and serves as a cell support and a storage of growth factors that are needed for cell infiltration and proliferation [6,14]. There has been an enormous effort to develop tissue-engineered scaffolds for diabetic wound healing that can provide structural and functional support and withstand harsh wound microenvironment. There are just a handful of biomaterials and skin substitutes that have gained FDA approval for treatment of chronic wounds, including Regranex (sodium carboxymethyl cellulose gel containing becaplermin), Apligraft, and Dermagraft. These tissue-engineered products have shown efficacy in several studies, and have also been able to reduce healing times [8,30]. However, these products have not been 100% successful in treating the chronic diabetic ulcers and have several drawbacks, including the possible risk of cancer associated with the use of three or more becaplermin gel tubes, shorter half-life, high cost, and stricter and limited storage conditions [30–32]. Therefore, there is a critical need for other alternatives and biotechnology solutions for treatment strategies of diabetic ulcers. Following section of this chapter will elaborate on different types of biomaterials used, their potentials, and current status in diabetic wound healing.

3. Biomaterials for dermal tissue engineering and regeneration The concept of tissue-engineered biomaterials was first introduced in the early 1990s [33,34], and since then, this concept has expanded to include genes, cells, growth factors, and other biomolecules. When biomaterials are used as part of the regenerative strategy, the approach usually requires several key characteristics and properties to be fulfilled. These include biocompatibility, biodegradability, bioavailability, porosity, topography,

Biomaterials for diabetic wound-healing therapies

and suitable mechanical properties. The most common biomaterials are composed of synthetic or natural polymers, ceramics, metals, or any combination of these.

3.1 Acellular biomaterials Acellular biomaterials provide an ECM devoid of cells where endogenous cells can migrate and initiate tissue regeneration. Acellular skin substitutes that are made from natural biological materials are the most common available skin substitutes for the treatment of chronic ulcers. Acellular materials include synthetic materials (see Tables 1 and 2), a combination of synthetic and natural (see Table 2), and decellularized human cadaver, human amniotic membranes, and animal tissues. 3.1.1 Synthetic polymeric biomaterials Synthetic polymers are gaining interest in wound healing because their physical and chemical properties can be tuned, and because these materials are capable of delivering bioactive biomolecules such as growth factors into the wound. Conventional synthetic polymers are polyesters-based and have better functionality and controlled shelf life as compared to natural polymers. These materials also have excellent and controllable tensile and compressive mechanical strength and degradation profile. Degradation of these polymers can be manipulated depending on the application need and typically utilizes hydrolytic cleavage of the polymer chain. However, most synthetic polymers are not bioactive and therefore are often used in combination with natural or synthetic polymers to improve functionality and to enhance cell adhesion, proliferation, and scaffold Table 1 Physical and chemical properties of different synthetic polymers. Synthetic polymer

Melting temperature (°C)

Glass transition temperature (°C)

FDA approval

PCL

59–64


60

Yes

[35– 37]

PLGA

>200

35–40

Yes

[36,37]

PLA

175–178

60–65

Yes

[37,38]

PU

>200


65

N/A

[39]

Chemical structure

Citation

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Table 2 Synthetic and combined (natural and synthetic) polymers and acellular skin substitutes for diabetic wound-healing applications. Biomaterials

Experimental model

Polyurethane

PU foam dressing; human

Polyglycolic acid and polylactic acid

PGA/PLA; human

Amino acid-based poly(ester amine)

Lyophilized monomers; diabetic rat

Poly (εcaprolactone) (PCL)/gelatin/ silicate Chitosan/ polyvinyl alcohol/zinc oxide Konjac glucomannankeratin—Avena sativa PLGA and vascular endothelial growth factor

Nanofibrous composite scaffold; diabetic mice Nanofibrous mats; diabetic rabbits

Desferrioxamine (DFO) and bioglass

Hydrogel scaffold; diabetic rats

Nanoparticles; diabetic and nondiabetic mice

Hydrogel; diabetic rats

Findings

Citations

Healing rate was accelerated, good absorption of wound exudate, a significant reduction in the wound area Significantly fewer adverse events; 30% wounds showed complete wound closure by 12 weeks Increase collagen deposition and angiogenesis; prohealing wound microenvironment with improved re-epithelialization of wound healing Significantly improved epidermal regeneration, angiogenesis, collagen deposition and decreased the inflammatory response Accelerated wound healing

[40,41]

Significantly accelerated wound healing; formation of an epidermis layer and blood vessels, and collagen deposition No effect on neutrophil infiltration and myeloperoxidase release; well-developed and -organized epithelium; enhanced granulation tissue formation; synthesis of a compact and denser collagen and better collagen alignment Upregulate the gene expression of VEGF

Other synthetic and combined (synthetic and natural) skin substitutes: 1. Hyalomatrix tissue construction matrix 2. Restrata 3. Integra bilayer matrix wound dressing 4. Integra dermal regeneration template 5. Integra omnigraft regeneration template 6. Integra flowable wound matrix

[42]

[43]

[44]

[45]

[46]

[47]

[48]

Biomaterials for diabetic wound-healing therapies

degradability [14,34,49]. Two most well-known skin substitute products that are made from synthetic materials and are designed to mimic skin morphology and structural properties include Hyalomatrix tissue reconstruction matrix (Medline, Mundelein, IL) and Restrata (Acera Surgical Inc., St. Louis, MO). Hyalomatrix is composed of hyaluronic acid (HA) derivatives in the fibrous form with an outer layer composed of a semipermeable silicone. Restrata is made from bioabsorbable polyglactin 910 and polydioxanone. In this section, recent efforts in the development of synthetic polymeric scaffolds for wound-healing applications will be addressed. Synthetic polymers that are most widely used due to their excellent biocompatibility and biodegradability include poly (ε-caprolactone) (PCL), polylactic acid (PLA), and poly (lactic-co-glycolic acid) (PLGA). Their chemical structure, melting temperature, and glass transition temperature are listed in Table 1. These polymers are FDA approved for use in different biomedical devices and are discussed in detail for its use in diabetic wound healing. 3.1.1.1 Poly (ε-caprolactone) PCL is a polyester (aliphatic synthetic polymer, semicrystalline) with a functional ester bond and is commonly used in preclinical trials for drug delivery into diabetic wounds [14,50]. These ester bonds usually degrade onto nontoxic byproducts as compared with other polyesters [51]. Moreover, PCL possesses unique properties such as low degradation rate, high drug permeability, and less acidic byproducts [52]. It is one of the preferred synthetic polymers because of its low melting temperature, viscoelastic properties, and it can be processed into any desired shapes and sizes for implant [51,53]. On the other hand, due to its hydrophobic in nature, PCL lacks desirable cell affinity and cell recognition sites, which results in impaired cell proliferation, cell adhesion, migration, and differentiation [50,54]. To overcome this problem, PCL is often combined with other hydrophilic natural polymers such as gelatin, chitosan, and collagen. Mixing of two different copolymers allows one to improve scaffold properties such as cell adhesion and strength. For example, research by Powell and Boyce showed that PCL could be combined with collagen to enhance the strength of the scaffold [55]. Thus, Powell et al. [55] showed that the 10%–100% PCL concentration significantly enhanced strength and stiffness of acellular scaffolds. However, they also reported that 30% of PCL showed reduced cell viability and reduced mechanical strength [55]. A study by Fang and coworkers [44] described the use of PCL/gelatin/silicate based nanofiber for full-thickness wound healing in diabetic mice model. The study reported that the use of PCLbased biomaterial scaffold significantly enhanced epidermal regeneration, angiogenesis, and collagen deposition, and reduced the inflammatory response. 3.1.1.2 Poly (lactic-co-glycolic acid) PLGA, a polyester, is a copolymer of PLA and polyglycolic acid (PGA). PLGA is also one of the few biodegradable, biocompatible FDA-approved polymers that have been used

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primarily for drug-delivery applications and nanofibrous scaffold fabrication [56]. Degradation process in PLGA occurs by hydrolysis or biodegradation through cleavage of the backbone ester linkages [57,58]. The degradation rate of this polymer can be modified by changing the chemical composition of lactic acid and glycolic acid, where reduction of glycolic acid ratio and higher molecular weight of the polymer leads to the slower polymer degradation rate [56,58]. However, if the chemical composition of PLGA is 50:50 (PLA:PGA), the rate of polymer degradation is fastest. One of the degradation products of PLGA is lactase. This byproduct is known to promote migration of endothelial cells and capillary-like tube formation in vitro [59,60]. Porporato et al. showed that subcutaneous implantation of the PLGA promoted angiogenesis and accelerated healing of the excisional skin wounds in mice [61]. Furthermore, a recent study by Chereddy et al. [47] showed that the PLGA could be used as a delivery vehicle of VEGF for wound healing in the diabetic mouse model. They reported that this approach resulted in well-developed and organized epithelium, enhanced granulation tissue formation and synthesis of a compact ECM with denser collagen alignment, while no effects on neutrophil infiltration and myeloperoxidase release were observed [47]. 3.1.1.3 Polylactic acid PLA is a bioactive, bioresorbable, and biodegradable thermoplastic aliphatic polyester. This polymer is derived from natural resources and is widely used in different biomedical applications, including but not limited to stents, sutures, and drug-delivery systems. Degradation process occurs via nonenzymatic hydrolysis. When the polymer comes in contact with an aqueous solution, such as water, ester bonds are attacked, and long chains are broken down into smaller chains. Degradation is primarily dependent on molecular weight, temperature, and pH of the medium. Recently, PLA in the form of electrospun fibers has been gaining interest for wound-healing applications, because different therapeutic molecules and growth factors could be entrapped for release. For example, hydrophobic drugs can be directly mixed into a solution of PLA, and electrospun into the fibrous scaffold and hydrophilic drugs are incorporated into PLA via water/oil emulsion techniques [62]. This nanofiber structure prevents loss of proteins, nutrients, moisture, thus facilitating proper healing and mimic the native topography of the wound bed [62]. 3.1.1.4 Polyurethane Polyurethane (PU) is composed of organic units that are connected by urethane links and comprises alternating soft and hard segments. Most of PUs are thermosetting polymers, i. e., they do not melt when heated. PU is one of the early synthetic polymers that have been developed as an attempted engineered skin replacement to treat full-thickness wounds. Today, this polymer is commonly used as a wound dressing due to its nontoxicity, good oxygen permeability, nonadhesive, and nonallergenic properties. Degradation and porosity of PU can be modified by replacing the soft segments with other copolymers such as PCL, PGA, PEG, and/or lactic acid, and also by varying manufacturing

Biomaterials for diabetic wound-healing therapies

techniques. Generally, for applications in diabetic wound healing, PU is loaded with different bioactive compounds such as silver and growth factors [63,64]. For example, Choi and coworkers reported that combining silver nanoparticles and recombinant human epidermal growth factor with PU foam improved healing of full-thickness diabetic wounds and resulted in proper re-epithelization and collagen deposition [63]. 3.1.2 Natural animal-derived polymers used as biomaterials Other class of biomaterials used for diabetic wound healing comprises natural polymers, which are naturally derived, and can be both ECM- and non-ECM derived. Unlike synthetic polymers, natural polymers closely resemble the components present in our biological system and are found abundantly. Most natural polymers are readily accepted by our immune system and are highly biocompatible, biodegradable, and nontoxic. These biomaterials support enhanced cell adhesion, proliferation, and differentiation. Despite having numerous positive attributes, these polymers, when processed, lack sufficient mechanical properties, and possess uncontrollable degradation rates. In addition, there is significant batch-to-batch variability of the properties of these polymers. This issue can be resolved by incorporating other synthetic or natural polymers. Natural polymers can be divided into polysaccharides, which include chitosan, alginate, HA; proteins, which include collagen, fibrin; and polyesters [65]. This section discusses different types of commonly used natural biomaterials (collagen, gelatin, chitosan, alginate, HA, and self-assembling peptides), their use in diabetic wound-healing applications. This section also provides list of biomaterials for wound treatment made from decellularized human cadaver, human amniotic membranes, and animal tissues (see Table 3). Table 3 Acellular skin substitutes from natural derived (human cadaver, human amniotic, and animal tissue). Source

Product

Skin substitute and outcomes

Citations

Allograft

1. EpiFix (dehydrated human amniotic membrane) 2. Grafix(placental membrane) 3. AMINOEXCEL (amniotic membrane) 4. TheraSkin (cryopreserved split-thickness human skin)

1. Healing rate of 77% and 92% after 4 and 6 weeks, respectively 2. Significantly higher wound closure (62% vs 21%), the median time to heal was also significantly improved, with reduced infection and no observed adverse events 3. Complete wound closure before 6 weeks and no treatment-related adverse event 4. Diabetic foot ulcers closed 60.38% of the time after 12 weeks and 74.1% after 20 weeks, an equivalent amount of collagen I and III present as compared to fresh skin

[66–71]

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Table 3 Acellular skin substitutes from natural derived (human cadaver, human amniotic, and animal tissue)—cont’d Sources

Devices

Human cadaver

AlloPatch HD Acellular Dermal Matrix AlloPatch Pliable Alloskin AC Acellular Dermal Matrix AlloSkin RT Dermacell Human Acellular Dermal Matrix and Dermacell AWM DermaSpan Acellular Dermal Matrix GraftJacket RTM, InteguPly, FlowerDerm Matrix HD Allograft AlloWrap, AmnioBand Allograft Placental Matrix, Amnioexcel AmnioFill Human Placental Tissue Allograft AmnioFix Amnion/Chorion Membrane Allograft Amniomatrix Human Amniotic Suspension Allograft Artacent Wound, BioDFactor Viable Tissue Matrix, Biovance Amniotic Membrane Allograf, Cygnus Amnion Patch Allografts Dermavest and Plurivest Human Placental Connective Tissue Matrix Epicord, Epifix, Integra BioFix Amniotic Membrane Allograft Interfyl Human Connective Tissue Matrix Xwrap Amniotic MembraneDerived Allograft Architect stabilized collagen matrix Bio-ConneKt Wound Matrix Colla-pad, Collexa CollaSorb collagen dressing Cytal wound matrix Endoform dermal template Integra Matrix Wound Dressing; originally Avagen wound dressing EZ Derm, Excellagen, Miroderm, Helicoll ologen Collagen Matrix Kerecis Omega3 Wound (originally Merigen wound dressing) Oasis Wound Matrix PriMatrix Dermal Repair Scaffold PuraPly Antimicrobial (PuraPly AM) Wound Matrix (formally called FortaDerm) TheraForm Standard/Sheet Absorbable Collagen Membrane

Human amniotic membrane

Animal tissue

3.1.2.1 Collagen Collagen is one of the most abundant proteins of ECM: it represents 25% of total body protein content. The primary function of the collagen is to provide mechanical support and maintain the integrity of the tissue. Collagen participates in cell signaling via interactions with cells in the wound, such as keratinocytes, endothelial cells, and fibroblasts, and therefore helps regulate a variety of processes, including cell anchorage, migration, differentiation, proliferation, and survival [72–74]. Furthermore, collagen is susceptible to proteolytic degradation, and collagen contraction after implantation is a significant issue that needs to be addressed.

Biomaterials for diabetic wound-healing therapies

Collagen is often chosen as an “ideal” biomaterial for wound-healing dressings mainly because under certain conditions, it can accelerate wound healing and granulation tissue formation, support neovascularization, and reduce bacterial infection in chronic wounds [74–77]. Singh et al. [75] compared the use of collagen dressing versus conventional dressing (silver sulfadiazine, nadifloxacin, povidone iodine, or honey) in burn and chronic wounds. They found out that healthy granulation tissue was present at a significant level for collagen-dressing-treated group. Collagen-dressing-treated patients also avoided the need for skin graft and enjoyed additional benefits such as ease of comfort and mobility. Another study by Kanda et al. [77] showed that the use of collagen-based scaffold loaded with FGF can lead to the improved wound area, the formation of dermis like architecture and capillaries in a diabetic mouse model. One of the collagen-based biomaterials currently on the market for diabetic and chronic foot ulcers is Apligraf. This material is composed of allogeneic human fibroblasts and/or keratinocytes and partially denatured collagen. The primary purpose of this material is to deliver growth factors and ECM to promote autologous healing. Falanga and Sabolinski [78] performed a randomized, controlled study by using Apligraf versus standard compression therapy in 120 patients that had hard-to-heal venous ulcers. They reported that the use of Apligraf significantly improved the time that took for complete wound closure, percent of patient healed by 6 months [78]. Potential disadvantages include high cost of the material and need for multiple applications. Studies that used selective collagen-based acellular skin substitutes for diabetic wound healing are listed in Table 4.

Table 4 Acellular collagen-based skin substitutes and clinical outcomes in treatment of diabetic ulcers. Biomaterials

Products

Outcomes

References

Collagen

1. Collagen-based dressing 2. Collagen matrix and negative pressure (Integra) 3. PuraPly (collagen sheets) 4. Promogran 5. ORCEL 6. Graftskin 7. OASIS (collagenbased ECM)

1. Appearance of healthy granulation tissue, reduction in infection and 60% wound healed within 2 weeks 2. Granulation tissue formation and limb salvation rate of 46% 3. N/A 4. Benefit of marginal significance to ulcers with duration >6 months 5. Promoted rapid healing, reduced scarring 6. Improved healing, nontoxic and was not clinically rejected 7. Complete wound closure in 49% as compared to 28% for patients treated with Regranex gel

[8,75,79– 84]

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3.1.2.2 Hyaluronic acid HA is a natural polysaccharide that is composed of N-acetyl-D-glucosamine and β-glucuronic acid disaccharide units. HA possesses versatile properties such as biocompatibility, biodegradability, viscoelasticity, nontoxic, and nonallergic and is readily available commercially [85]. HA is synthesized from synovial fluid, rooster combs, vitreous humor, and umbilical cord and bacteria. HA is found mostly in all vertebrate organs, is abundant in ECM of soft connective tissues, and is present in both dermis and epidermis layers. HA is used as a biomaterial for diabetic wound healing because it has been shown to promote epithelial and mesenchymal cell differentiation and migration [74,85]. HA degrades by hyaluronidase (metabolic degradation), with the degradation byproducts shown to support endothelial cell proliferation, stimulate angiogenesis, and modulate inflammatory process [86–89]. One of the HA-based biomaterials currently in the market for diabetic foot ulcer is Hyalofill. A study by Vazquez et al. [87] showed that the use of hyaluronan dressing in diabetic foot ulcers reduced the duration of granulation tissue formation by 29% and reduced the total time to heal by 31%. Another study by Lobmann et al. [90] used autologous human keratinocytes cultured HA graft to study healing in diabetic foot lesion. They reported that 79% of diabetic foot ulcers treated with HA graft healed fully between 7 and 64 days [90]. They also reported faster-wound closure and reduced hospital stay [90]. Selective HA-based acellular skin substitutes are listed in Table 5 with clinical outcomes.

Table 5 Acellular HA-based skin substitutes and clinical outcomes in treatment of diabetic ulcers. Biomaterials

Products

Outcomes

References

Hyaluronic

1. LaserSkin 2. Vulnamin (amino acid and HA) gel 3. Hyiodine 4. Hyaff (ester of hyaluronic acid) 5. Hyalograft 3D

1. Improves re-epithelization, faster closure of foot lesions, and reduced hospital stay 2. Reduction in ulcer area, significantly higher healing rate, and no observed infections or other adverse events 3. Complete healing within 6 to 20 weeks, no side effects or allergic reactions 4. 65.3% of the treatment group healed completely, significant healing of the dorsal ulcer; provides a granulation tissue and results in complete healing 5. Improved healing

[90–95]

Biomaterials for diabetic wound-healing therapies

3.1.2.3 Gelatin Gelatin is a unique biomaterial that is used for wound-healing applications in the form of hydrogel because of its biocompatibility and biodegradability in physiological pH. Gelatin is a collagen derivative, which possesses low antigenic properties. Gelatin, in an acidic form, is capable of releasing positively charged growth factors [77]. 3.1.2.4 Chitosan Chitosan, an N-deacetylated product, is derived from chitin. Chitin is a poly-N-acetylD-glucosamine and is the second most abundant polysaccharide macromolecule found in nature after cellulose [96–98]. It is found in various living organism such as shrimps, crabs, insects, and other arthropods. Chitosan is known to have several properties, including, but not limited to, its biodegradability, biocompatibility, nontoxicity, bioadhesiveness, antimicrobial, and nonantigenic properties—all of which offer advantages in biomedical applications [99]. Due to its unique properties, such as easy chemical modification of its functional group (amino and hydroxyl group), chitosan has been used in wound dressing, wound healing, drug delivery, and other biomedical and tissue engineering applications [100]. Structurally, chitosan is a linear polysaccharide composed of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine units. The biopolymer is defined primarily based on the fraction of D-glucosamine units, commonly defined as the degree of deacetylation (DD), and the term “chitosan” is used when DD is greater than 50%v [101]. Chitosan is known to have poor solubility in common organic solvents due to the rigid D-glucosamine structure, high crystallinity, and capacity to form an intermolecular hydrogen bond [100]. Chitosan is the only naturally occurring positively charged polysaccharide that contains primary aliphatic amines in its structure [102]. These primary aliphatic amines can be protonated under acidic conditions (amine pKa is 6.3), which makes chitosan a cationic polyelectrolyte [103]. The cationic nature of the polymer allows it to become water soluble after the formation of carboxylate salts, such as formate, acetate, lactate, malate, citrate, glyoxylate, pyruvate, glycolate, and ascorbate [104,105]. Moreover, chitosan is soluble in weak organic acids allowing it to interact with negatively charged molecules such as proteins, nucleic acids [106]. This interaction causes chitosan to adhere to the wound tissues and promote blood coagulation and wound healing [107,108]. Chitosan-based materials can be synthesized in different forms such as membranes, gels, scaffolds, nano- and microparticles, with several of these utilized in wound healing of chronic ulcers. One of the FDA-approved chitosan-based dressing is HemCon Bandage [109]. This material is able to stop blood loss after injury and regenerate the tissue (granular and fibrous tissue formation) [109,110]. Chitosan is also known to stimulate angiogenesis, promote tissue granulation, synthesis of the collagen matrix, and provoke inflammatory function of macrophage and re-epithelization of tissue [108,110,111].

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3.1.2.5 Alginate Alginate, a natural polymer, linear unbranched copolymer, is composed of α-L-guluronate (G) and 1,4-linked β-D-mannuronate (M) residues. The number of G and M unit present varies depending on the manufacturing process and the original source of the alginate. G block is capable of binding to the divalent cation to create a hydrogel. Alginate has shown great promise in the field of neuronal repair as an axonal regrowth scaffold, cell growth matrix, and neural drug-delivery device [112–114]. Alginate has been widely used for various biomedical applications because of its proven biocompatibility, natural origin, and its low cost. Various complications arise when treating diabetic wounds. One such complication is wound exudates. Diabetic wound exudates have been found to contain increased inflammatory mediators and MMPs leading to the damage of wound bed and ECM [115]. Therefore, managing wound exudates to reduce healing times and prevent infection is one of the key aspects for a successful diabetic ulcer therapy. One potential solution is to use alginate-based scaffolds, which can absorb 15–20 times their weight. This occurs because blood exudates consist of sodium ions, whereas the alginate dressing consists of divalent cations. As a result, ion exchange occurs between the dressing and exudates, resulting in the formation of gel [116]. A recent study by Wang et al. reported that the use of calcium alginate improved type I collagen synthesis, accelerated wound-healing process in diabetic rats, and faster epithelization [117]. The alginate-based scaffolds have gained great interest in therapeutic delivery applications, including cells, small and macromolecular drugs, proteins, DNA. Alginate’s potential in drug delivery is very promising for multiple reasons: it forms a nonantigenic hydrogel on contact with divalent cations such as Ca2+, Mg2+, or Zn2+; it does not require harsh chemical crosslinking and/or high-temperature agents; and it can be converted back to solution by chelating the divalent cations using EDTA or sodium [14,118–121]. A recent study by Murat et al. reported that the use of alginate-based microspheres loaded with VEGF showed significant VEGF release and neovascularization in the rat model. The unique encapsulation properties of alginate for delivery of various growth factors, drugs, and cells are of particular importance for the treatment of diabetic wounds. Table 6 presents examples of the acellular-based natural polymers used in vitro, in vivo, and as a skin substitute for treatment of diabetic wounds. 3.1.2.6 Self-assembling peptides Another class of biomaterial that can be used for diabetic wound-healing therapeutic applications is represented by self-assembled peptides. These polymers are self-complementary amphiphilic oligopeptides that consist of 8–16 amino acids residues. These residues are arranged in such a fashion that allows the amino acid chains to alternate in hydrophobic and hydrophilic groups. These oligopeptides, self-assembled peptides, arrange in beta sheets [132] when in contact with water and form stable 3D hydrogel networks when

Biomaterials for diabetic wound-healing therapies

Table 6 Natural polymer-based therapeutic applications for diabetic wound healing. Biomaterials

Experimental model

Periostin/collagen and CCN2/collagen

Electrospun scaffold; diabetic mice

Chitosan/alginate/ maltodextrin/pluronicbased mixed polymeric micelles

Polymeric micelles; diabetic rats

Chitosan/polyvinyl alcohol/zinc oxide

Nanofibrous mats; diabetic rabbits Hydrogel, diabetic rabbits

Plasma-modified collagen Konjac glucomannankeratin—Avena sativa

Hydrogel scaffold, diabetic rats

Crosslinked hyaluronic acid-based biomaterial (CMHA)

Gel; normal horses

Collagen-lamininhyaluronic acid-DPPC micro particles

Lyophilized matrix with micro particles; diabetic rats Nano-bio composites, diabetic mice

Bamboo cellulose nanocrystals impregnated with silver nanoparticles 3D chitosan scaffold

3D scaffold; diabetic rats

Findings

Citations

Increased excisional wound closure rates; reduced neutrophil infiltration; increase in mesenchymal cell infiltration; upregulation of gene clusters associated with wound contraction, cell differentiation, and suppression of PPARγ signaling Recovered the pancreatic β cells that were damaged by “Bisphenol A”; reduced scar formation and fast wound closure Accelerated wound healing

[122]

[123]

[45]

No additional inflammatory responses; promotion of angiogenesis Significantly accelerated wound healing; formation of an epidermis layer, blood vessel, and collagen deposition Wounds decreased to half their original size significantly faster; healed with higher quality, less fragile epithelium Enhanced wound healing

[124]

A decrease in the levels of proinflammatory cytokines IL-6 and TNF-α; improved reepithelialization, and collagen deposition Improved quality of the restored tissue

[127]

[46]

[125]

[126]

[128] Continued

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Table 6 Natural polymer-based therapeutic applications for diabetic wound healing—cont’d Biomaterials

Experimental model

Gelatin microcryogels

Gel; diabetic mice

Chitosan (skin substitute)

Chitosan plus isosorbide dinitrate spray; human Alginate hydrogel containing phenytoin; human

Alginate (skin substitute)

Findings

Citations

Improved cell retention and secretion of multiple growth factors Accelerated healing and signification regeneration

[129]

No statistically significant difference in complete healing between groups

[131]

[130]

in contact with physiological pH. These self-assembled proteins have excellent stability and are resistant to a temperature of up to 90°C [133,134]. They assemble into interwoven nanofibers structure with fiber diameter close to 10 nm and pore size of 5 to 200 nm [132,135,136]. This pore size resembles the structure of native ECM allowing diffusion of oxygen, nutrients, and various growth factors essential for diabetic wound healing. Self-assembling peptide nanofibers are gaining interest recently in the tissue-engineering field. Due to their cell-native amino-acid composition, these biomaterials are biocompatible and are able to support cell growth, differentiation, and cell attachment. Also, their mechanical properties and cell attachment sites can be controlled by design. For example, one particular sequence, RAD16-II nanofibers (RARADADA)2, has been shown to induce angiogenesis in vitro and in in vivo model of diabetic wound healing without any modification [137,138]. Recent study by Narmoneva et al. has demonstrated that use of RAD16-II promotes angiogenesis by enhancing survival and proliferation of diabetic endothelial cells in vitro, enhances endothelial cell recruitment and neovascularization in diabetic wound healing in vivo, and significantly accelerated wound closure, neovascularization, and overall wound healing with enhanced repair tissue strength in diabetic mouse model (see Fig. 3) [137–142]. Importantly, the amino acid sequence of RAD16-II is resistant to proteolytic degradation because it lacks MMP cleavages sites [132], and thus can withstand the harsh proteolytic environment of the diabetic wound while providing a stable proangiogenic, proregeneration stimuli. Furthermore, a recent study by Bradshaw et al. [143] reported the modification of RAD16-I (RADARADA)2 peptide nanofiber by adding two functional motifs: fibronectin motif (RGDS) and collagen type I motif (FPGERGVEGPGP). This modification allowed for improvement of keratinocytes and fibroblasts migration and proliferation, which is crucial for wound closure and full re-epithelization. Hartgerink et al. have demonstrated that another nanofiber hydrogel (multidomain peptide K2(SL)6K2) made using

Biomaterials for diabetic wound-healing therapies

Fig. 3 (A) Histologic appearance of the wound repair tissue at day 28 postwounding (H&E staining) in the db/db mouse model. Wounds treated with the nanofiber hydrogel show presence of In Situ Tissue Engineered Provisional Matrix (ISTEPM), significantly reduced scar width (B) and thickness of the wound similar to the unwounded skin, in contrast to a wider and thinner scar in the PBS and hyaluronic acid (HA) controls (n ¼ 7 animals/group). The ISTEPM wounds are not raised and the thickness of the repaired epithelium is similar to that of the native skin, indicating the absence of a hypertrophic response. There is evidence of hair follicle formation in the ISTEPM wounds, which is not observed in the controls. High magnification panels show the native skin hair follicle bulge and the characteristic brown staining at the edge of the wound as a reference (panel 1), with similar brown staining observed in the repaired area in the ISTEPM wounds (panel 2). ISTEPM formation also results in significant increase in the wound apparent stiffness (C) and maximum tensile load (D), as compared with PBS or HA control treatments (n ¼ 7 animals/group). Bar plots represent average SD. (From (A) Balaji S, et al. Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing. Wound Repair Regen 2012;20(1):15–27.)

the same principle of self-assembly can accelerate the healing of diabetic wounds by increasing granulation tissue formation, re-epithelialization, vascularization, and innervation [144]. Overall, use of novel self-assembling peptide nanoscaffolds is promising because it allows in situ modification of diabetic wound environment by providing missing cues for cellular response and direct chronic wounds toward regenerative healing.

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3.2 Cell-based skin substitutes Wound healing is a complex process that requires orchestrated interactions of various cellular network, cell-ECM signaling, and cell-cell communication. Under diabetic conditions, these interactions become disrupted. The resident cells in the diabetic wounds are nonresponsive and are critically impaired [145,146], and the wound microenvironment itself is altered due to hyperglycemia, neuropathy, inflammation, hypoxia, and circulatory dysfunction. Primary cells to be affected by the chronic condition are neutrophils and macrophages. Because of the impaired microenvironment, neutrophils and macrophages cannot function normally and fail to clear pathogens, fail to support cell adhesion and proliferation. This causes to recruit more inflammatory cells leading to prolonged inflammation [145]. Increased presence of inflammatory cells leads to upregulation of TNF-α and IL-1 and increased production of MMPs [145,147,148]. Chronic wounds are found to have a concentration of MMPs 60 times higher than the normal wounds [147]. This increased concentration of MMPs causes significant impairment in ECM regulation and affects production of key growth factors such as transforming growth factor β1, platelet growth factor, and VEGF. Diabetic conditions also have detrimental effects on other cells in the wound, including fibroblasts, keratinocytes, and endothelial cells, which are collectively responsible for ECM deposition, vascularization, and re-epithelialization. Another notable effect of diabetes on wound-healing process is the insufficient recruitment of other progenitor and stem cell population. There have been numerous studies in animal models as well as human clinical trials, but much still needs to be done to understand the critical roles of each cell type fully and to overcome the regulatory hurdles and to conduct large clinical trials. This section will discuss different cell-based approaches for diabetic wound-healing therapy, including cellular skin substitutes based on natural and synthetic polymers, human amniotic membrane, human cadaver skin, and animal sources, as well as the roles of specific cell types such as multipotent stem cells, embryonic stem cells (ESCs), and pluripotent stem cells (PSCs).

3.2.1 Cellular skin substitutes based in a combination of natural and synthetic biomaterials There is one skin substitute containing cells that is made of natural and synthetic biomaterial. Dermagraft, manufactured by Organogenesis, is a human fibroblast-derived dermal substitute. Fibroblast cells are first isolated from human foreskin and cultured in a bioabsorbable polyglactin mesh scaffold. Once cultured, fibroblasts cells secrete different cytokines to form metabolically active dermal substitutes. Dermagraft is approved for treating diabetic foot ulcers greater than 6-week duration [18,94].

Biomaterials for diabetic wound-healing therapies

3.2.2 Cellular skin substitutes from human amniotic membrane There are several skin substitutes made from human amniotic membrane that are available in the market, including Affinity Human Amniotic Allograft, Fl oGraft Amniotic Fluid-Derived Allograft, Grafix, and GrafixPL Prime. 3.2.3 Cellular skin substitutes from human cadaver skin Theraskin is the only skin substitute derived from human cadaver skin. Theraskin is a cryopreserved human, living, split-thickness allograft that contains two different types of cells, fibroblasts and keratinocytes [94,149]. Fibroblasts and keratinocyte cells survive through harvesting, cryopreservation, and thawing [149]. Theraskin is usually collected within 24-h postmortem from an organ donor, washed with antibiotics, and cryopreserved [94]. This product is regulated by FDA as a human tissue for transplantation. 3.2.4 Cellular skin substitutes based on combined human and animal sources Apligraf is one of the skin substitute bases on combined human and animal source that is approved for treating diabetic foot ulcers and venous leg ulcers [18,94,149]. Apligraf consists of two different layers, type I bovine collagen gel with neonatal human fibroblast and neonatal keratinocytes [18,94]. This bioengineered skin substitute actively secretes growth factors, cytokines, and ECM proteins necessary for tissue regeneration [18,94,149]. 3.2.5 Stem-cell-based substitutes for regenerative healing of diabetic wounds 3.2.5.1 Induced pluripotent stem cells Induced pluripotent cells (iPSCs) have high differentiation potential and are multipotent with self-renewal capabilities. iPSCs are derived from adult somatic cells and can differentiate into any adult cell types (i.e., pluripotent), thus limiting the concerns associated with ethical dilemmas [150]. Unlike other cell types, these cells do not have to go through procedures such as bone marrow or adipose tissue biopsies [151], e.g., the process is noninvasive. iPSCs are known to differentiate into all three germ layers, and these terminally differentiated cells can enhance the diabetic wound healing through paracrine and direct cellular effects [151,152]. iPSCs are known to secrete different growth factors, cytokines, promote angiogenesis, and mitigate the impaired homing potential of progenitor cells [151,153,154]. These cell types, when used therapeutically, reduce the chance of immune rejection. There are various sources of iPSCs, such as human-induced pluripotent stem-cellderived endothelial cells (hiPSC-EC), human-induced pluripotent-derived fibroblast (hiPSC-F), human-induced pluripotent-derived mesenchymal stem cells (hiPSCMSC), and human-induced pluripotent stem cell-derived extracellular vesicles (hiPSC-EV). A recent study by Shen et al. [155] reported application of vascular

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Table 7 Advantages and disadvantages of embryonic, adult, and induced pluripotent stem cells for diabetic wound-healing applications. Stem cells

Embryonic

Adult

Induced pluripotent

Advantages

– High differentiation potential – Retain their potency – Provide consistent phenotype

– High differentiation potential – No ethical concerns – Donor-specific therapy – Low cell harvesting procedure risk

Disadvantages

– Ethical concerns – High malignancy risk

– Self-renewal and differentiation – Donor specific therapy – Lower malignancy risk – No ethical concerns on BM-MSC, ADSC – Targeted differentiation – Simple cell harvesting protocol – Disposable tissue – Biopsy high surgical risk – Low stem cell concentration in BM-MSCs – Limited differentiation potential

– High malignancy risk – Complex induction protocol – Biased differentiation

constructs containing hiPSC-EC for diabetic wound healing in diabetic mice model. Authors reported that this method resulted in rapid granulation tissue formation, macrophage infiltration, and better facilitation of neovascularization [155]. See Table 7 for list of advantages and disadvantages of iPSCs. 3.2.5.2 Mesenchymal stem cells Mesenchymal stem cells (MSCs) are also known as adult stem cells (fibroblast-like) that are capable of self-renewal and differentiation. Some of the sources of these cell types include bone marrow (BM-MSCs), adipose tissue (ADSCs), and umbilical cord blood (hUC-MSC). Unlike ESCs, the use of MSCs may reduce or address the issue of ethical concerns. BM-MSCs-based therapy has been used frequently in various preclinical and clinical studies [156]. A recent study by Kuo et al. showed that use of BM-MSCs in diabetic rat model caused significant reduction in wound size, shorter healing times (see Fig. 4), a significant reduction in topical proinflammatory reaction, suppression of CD45 expression, and upregulation of cellular proliferation and regeneration [157]. MSCs are gaining interest in the field of diabetic wound healing because of their ability to migrate toward injury site and activate resident progenitor cells, participate in the regeneration of damaged tissue and secrete essential growth factors and cytokines, and exert an antiinflammatory response [157]. A study by Dash et al. showed the use of autologous cultured BM-derived MSCs in a patient with nonhealing lower-extremities ulcer

Biomaterials for diabetic wound-healing therapies

NC

C

MSC-1

MSC-2

1 week

5 week

Wound healing time (week)

12 10 8 6 4 2 0 NC

C

MSC-1

MSC-2

Fig. 4 Topical mesenchymal stem cell injection in the wound edge decreases the total time course for wound healing in the diabetic rats. The experimental results indicated that the wound size was significantly reduced in mesenchymal stem cell-1 (#P < .001) and mesenchymal stem cell-2 (*P < .001) groups as compared with the controls. NC, normal control; C, control (diabetes control without treatment); MSC-1, one session of mesenchymal stem cells; MSC-2, two sessions of mesenchymal stem cells. (From Kuo YR, et al. Bone marrow-derived mesenchymal stem cells enhanced diabetic wound healing through recruitment of tissue regeneration in a rat model of streptozotocin-induced diabetes. Plast Reconstr Surg 2011;128(4):872–80.)

significantly improved pain-free walking distance, reduced the size of ulcer with no significant alteration in biochemical parameters [158]. Similarly, adipose-derived stem cells (ADSCs) are another type of MSCs that are multipotent in nature. These can differentiate into various lineages such as muscles, fat, bone, and cartilage [150,159]. These types of cells can be harvested through minimal invasiveness, thus becoming attractive to the field of regenerative medicine. Lee et al. reported the formation of blood vessels in patients with critical limb ischemia when adipose-tissuederived stem cells were implanted intramuscularly [160]. See Table 7 for list of advantages and disadvantages of MSCs for diabetic wound healing applications. 3.2.5.3 Embryonic stem cells ESCs are pluripotent cells that are located within blastocysts and have the potential to differentiate into any of the three primary germ layers: endoderm, ectoderm, or mesoderm. These cells are superior to adult-derived stem cells because ESCs can retain their potency, provide consistence phenotype, and have the high proliferative ability [161,162]. However, the use of these cells for diabetic wound healing is limited mainly

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due to ethical concerns, invasive harvesting techniques, and limited cell survival [162]. A study by Lee et al. used ESC in diabetic rat wound healing, and reported a reduction in wound size, a significant increase in EGF, and VEGF levels at days 5 and 10 [163]. See Table 7 for list of advantages and disadvantages of ESCs.

4. Emerging biomaterial technologies for treatment of diabetic wounds Chronic diabetic ulcers represent a fast growing health burden for patients with diabetes, often leading to limb amputation in as many as 12%–24% of cases [164–166], which underscores an ever-increasing demand for better and clinically effective therapies. This section provides an overview of several exciting novel biomaterial technologies that are promising for the treatment of chronic ulcers, including microRNA-conjugated nanoparticles, multifunctional alginate, peptide and DNA hydrogels, in situ-formed stemcell-based skin substitutes, noncontact biophysical therapies, as well as therapeutics derived from scarless fetal healing [11,164–172]. An exciting novel theme in biomaterials in wound-healing research is development of a combination therapies based on microRNA (miR) incorporation. These molecules are known regulators of the production of proinflammatory cytokines at the posttranscription level [11,173], where microRNA binds to the target messenger RNAs leading to mRNA degradation and gene activation. Recently, Zgheib et al. used cerium oxide nanoparticles conjugated with miR-146a for diabetic wound healing in the mouse model [11]. This therapy resulted in improved biomechanical properties (maximum load and tensile modulus) of the wound post healing, improved healing time, decreased inflammation, and increased angiogenesis. Another study by Orgill et al. reported that the expression of miR-26a is increasingly induced in response to diabetes 4 days postwounding [167]. To control the increased expression, they injected miR26a inhibitor and found 80% increase in angiogenesis, 2.5-fold increase in granulation tissue thickness and accelerated wound closure, which was associated with induction of BMP/SMAD1 signaling in endothelial cells [167]. Another study [171] by the same group reported that the miR-615-5p inhibited the VEGF-AKT/eNOS signaling pathway in endothelial cells. Upon delivery of miR-615-5p inhibitor, there was a significant increase in angiogenesis, granulation tissue thickness, and wound closure in diabetic mice [171]. Overall, these findings demonstrate that miRNA-based materials can regulate angiogenesis and inflammation in diabetic wound healing and therefore provide promising strategies to ameliorate diabetes-related complications in the wounds. Hydrogel-based materials that can be used as a therapy-delivery system into the wound represent an important class of rapidly developing therapeutic tools. For example, a number of alginate-based hydrogels have been developed for controlled and localized delivery of different-sized molecules and drugs into the wound. Veves et al. designed a

Biomaterials for diabetic wound-healing therapies

novel biomaterial that consists of alginate and deoxyribonucleic acid and incorporates active endothelial cells and tested this approach in the mouse models of DM [168]. This biomaterial was found to be biocompatible, easily injectable into the wounds, and was demonstrated to accelerate wound closure. Another example is hydrogel with sodium alginate enriched with fatty acids and vitamins A and E that has shown promising results in the healing of foot wounds in diabetic patients [174]. Self-assembling peptide hydrogels represent another unique group of biomaterials that are emerging for applications in wound healing (discussed in more detail in Section 3.1.2.6). The material and biochemical properties of the nanofiber hydrogels can be easily tailored via sequence alterations and peptide concentration [132]. This unique quality of the self-assembling peptide hydrogels can be used effectively to modulate wound microenvironment and direct cell behavior within the wound via the novel mechanism that involves integrin activation on the cell surface via nonspecific interactions with the particular motifs on the nanofibers [138–141]. Importantly, these materials confer their therapeutic effects by modulating cell behavior via cell-biomaterial interactions in the absence of exogenous pharmacological agents, cells or gene therapy, which removes potential hurdles associated with FDA approval. In addition to noncellular biomaterial therapies discussed, stem cell delivery and skin substitutes are emerging as a frontrunner for diabetic wound healing research. Stem cells are capable of inherent regeneration via paracrine effects [44,175,176]. Gurtner et al. developed an injectable poly(ethylene glycol)-gelatin based hydrogel system to deliver ASCs into diabetic wounds [164]. Their findings showed decreased inflammatory cell infiltration, enhanced blood vessel formation, and accelerated wound closure in the mouse model. In another study, PEG-gelatin-based hydrogel was able to enhance ASCs viability, enhance stem cell retention, and maintain stem cell properties [56]. However, clinical application of stem cell therapies has been limited due to ethical concerns, reduced potency during ex vivo expansion, and low survival after implantation to the wounds [164,176,177], and further exploration is needed. Other attractive emerging therapy for diabetic wound healing involves applying noninvasive biophysical stimulations, such as electric field or ultrasound treatment, to the wound, where mechanical or electrical potential is converted into a biochemical response at the cellular level. Gurtner and coworkers reported using noncontact, low-frequency ultrasound (40 kHz, displacement of 65 μm) for diabetic wound healing in mice [178]. This therapy showed increased expression of prohealing factors, including stromal-cell-derived factor 1, VEGF and CD31, and significant reduction in the mean wound size. Another study reported that a novel dressing that utilizes wireless nonthermal wound stimulation by highfrequency electric field in the diabetic (db/db) mouse model results in enhanced angiogenesis and VEGF release and improved healing via frequency-dependent MAPK/ERK pathway activation [172]. In yet another development, several studies reported using either electric fields or currents in patterned electroceutical dressings to disrupt biofilms in the

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chronic wounds while accelerating wound healing [179–181]. Overall, these results illustrate the breadth of the field of biomaterial-related wound healing therapies that are currently under investigation. Recently, scarless (regenerative) wound healing that is observed in the fetal dermal wound has been gaining interest [182,183]. The differential regulation of inflammatory response and mechanical stress, as well as differences in collagen and HA ECM composition between fetal and adult wound healing, have been shown to play a critical role in promoting regeneration in the fetus while resulting in scar formation in the adult [183]. From the therapeutic standpoint, the specific focus has been on inflammatory regulator interleukin 10 (IL-10), which is one of the key players seen in fetal scarless wound healing [182,184]. A recent study by Balaji et al. reported that the IL-10 activates signal transducer and activator of transcription 3 (STAT3) [185]. This activation of STAT3 and use of IL-10 as a biomaterial causes to regulate fibroblast-specific formation and synthesis of HA-rich wound ECM [184,185]. However, there is little information available regarding an endogenous role for IL-10 in diabetic ulcers, and more studies are needed to test the potential effectiveness of IL-10-based therapies in chronic wounds.

5. Conclusions Considering the exponential growth trend of DM and related complications, such as nonhealing ulcers, there is a critical need for the development of effective bioactive biomaterials for new therapies to treat chronic diabetic ulcers. Recent advances in the biomaterials research discussed in this chapter demonstrate great potential of many approaches currently under development and/or in preclinical testing, especially those that include bioactive compounds or encapsulated cell therapies, to find the right solution for seemingly intractable problem of nonhealing diabetic ulcers. However, in addition to therapeutic potential of any given biomaterial, there are additional considerations that will ultimately determine whether a promising therapy will make it to the market. These include ease of manufacturing, storage without loss of viability, cost-benefit relationship, and suitability for application in diabetic individuals, in particular, those with uncontrolled hyperglycemia. Nevertheless, given the multitude of approaches being investigated (e.g., polymers and composites, self-assembling peptides, hydrogels and nanoparticles, microRNAs, alternative biophysical therapies, cell- and drug-delivery therapies), along with the explosive pace of the biomaterial technology development, the analysis presented in this review warrants cautious optimism for a breakthrough that would enable the regenerative healing of a diabetic wound and provide much needed cure for chronic diabetic ulcers.

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[128] Intini C, et al. 3D-printed chitosan-based scaffolds: an in vitro study of human skin cell growth and an in-vivo wound healing evaluation in experimental diabetes in rats. Carbohydr Polym 2018;199:593–602. [129] Zeng Y, et al. Preformed gelatin microcryogels as injectable cell carriers for enhanced skin wound healing. Acta Biomater 2015;25:291–303. [130] Totsuka Sutto SE, et al. Efficacy and safety of the combination of isosorbide dinitrate spray and chitosan gel for the treatment of diabetic foot ulcers: a double-blind, randomized, clinical trial. Diab Vasc Dis Res 2018;15(4):348–51. [131] Shaw J, et al. The effect of topical phenytoin on healing in diabetic foot ulcers: a randomized controlled trial. Diabet Med 2011;28(10):1154–7. [132] Zhang S. Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol 2003;21 (10):1171–8. [133] Petka WA, et al. Reversible hydrogels from self-assembling artificial proteins. Science 1998;281 (5375):389–92. [134] Schneider JP, et al. Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide. J Am Chem Soc 2002;124(50):15030–7. [135] Zhang S, et al. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci USA 1993;90(8):3334–8. [136] Holmes TC, et al. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc Natl Acad Sci USA 2000;97(12):6728–33. [137] Davis ME, et al. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation 2005;111(4):442–50. [138] Cho H, et al. Regulation of endothelial cell activation and angiogenesis by injectable peptide nanofibers. Acta Biomater 2012;8(1):154–64. [139] Balaji S, et al. Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing. Wound Repair Regen 2012;20(1):15–27. [140] Narmoneva DA, et al. Self-assembling short oligopeptides and the promotion of angiogenesis. Biomaterials 2005;26(23):4837–46. [141] Hurley JR, et al. Nanofiber microenvironment effectively restores angiogenic potential of diabetic endothelial cells. Adv Wound Care 2014;3(11):717–28. [142] Sheikh AQ, et al. Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions. Am J Physiol Cell Physiol 2014;306(8):C768–78. [143] Bradshaw M, et al. Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration. Sci Rep 2014;4:6903. [144] Carrejo NC, et al. Multidomain peptide hydrogel accelerates healing of full-thickness wounds in diabetic mice. ACS Biomater Sci Eng 2018;4(4):1386–96. [145] Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med 2014;6(265):265sr6. [146] Ramirez HA, et al. Comparative genomic, microRNA, and tissue analyses reveal subtle differences between non-diabetic and diabetic foot skin. PLoS One 2015;10(8):e0137133. [147] Trengove NJ, et al. Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 1999;7(6):442–52. [148] Kim KA, et al. Dysfunction of endothelial progenitor cells under diabetic conditions and its underlying mechanisms. Arch Pharm Res 2012;35(2):223–34. [149] Towler MA, et al. Randomized, prospective, blinded-enrollment, head-to-head venous leg ulcer healing trial comparing living, bioengineered skin graft substitute (Apligraf ) with living, cryopreserved, human skin allograft (TheraSkin). Clin Podiatr Med Surg 2018;35(3):357–65. [150] Kanji S, Das H. Advances of stem cell therapeutics in cutaneous wound healing and regeneration. Mediat Inflamm 2017;2017:5217967. [151] Baraniak PR, McDevitt TC. Stem cell paracrine actions and tissue regeneration. Regen Med 2010;5 (1):121–43. [152] Singh VK, et al. Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev Biol 2015;3:2.

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22):2478–85. [155] Shen YI, et al. Engineered human vascularized constructs accelerate diabetic wound healing. Biomaterials 2016;102:107–19. [156] Lopes L, et al. Stem cell therapy for diabetic foot ulcers: a review of preclinical and clinical research. Stem Cell Res Ther 2018;9(1):188. [157] Kuo YR, et al. Bone marrow-derived mesenchymal stem cells enhanced diabetic wound healing through recruitment of tissue regeneration in a rat model of streptozotocin-induced diabetes. Plast Reconstr Surg 2011;128(4):872–80. [158] Dash NR, et al. Targeting nonhealing ulcers of lower extremity in human through autologous bone marrow-derived mesenchymal stem cells. Rejuvenation Res 2009;12(5):359–66. [159] Zuk PA, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7(2):211–28. [160] Lee HC, et al. Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: a pilot study. Circ J 2012;76(7):1750–60. [161] Wu DC, Boyd AS, Wood KJ. Embryonic stem cell transplantation: potential applicability in cell replacement therapy and regenerative medicine. Front Biosci 2007;12:4525–35. [162] Gorecka J, et al. The potential and limitations of induced pluripotent stem cells to achieve wound healing. Stem Cell Res Ther 2019;10(1):87. [163] Lee K-B, et al. Topical embryonic stem cells enhance wound healing in diabetic rats. J Orthop Res 2011;29(10):1554–62. [164] Dong Y, et al. Acceleration of diabetic wound regeneration using an in situ–formed stem-cell-based skin substitute. Adv Healthc Mater 2018;7(17):1800432. [165] Gregg EW, et al. Changes in diabetes-related complications in the United States, 1990-2010. N Engl J Med 2014;370(16):1514–23. [166] Faglia E, et al. Influence of osteomyelitis location in the foot of diabetic patients with transtibial amputation. Foot Ankle Int 2013;34(2):222–7. [167] Icli B, et al. Regulation of impaired angiogenesis in diabetic dermal wound healing by microRNA26a. J Mol Cell Cardiol 2016;91:151–9. [168] Tellechea A, et al. Alginate and DNA gels are suitable delivery systems for diabetic wound healing. Int J Low Extrem Wounds 2015;14(2):146–53. [169] Pradhan Nabzdyk L, et al. Expression of neuropeptides and cytokines in a rabbit model of diabetic neuroischemic wound healing. J Vasc Surg 2013;58(3):766–75.e12. [170] Tecilazich F, Dinh T, Veves A. Treating diabetic ulcers. Expert Opin Pharmacother 2011;12 (4):593–606. [171] Icli B, et al. MicroRNA-615-5p regulates angiogenesis and tissue repair by targeting AKT/eNOS (endothelial NO synthase) signaling in endothelial cells. Arterioscler Thromb Vasc Biol 2019. Atvbaha119312726. [172] Sheikh AQ, et al. Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field. J R Soc Interface 2013;10(78):20120548. [173] Moura J, Borsheim E, Carvalho E. The role of microRNAs in diabetic complications-special emphasis on wound healing. Genes (Basel) 2014;5(4):926–56. [174] Gustinelli Barbosa MA, et al. Effects of hydrogel with enriched sodium alginate in wounds of diabetic patients. Plast Surg Nurs 2018;38(3):133–8. [175] Cerqueira MT, Pirraco RP, Marques AP. Stem cells in skin wound healing: are we there yet? Adv Wound Care 2016;5(4):164–75. [176] Duscher D, et al. Stem cells in wound healing: the future of regenerative medicine? A mini-review. Gerontology 2016;62(2):216–25.

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[177] Nuschke A. Activity of mesenchymal stem cells in therapies for chronic skin wound healing. Organogenesis 2014;10(1):29–37. [178] Maan ZN, et al. Noncontact, low-frequency ultrasound therapy enhances neovascularization and wound healing in diabetic mice. Plast Reconstr Surg 2014;134(3):402e–411e. [179] Roy S, et al. Disposable patterned electroceutical dressing (PED-10) is safe for treatment of open clinical chronic wounds. Adv Wound Care 2019;8(4):149–59. [180] Barki KG, et al. Electric field based dressing disrupts mixed-species bacterial biofilm infection and restores functional wound healing. Ann Surg 2019;269(4):756–66. [181] Banerjee J, et al. Silver-zinc redox-coupled electroceutical wound dressing disrupts bacterial biofilm. PLoS One 2015;10(3):e0119531. [182] Liechty KW, et al. Fetal wound repair results in scar formation in interleukin-10-deficient mice in a syngeneic murine model of scarless fetal wound repair. J Pediatr Surg 2000;35(6):866–72 [discussion 872-3]. [183] Zgheib C, Xu J, Liechty KW. Targeting inflammatory cytokines and extracellular matrix composition to promote wound regeneration. Adv Wound Care 2014;3(4):344–55. [184] King A, et al. Interleukin-10 regulates fetal extracellular matrix hyaluronan production. J Pediatr Surg 2013;48(6):1211–7. [185] Balaji S, et al. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling. FASEB J 2017;31(3):868–81.

CHAPTER 15

Photobiomodulation therapy in diabetic wound healing Sasikumar Ponnusamy, Rodrigo Mosca, Karishma Desai, Praveen Arany Oral Biology and Biomedical Engineering, University at Buffalo, Buffalo, NY, United States

Diabetes mellitus (DM) is a complex metabolic chronic disorder and devastates the lives of affected individuals [1]. According to World Health Organization (WHO) [2], the number of people with diabetes has increased from 108 million in 1980 to 422 million in 2014. The global prevalence of diabetes among adults over 18 years of age has risen from 4.7% in 1980 to 8.5% in 2014 [3]. In 2016, according to the WHO, an estimated 1.6 million deaths were directly caused by diabetes and another 2.2 million deaths were attributable to high blood glucose in 2012 (WHO). Diabetes contributes to multiorgan dysfunction, including cardiovascular disease, chronic kidney disease, and blindness among others. Impaired wound healing is a major complication affecting multiple anatomical sites and is a serious problem in clinical practice [4]. It was estimated that 15% of individuals with DM will develop foot ulceration and wounds, and 3% will require lower-extremity amputation [5]. In addition, an increased incidence of wound complications in surgical patients with DM increases the general surgical risks due to the metabolic abnormalities associated with DM [6]. Normal wound healing is a well-regulated process, which includes hemostasis, inflammatory, proliferative, and remodeling phases [7, 8]. However, wound healing in diabetes mellitus is often impaired and results in nonhealing or protracted, chronic skin ulcers [9]. In diabetes, high levels of glycation products blood result in widespread inflammation and narrowing of blood vessels, prompting poor circulatory system and decreased neurovascular functions [10]. Due to the poor circulatory system, diabetic wound demonstrates a persistent inflammatory phase. Thus, the impaired diabetic healing response is due to complex underlying pathology involving poor circulation and a protracted inflammatory response that result in reduced cellular proliferation and elaboration of growth factors [11, 12]. Current treatment of the diabetic wound includes systemic glycemic control, local wound care and infection control, revascularization, and pressure-relieving strategies. Most of these individual strategies are empirical and are driven by general wound care, a rationalized approach to the underlying pathophysiology in diabetes are currently being pursed. Nonetheless, despite these current multidisciplinary treatments, clinical Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00015-0

© 2020 Elsevier Inc. All rights reserved.

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outcomes in diabetic wounds remain often unsatisfactory [9]. Novel approaches beyond the routine surgical and pharmacological treatment are a significant current need. These include innovative biotechnology (biomaterials, nanotechnology) and biophysical (ultrasound, light, radiofrequency, pressure) treatment approaches.

1. Light as a therapeutic intervention Anecdotal use of various forms of light for human health dates back to several ancient civilizations. The use of light therapy in modern medicine can be traced to a Nobel Prize in Medicine in 1903 for the use of antimicrobial effects of sunlight. A striking therapeutic benefit of using low-dose laser treatments to improve wound healing was noted in the 1960s [13–15]. This innovative treatment modality has been referred to with several terms such as photostimulation, cold laser treatments, soft laser therapy, and most popularly, low-level light/laser treatments (LLLT), among many others. A recent consensus on terminology outlined the preferred term as Photobiomodulation (PBM) Therapy [16]. This is defined as a form of light treatment that utilizes nonionizing light sources, including Lasers, LEDs, and broadband light, in the visible and infrared spectrum. This process involves a nonthermal process with endogenous chromophores eliciting photophysical (i.e., linear and nonlinear) and photochemical events at various biological scales. This treatment results in beneficial therapeutic outcomes, including, but not limited, to the alleviation of pain or inflammation, immunomodulation, and promotion of wound healing and tissue regeneration. Unlike biological agents used in wound care, light is a physical form of energy and a better understanding of its biophysical nature is important to appreciate light-tissue interactions. Light interaction with matter is broadly categorized into four processes, namely, absorption, reflection, scattering, and transmission. For the purposes of the therapeutic benefits from light treatments, these interactions can be broadly classified into productive (absorption and scattering) versus nonproductive (reflection and transmission). The latter is not relevant for the biological mechanism per se but is a key aspect of safety and clinical efficacy during treatment delivery. A predominant aspect of productive, therapeutic light-tissue interaction is based on the process of absorption by a selective chromophore. If there are no relevant wavelength-specific chromophores in the tissues, the photons pass through the tissue as total transmission without producing any biological (nonproductive) effects. The effectiveness of light tissue penetration in human skin is associated with the absorption spectra of three major biological chromophores, namely, melanin in epidermis, hemoglobin (oxy- and deoxy-) in blood within the dermis, and water throughout tissues (Fig. 1). Scattering occurs when the incident photon changes its direction of propagation due to differences in refractive indices. Scattering enables the incident light to spread out and progressively reduces penetration thereby limiting depth of treatments.

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Fig. 1 The blue (435–500 nm) wavelength is absorbed by melanin, blood, and porphyrins, while red (620–750 nm) penetrates deeper as it is less absorbed by blood and melanin. The least absorption by these major chromophores occurs in the near-infrared (NIR, 750–950 nm) where water becomes most important. Therefore, the penetration depth of a given light source is a result of both its inherent wavelength-dependent photon energy (more for blue than red or near-infrared) but also largely dependent on presence of relevant biological wavelength-specific chromophores (also more for blue-red than NIR). This implies that effective light penetration is equal to the inverse of the wavelength-specific tissue absorption coefficient. (Based on Mosca RC, et al., Adv Skin Wound Care 2019;32(4):157.)

2. Mechanisms of photobiomodulation therapy Photobiomodulation (PBM), also known as low-level light therapy, is application of light (usually visible to infrared wavelengths) to stimulate or inhibit cellular function leading to beneficial effects [17]. Photobiomodulation has been used to improve many other human diseases, including skin antiaging, osteogenic differentiation and recently used in various neurological and psychological conditions, including ischemic stroke, chronic traumatic brain injuries, depression, and dentin formation. PBM has been used for more than 40 years to promote wound healing, reduce pain and inflammation, and promote tissue healing. There are three well-described PBM molecular mechanisms that appear to operate within discrete cellular compartments. These are briefly described in the following sections.

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2.1 Energizing the powerhouse—The mitochondria: The performance mechanism The first involves absorption of specific wavelengths of light by a key enzyme of the respiratory chain within the mitochondria, cytochrome C oxidase (COX) that is central to aerobic metabolism of animals and is a critical element of energy production in all organisms [18]. Embedded in the inner mitochondrial membrane, COX is the terminal enzyme of the electron transport system, accepts electrons from cytochrome C, and subsequently transfers them to molecular oxygen to generate water. The action spectrum of COX runs from the 580–700 nm wavelengths 635 nm (red) and 730 nm (IR) [19, 20]. Absorption of the incident photons by the COX initiates a photochemical cascade increasing adenosine triphosphate (ATP). The light-induced increase in ATP synthesis and increased proton gradient lead to an increasing activity of the Na+/H+ and Ca2+/Na+ antiporters, and of all the ATP-driven carriers for ions, such as Na+/K+ ATPase and Ca2+ pumps. ATP is the substrate for adenylcyclase, and therefore the ATP level controls the level of cAMP. Both Ca2+ and cAMP are very important second messengers [21]. The activity of COX is inhibited by bound nitric oxide (NO). PBM can reverse this inhibition by photodissociation of NO from its binding sites, increasing the respiration rate on mitochondria cell and also, protecting cells against NO-induced cell death. Besides increasing the ATP, other molecules are involved in the signaling pathways from mitochondria to nuclei: reactive oxygen species (ROS) and reactive nitrogen species (RNS) [22]. ROS are small oxygen-derived molecules, which have been reduced with added electrons to become highly reactive that are either oxidizing agents or are easily converted into oxygen radical such as superoxide ð O2  Þ, hydrogen peroxide (H2O2), hydroxyl radicals ( OH), Hydroxyl ion (OH) playing beneficial roles [23, 24]. These radicals play key roles in inflammation as direct germicidal agents used by neutrophils and macrophages. The concerted production of large amounts of ROS by immune cells is of great importance for effective host defense. Inside phagosomes, high concentrations of ROS create a toxic oxidative stress environment for phagocytized microbes leading to DNA damage, lipid peroxidation, and oxidation of amino acids [23]. In addition, ROS (in low concentrations) are involved in a myriad of physiological cell-signaling pathways, referred to as redox-signaling pathways. There is also increasing evidence that ROS are crucial for wound repair, not only as germicides but also for cellular signaling. PBM produce a shift in overall cell redox potential in the direction of greater oxidation and increased ROS generation and cell redox activity, increasing in ATP production, which implies more efficient electron flow through the electron transport chain [22, 25]. However, the overall effect of violetblue light PBM (395–470 nm wavelength) is to increase the level of cellular ROS and can even produce apoptosis and outright cytotoxicity if the doses are high enough. The Violet light (395–425 nm) uses the chromophore responsible for this action and







Photobiomodulation therapy in diabetic wound healing

is thought to be generation of ROS via photoexcitation of metal free porphyrins that have a large Soret band at about 405 nm. Royal blue light (430–470 nm) is also known to generate ROS and in this case the chromophores are thought to be flavins, flavoproteins, or cryptochromes [26].

2.2 Lighting up beacons—Photosensitive membrane receptors and channels: Analgesia mechanism The second PBM mechanism involves light-modulated cell membrane receptors and transporters such as the opsins (Melanopsin—opsin4; Opn4 and Encephalopsin—Opsin 3; Opn3), transient receptor potential V1, and aryl hydrocarbon receptor (AHR) [27, 28]. AHR are important for normal cell development and immune responses and serve not only as an internal oxygen and redox status sensor, but also recognize low-molecularweight compounds and light with endogenous ligands derived from tryptophan due to UV or visible light exposure-induced photolytic destruction/photo-oxidation. Opn4, a nonimage-forming opsin, found in the retina, has been linked to a number of behavioral responses to light, including circadian photoentrainment and also, regulating blood vessel function, particularly in the context of photorelaxation. The Encephalopsin (Opsin 3) is present in the pulmonary vasculature and is localized to the smooth muscle, and has peaking wavelength spectral sensitivity ranges from 420 to 500 nm (blue-cyan range) and unidentified light-dependent vasorelaxation pathway. Neuropsin (Opn5) is an atypical opsin known to respond to near-UV photons (λmax of 380 nm) and is expressed in retinal ganglion cells (RGCs) in adult mice. It regulates seasonal breeding behavior in birds and the activity cycle in mice, but also mediates photoentrainment of the retinal circadian clock, and that regulates vascular regression timing [29].

2.3 Harnessing endogenous stem cells for regeneration—TGF-β activation: Healing mechanism A third mechanism that was recently elucidated by our group involves direct activation of potent molecule, Transforming Growth Factor (TGF-β1) by PBM treatments [17]. TGF-β is one of the most important growth factors at wound healing. It exerts pleiotropic effects on wound healing by regulating cell proliferation, differentiation, extracellular matrix (ECM) production, and modulating the immune response [30]. Following injury, large amounts of TGF-βs are released into the wounded tissue by platelets that recruit innate immune cells and also attract fibroblasts and stimulates proliferation. There are three mammalian isoforms of TGF-β (TGF-β1, -β2, and -β3). TGF-β2 is needed to infiltrate immune cells in general and of monocytes and macrophages in particular, and also, markedly the deposition of collagen types I and III and fibronectin, which resulted in an improvement in scar formation. TGF-β3 showed to have a TGF-β1-antagonistic effect in scar formation. In wounds with minimal or no scar formation, such as in the oral

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mucosa, expression of TGF-β1 decreases along with a significant increase in the ratio of TGF-β3 to TGF-β1, reduced collagen type I deposition by restricting myofibroblast differentiation and promoting collagen degradation by MMP-9 [31]. TGF-β isoforms are synthesized as homodimeric pro-proteins and undergo proteolytic cleavage in the trans-Golgi network by furin-like pro-protein convertases, resulting in the formation of the mature TGF-β dimer. This dimer remains noncovalently associated with its pro-peptide, termed the latency-associated peptide (LAP), rendering the growth factor latent [32]. It has been shown that Integrin activates latent TGF-β1 (LTGF-β1) through two different mechanisms through a conformational change to the LTGF-β1 complex. This causes the release of active TGF-β1, which becomes available to interact with its receptor. Other mechanisms of LTGF-β1 activation involving Integrin in wounds involve a protease-dependent activation. In these cases, Integrin could simultaneously bind the LTGF-β1 complex and proteinases (like MMP-2 and MMP-9), and thereby the MMPs cleave the active TGF-β1 from its inactive form. PBM treatments have been shown to induce low amounts of ROS that act on a specific amino acid residue of the LTFG-β1 complex resulting in a conformational change activating it [17]. Thus, activation of TGF-β1 via PBM treatment allows exquisite spatial and temporal control of its pathophysiological roles. TGF-β1, in early stages of the healing process, prompts recruitment of inflammatory cell into the injury site, which are later involved in a negative feedback via release of superoxide from macrophages. It has also been shown that TGF-β1 can induce its own expression, thus prolonging its biological actions beyond its initial activation. During this interim stage, granulation tissues are gradually formed and TGF-β1 prompts the expression of key components of ECM. Further, TGF-β1 improves the angiogenic properties of endothelial progenitor cells to facilitate blood supply to the injured site and stimulates contraction of fibroblasts to enable wound closure. Keratinocyte migration is also promoted by TGF-β1 via regulation of cell migrationassociated Integrin and is one the main collagen-stimulating factors, especially type I in fibroblasts [33]. PBM has the ability to activate endogenous components, by postwounding hemostatic milieu, in a controlled, self-limiting manner on a critical aspect of this potential therapeutic modality because both ROS and TGF-β1 in excessive amounts are potently deleterious [17]. Further, the early wound environment contains abundant plateletderived and serum latent transforming growth factor β (LTGF-β1). In later phases of healing, the predominant source of LTGF-β1 is inflammatory cells, especially the macrophages. Nonetheless, PBM extends well beyond the local exposure site and could potentially activate other sources, such as cell-secreted and extracellular of LTGF-β1 [17, 34]. Overall, PBM treatments can harness the wound-promoting responses of TGF-β1 in various healing phases from repetitive PBM treatments.

Photobiomodulation therapy in diabetic wound healing

3. PBM therapy in diabetic wound healing Diabetes is known to be associated with poor wound healing and is responsible for 50%– 70% of all nontraumatic amputations and it is estimated that 15% of all diabetic patients will develop an ulcer on the feet or ankles at some time during the disease course [35]. The characteristic of diabetic wounds such as peripheral neuropathy, structural deformity, altered immune function or increased susceptibility to infection and hypoxia/ischemia often delayed the healing processes [36]. Even though numerous treatment options available for diabetic wounds treating, which includes debridement, mechanical load relief, topical antibiotics and dressings, newer developments include the use of bioengineered skin equivalents, growth-factor therapy, and hyperbaric oxygen treatment, but none of them showed effective healing. Alternative treatment for promoting diabetic wound healing is essential. The search for potent treatment agents and strategies with less or no side effects has attracted many researchers.

3.1 PBM and redox modulation in diabetic wounds Some of the animal studies showed that PBM increased the wound-healing process in diabetic rat model. The diabetic wound rat cotreated with quercetin and laser (632.8 nm) shows enhancing wound healing in nondiabetic and diabetic rats by limiting chronic inflammation, improving glycemic state, increasing insulin level, suppressing oxidative stress, and enhancing the antioxidant defense system [37]. Wounded diabetic animals treated with 904-nm laser triggering the microvasculature, with lowered levels of nitrite, and increased protection against oxidative damage in lipid membranes. The increased production of collagen and decreased oxidative and nitrosative stress suggest that PBM may be a viable therapeutic alternative in diabetic wound healing [38]. In a study by Byrnes et al., 632-nm laser treatments improved cutaneous wound healing in an animal model with type II diabetes [39]. Hypoxic wounded and diabetic hypoxic wounded models responded positively to PBM therapy where it has a stimulatory effect on stressed cell viability and promotes proliferation to aid repair and wound healing [40]. This suggests that the more stressed the cells are, the better they appear to respond to PBM treatments. Soleimani et al. revealed PBM using 890-nm laser light significantly accelerated the wound-healing process in the streptozotocin-induced type one diabetes mellitus in an experimental rat model [41]. Overall PBM treatments were noted to improve wound closure by cell viability, proliferation, and collagen production by modulating cytokine expression.

4. Optimizing PBM treatments to dysregulated signaling pathways in diabetic wound healing PBM therapy has been used in several fields to promote healing but remains controversial due to lack of robust and reproducible clinical outcomes. This has been largely attributed

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TGF-β VEGF bFGF SDF TNF-α EGF Interleukins Estrogen receptors

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Fig. 2 Signal transduction pathways induced by PBM treatments that operate directly at the ligand and induce downstream cytoplasmic signaling intermediates. Integration and cooperation (crosstalk) of these pathways are critical for directed therapeutic biological responses such as proliferation, migration, or differentiation.

to a lack of understanding of the underlying cellular and molecular mechanisms that has prevented development of rigorous clinical treatment protocols. In poorly healing diabetic wounds, alteration of cellular and molecular signals required for the normal wound-healing process such as angiogenesis, granulation tissue formation, epithelialization, and remodeling has been observed. Cytokine signaling pathways have been shown to drive several of these processes and are noted to be dysregulated in diabetic wounds. Hence, one rationale for the use of PBM treatment to promote diabetic wound healing could be based on optimizing PBM treatments to evoked signaling pathways (Fig. 2). The following section focuses on specific molecular signaling pathways noted to be triggered by PBM treatments in diabetic wound healing both in vitro and in vivo.

4.1 JAK/STAT signaling In a study with 660-nm laser treatments at 5 J/cm2 in diabetic wounded cells, significant increase in epidermal growth factor (EGF), and activation of its receptor phosphorylated EGF receptor (p-EGFR) by activation of Janus kinase/Signal transducer and activators of

Photobiomodulation therapy in diabetic wound healing

transcription (JAK/STAT), signaling pathway was observed [42]. The activation of JAK/STAT (p-JAK2, p-STAT1, and p-STAT5) was noted to stimulate cell proliferation and migration and suggests PBM treatments directly modulate cellular autocrine signaling. In a follow-up study, Houreld et al showed that PBM at 660 nm laser modulated various cell adhesion molecules (CAMs) contributing to the increased healing in diabetic wound [43]. They noted upregulation of 10 gene and downregulation of 25 genes related to CAMs in diabetic wounded cells. In another study by the same group, PBM induced a stimulatory effect on various CAMs, namely, cadherins, integrins, selectins, and immunoglobulins [44]. They also discuss the role of matrix metalloproteinases in diabetic wound healing modulated by PBM treatments.

4.2 Interleukins, bFGF, and TNFα A few studies have shown that PBM therapy alters cytokine production and helps promote wound healing in diabetic condition. The diabetic wounded cells irradiated with He-Ne laser (632.8 nm, 2.2 mW/cm2, and 5 J/cm2) stimulated the cytokines IL-6 expression, increased proliferation, and stimulated cellular migration in diabetic wounded fibroblast cells [45]. This study suggests that PBM appears to be beneficial in diabetic wound healing. Low-level laser irradiation of 2 J/cm2 in human skin fibroblasts (HSFs) cells cultured in high glucose concentration medium showed that PBM stimulated the release of IL-6 and basic fibroblast growth factor (bFGF) [46]. Since endothelial cells play an important role in the pathogenesis of diabetes, Goralczyk et al. examined the effects of 830-nm laser on human umbilical vein endothelial cells (HUVEC). Laser irradiation significantly increased the IL-6 production and reduced the TNF-α production while enhancing the cell proliferation under hyperglycemic condition [47]. Another study shows that irradiation of diabetic wounded fibroblast cells at 830 nm with 5 J/cm2 has a positive effect on wound healing in vitro by reducing proinflammatory cytokines (IL-1β and TNF-α) and stimulating ROS and NO [48]. Diabetic human fibroblast cell line irradiated with power density of 5 J/cm2 with 636 nm of diode laser resulted in increased wound closure, proliferation and decrease in apoptosis, and reduction in proinflammatory cytokines IL-1β, 6, and TNF-α; overall this directs the cells into the cell survival pathway [49].

4.3 VEGF and SDF-1α In diabetic rat model experiment showed healing of the wound because of collagen fibers in diabetic wounded skin increased by 904-nm laser but not by 850-nm LED and both laser and LED groups increased the blood vessels. Vascular endothelial growth factor (VEGF) was higher and cyclooxygenase (COX2) was lower in the LED group. Whereas mitochondrial fusion was higher in laser group and mitochondrial fusion was lower in LED and suggest that 904-nm laser treatment increased the wound healing [50]. PBM

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of 890-nm laser significantly increased the skin injury repair process to more rapidly reach the proliferation phase of the wound healing in type one diabetes mellitus rats by activating basic fibroblast growth factor (bFGF) and stromal-cell-derived factor-1α (SDF-1α) [51]. Regulation of the levels of TGF-β and VEGF and decreased activation of MMPs was also noted after PBM [43, 52, 53]. Rat models treated with PBM also showed an increase in collagen production and accelerated healing [54, 55].

4.4 TGF-β Transforming growth factor-beta (TGF-β) is a central player in wound healing modulating multiple cell types in a spatiotemporal manner [31]. A prior study noted the ability of PBM treatments to improve wound healing that correlated with increased TGF-β1 expression [56]. This study was based on significant similarities observed in wounds treated with recombinant TGF-β1 and PBM therapy, including elaboration of ECM (collagen and fibronectin), myofibroblast transformation, angiogenesis, and modulation of the inflammatory responses among many others. Dang et al. observed that PBM treatments at a wavelength of 800 nm enhanced skin structure and expression of new collagen via activation of the TGF-β/Smad signaling pathway [57]. Keskiner et al. examined PBM treatments at 1064 nm on wound healing in the palatal area following free gingival graft harvesting in patients recruited to a doubleblind, randomized controlled clinical study [58]. Their results noted significant increased expression of TGF-β1, PDGF-BB, and IL-8 levels in subjects treated with PBM at day 7 compared to untreated controls. On day 12, TGF-β1 remained significantly higher while no differences in PDGF-BB and IL-8 levels were observed. Keshri et al. irradiated dermal wounds in hydrocortisone-induced immunosuppressed rats with an 810-nm diode laser and they noted a decrease in proinflammatory markers (NF-κB and TNF-α) and an increase in the wound contraction marker (α-SMA), ECM deposition, and increased protein expression of fibroblast growth factor receptor-1 (FGFR-1), FN and TGF-β2 [59].Tang et al. observed that NIR diode laser (810 nm) irradiation of oral keratinocytes and fibroblasts induced the expression of human β-defensin 2 (HBD-2) in fibroblasts [60]. They reported that the expression of HBD-2 was mediated via the TGF-β pathway stimulated by PBM treatments that have potent antimicrobial effects in wound healing. Ruh et al. noted that PBM treatments at 660 nm elevated TGF-β1 gene expression, and VEFG expression, and decreased TNF gene expression in diabetic patients with pressure ulcers and patients also shown an improvement in granulation tissue size [61]. Assis et al. also found that PBM (808 nm) combined with aerobic and aquatic exercise increased TGF-β and IL-10 expression and had a positive effect on the degenerative process related to osteoarthritis in the articular cartilage in rats [62]. Fekrazad et al. evaluated TGF-β expression in wounded diabetic rats following PBM at 660 and 808 nm, alone or in combination [63]. Interestingly, they observed decreased TGF-β1 levels in late stage healing suggesting

Photobiomodulation therapy in diabetic wound healing

the common fibrotic sequelae could be potentially avoided. Overall this literature indicates that the ability of PBM treatments to promote healing is mediated by the activation of TGF-β signaling pathway that provides a multifaceted therapeutic biological responses.

4.5 PI3K/AKT signaling via mTOR and GSK-3β The PI3K/AKT pathway plays a central role in tissue homeostasis, wound healing, and regeneration, while dysregulation has been noted during fibrosis and malignant transformation [64–67]. The PI3K/AKT/mTOR pathway is critical for cell migration and its reduced function prevents epithelial mesenchymal transition (EMT), cell proliferation, and wound healing. Stimulated AKT/mTOR promotes cellular growth, migration, angiogenesis, and collagen synthesis. Activated PI3K/AKT signaling is related to several activities that are linked to the positive influence of PBM, including proliferation, migration, angiogenesis and cell survival, and wound healing. Another AKT signaling substrate, the GSK-3β pathway, regulates various biological activities for cellular metabolism, migration, apoptosis, spreading, and inflammation. This pathway also plays a critical role in the development of diseases like diabetes and Alzheimer [68]. Inhibition of GSK-3β via its phosphorylation at position Ser9 by AKT is essential for cutaneous wound healing [69]. GSK-3β substrates, such as β-catenin and cyclin D1, are key proteins for promoting cell proliferation and survival. Perturbations of epidermal growth factor receptor (EGF) and extracellular-signal regulated kinases (ERK) modulating PI3K/AKT signaling have been observed in diabetes. This results in increased cellular apoptosis and decreased proliferation, leading to delayed wound healing [70]. Diabetic wounds have been noted to express varying levels of total and phosphorylated proteins in the AKT/mTOR and AKT/GSK-3β signaling pathways. Reduced expression of β-catenin and cyclin D1 is noted to be a critical mechanism of compromised wound healing [71, 72]. Huang et al. observed that PBM therapy with 633 nm induced the nuclear redistribution and transcriptional activation of nuclear estrogen receptors (ERs) in a ligand-independent manner through activation of PI3K/Akt pathway [73]. In another study, they observed PBM treatments promoted pancreatic-β cell replication and cellcycle progression through activation of Akt1/GSK-3β isoform-specific signaling axis [74]. Zhang et al. noted that PBM treatments with 632.8 nm promotes cell proliferation through PI3K/Akt activation in kidney fibroblast cell line (COS-7) [75]. Lu et al. demonstrated that PBM treatments with 632.8 nm regulated Src/PI3K/Vav1/Rac1 signaling pathway enhancing macrophage phagocytosis [76]. Shingyochi et al. examined the effects of PBM with a carbon dioxide (CO2) laser on fibroblast proliferation and migration in wound healing [77]. They observed increased fibroblast cell proliferation and migration, and an eventual wound size reduction induced via activated AKT, ERK, and JNK

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signaling pathways. Overall these observations suggest that Akt and PI3K signaling pathways are activated by PBM therapy in a cell-type-dependent manner and could be used therapeutically to direct response.

5. Implications for PBM therapy in diabetes wounds and associated fibrosis and cancers Oral conditions like dry mouth, glossitis, bacterial and fungal infections, gingival abscess, periodontitis, tooth loss, oral potentially malignant and malignant disorders can occur in diabetes [78, 79]. Diabetes is often diagnosed with comorbidities such as inflammatory lesions, organ fibrosis, or malignancies [80]. Incessant tissue injury in diabetes exacerbates changes in the preexisting inflammatory microenvironment that can lead to nonhealing of ulcers, fibrosis, and possible malignant change [81, 82]. A study by Dikshit et al. suggests that there may be an association between oral potentially malignant lesions and diabetes in women [83]. However, the exact mechanism for this association remains to be delineated. Additionally, higher risk for fibrosis and carcinoma of colon, liver, bladder, and kidneys has been noted in diabetes implicating Insulin and Insulin-dependent growth factor axis [80, 84–92]. The molecular mechanism of wound healing, fibrosis, and carcinoma progress in a continuum and are both based on the interplay of the cellular, intracellular, and extracellular matrix (ECM) components. Fibrosis, an altered wound-healing process, presents with peculiar molecular changes driven predominantly by TGF-β and other cytokines [81, 93]. TGFβ has an elaborate role in differentiation of fibroblast to myofibroblasts that have central roles in wound contraction and collagen overproduction. Overexpression of MMPs, with reduced activity of cell adhesion molecule and other growth factors, further leads to the accumulation of ECM and delayed healing. In diabetic individuals, these biomolecules have been studied in plasma and urine as potential biomarkers for fibrosis of liver and kidneys [84]. Furthermore, PBM therapy has been recently approved for routine treatment of oral mucositis in postoncotherapy [94]. It seems prudent to examine utility of PBM for treatments for premalignant and malignant lesions of the oral cavity associated with diabetes mellitus. In summary, wound healing is complex biological process involving several different players, including biochemical, cellular, and biophysical cues. While this process is compromised in diabetic wounds, lasers or light-emitting diodes (LEDs) have been shown to provide clinical benefits. Attention to the clinical protocol should be based on underlying pathology (metabolic, inflammation, or neurological) as well as optimized delivery regimens, including tissue surface or distance-to-target measurements. Further investigations into the cellular and molecular mechanisms of PBM therapy will improve the rationale and clinical applications of this innovative treatment.

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Therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts Jaideep Banerjee, Sandeep Dhall

Osiris Therapeutics (now part of Smith & Nephew), Columbia, MD, United States

1. History of placental tissue in wound repair The placenta is a provisional organ and becomes salvage material after delivery. Research however has identified regenerative properties of the placenta that may be beneficial for tissue injury repair. For decades, clinicians and researchers have worked on the application of the placenta for therapeutic purposes, in the form of extracts and cell or tissue transplants. A fair amount of data now exists on the therapeutic benefits of placental and umbilical tissue for wounds, specifically diabetic ulcers. This review aims at discussing the properties of human placenta that may prove beneficial for different aspects of chronic wound repair. Postpartum placenta has three major components—extracellular matrix, growth factors, and cells such as trophoblast cells, fibroblasts, epithelial cells, stem cells, and endothelial cells of vessels. The fetal part includes the chorion, amnion, and umbilical cord. The amnion and the chorion can be easily separated at the intermediate layer. The amnion is 100 μm thick and is composed of a single-layered epithelium and the amniotic mesenchyme. Chorionic membrane is composed of fibroblasts and trophoblast cells that is connected to the maternal decidua. Although both the amnion and chorion can be used interchangeably, the chorion is more conforming and softer to handle. The umbilical cord is much thicker and is covered by the amniotic epithelium and contains two arteries and one vein that are immersed in Wharton’s jelly (which contains a large amount of fibroblast cells and has an intercellular substance rich in hyaluronic acid). The cellular composition of the placenta and growth factors that can drive signaling, along with the extracellular matrix that provides a platform for host cells to migrate, has made it attractive for clinicians and researchers. The rich source of stem cells in placenta makes it particularly attractive for regenerative therapy. The placenta has been reported to contain a population of broadly multipotent stem cells that have a mesodermal phenotype and demonstrate a broad differentiation potential that is not limited to mesenchymal lineages, but extends also to hepatocytes, vascular endothelial, pancreatic and Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00016-2

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neuronal differentiation. Mesenchymal stem cells (MSCs) are also very good sensors of a tissue microenvironment and can react through paracrine mechanisms by packaging growth factors, microRNAs, and cytokines into exosomes and release them into the host tissue to trigger signaling and tissue regeneration. Amniotic and chorionic MSCs express low levels of HLA-ABC and no HLA-DR, making them immune-evasive and thus appropriate for placental tissue to be used as allografts without the risk of rejection. Amniotic membrane has been used in clinical practice to a higher extent in comparison to the other components of the placental complex. The first reported use of the amniotic membrane was performed in 1910 by J. Davis for the closing of skin defects; later, it was applied as the soft material in different fields of surgery [1] and then for treating skin wounds [2, 3]. In fact, intact amniotic tissues were applied to skin burns and ulcers and then covered with dressings. Upon removal of the dressings 2 days later, the authors reported that the amnion had integrated with the patient’s tissues. They also reported lack of infection, a significant decrease in pain, and an increased rate of re-epithelialization of the traumatized skin surface in patients treated with amnion. Furthermore, application of amniotic and chorionic membranes has been used for the treatment of nonhealing diabetic, vascular and pressure ulcers, vaginal reconstruction surgery, enterocutaneous fistula, prevention of adhesions, orthopedic pathology, replacement of the pelvic peritoneum, urology as some of the examples [4–7] (Fig. 1).

2. Preservation techniques of placental membranes for commercial use Placental tissue allografts are regulated by the FDA as human cell, tissue, and cellular and tissue-based products as defined in 21 CFR Part 1271 and Section 361 of the Public Health Services Act. When minimally manipulated and in their native format, placental tissue allografts are categorized as a “skin substitute” and intended for homologous use as a wound cover to aid in the repair of acute and chronic wounds without restriction by etiology and location [8]. Commercially available placental membrane products can contain different parts of the placenta—the amnion, chorion, umbilical cord, amniotic fluid, or a variety of their combinations—resulting in different product compositions and properties [8]. In order to use placental tissue therapeutically, it is important to preserve them optimally to retain the native components of the placenta while ensuring safety and their long shelf-life. Various methods of sterilization and preservation of fresh placental membranes render them safe off-the-shelf commercial products. However, these processes can lead to varying degrees of damage to the tissue components and therefore affect the functional properties of the placental tissue. Placental membranes are available as devitalized (i.e., containing dead cells that were killed during tissue preservation) or as tissue with viable cells. Some of the techniques by which placental membranes have been preserved are: (a) Amnion and chorion from

Fig. 1 Amnion, chorion, and umbilical tissue allografts.

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placenta donated following scheduled Caesarean sections are processed to gently cleanse the layers removing blood components while protecting the delicate scaffold of the amniotic membrane, leaving an intact extracellular matrix. The graft is then dehydrated or lyophilized under controlled drying conditions. The result is a durable graft with natural barrier properties that offers clinicians a clear advantage in soft-tissue applications [9]. (b) Chemical preservation utilizing a high salt or glycerol concentration to sequester and reduce free water. (c) Cryopreservation using glycerol and storing at 80°C. This procedure decellularizes the amniotic membrane, but the tissue morphology and structure are maintained. Biochemically, cryopreservation retains high-molecular-weight hyaluronic acid species, including the heavy chain-hyaluronic acid complex that is known to exert antiinflammatory and antiscarring effects [10–16]. (d) Cryopreservation performed in a dimethyl sulfoxide containing cryoprotectant solution at a controlled cooling rate and then storing the tissue at 80°C can preserve the tissue integrity, growth factors of the extracellular matrix and retain the native cells of the placenta in a viable form [8]. (e) A recently published lyopreservation technique has been shown to retain the structural integrity of the extracellular matrix, preserve the growth factors, and retain cell viability while allowing the tissue to be stored at room temperature in the shelf for over a year. Tissue samples were soaked in trehalose and then lyopreserved in a lyophilizer set to optimized parameters. This process is different than traditional dehydration, as it allows for tissue storage at ambient temperatures while retaining viable cells present in the native placenta [17] (Fig. 2). While dehydration may be a simpler way to ensure a shelf-stable and safe tissue for use as an allograft, limitations exist. Cryopreservation retains the delicate native architecture of the amniotic membrane (AM)/umbilical cord (UC) extracellular matrix, quantity and activity of key biological proteins. If optimally cryopreserved, viability of cells can also be retained that can stay alive up to 4–8 days in vivo after application to a mouse chronic wound. These cells were found to not proliferate within fresh placenta and the cryopreserved placental membrane; however, isolated cells proliferated in culture. Traditional lyophilization or dehydration dramatically impacts the tissue architecture, suggesting alteration of critical components within. Dehydrated placental tissues lack the heavy-chain hyaluronic acid/pentraxin 3 complex, a key antiinflammatory and signaling molecule for tissue regeneration that is present in fresh AM/UC. Terminal sterilization by gamma and electron beam irradiation has also been shown to damage the basement membrane, elastin, and collagen fibers that subsequently affects the quality of the graft’s structure and integrity. In another study, only collagen IV and fibronectin were detected in the basement membrane of dehydrated amnion, whereas cryopreserved amnion was found to retain detectable levels of collagens IV to VII, fibronectin, and laminins indicating that ECM proteins are better protected via cryopreservation [8, 14, 18–22].

Therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts

Fig. 2 Preservation methods for placental allografts.

3. Benefits of placental allografts in chronic wound repair The placental membrane has several properties that makes it especially suited to support wound healing. The amniotic tissue has been marketed as amnion and chorion separately or as being composed of both the amnion and chorion layers together. However, chorion includes the chorionic trophoblast layer, which contains high levels of MMPs and proinflammatory cytokines in the placenta at term. Also chorionic membrane containing the trophoblast layer elicits an immunogenic response, while a cleaned chorionic membrane did not [23]. Therefore when using chorion as a tissue allograft for wound repair, it may be beneficial to remove the trophoblast layer [23, 24]. The most important way by which placental membranes can facilitate wound repair is by providing a biological barrier. The extracellular matrix of the placental membrane provides a matrix for migration and proliferation of host cells to fill in the tissue deficit. The scaffold is biocompatible and biodegrades over time. There have been no reported findings of adverse events such as being toxic, injurious, carcinogenic, or eliciting an immunologic response in the recipient tissue. It also allows cell adhesion and stimulates host cell migration. Additionally, placental membranes contain a significant number of cytokines and essential growth factors, which stimulate cell signaling. Application of the placenta on a wound has demonstrated its ability to reduce inflammation, scarring, pain, and reduced

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biofilm occurrence. These properties are particularly beneficial for wounds in patients with comorbidities such as diabetes, where the body’s own regeneration potential is compromised. Chronic diabetic wounds are trapped in a persistent inflammatory state with elevated levels of proinflammatory cytokines and proteases together with impaired expression of growth factors. Increased numbers of PMNs and MoMΦs in nonhealing diabetic wounds have been demonstrated, and similarly an increased ratio of proinflammatory compared with antiinflammatory MoMΦs in diabetic skin. This imbalance of proinflammatory and antiinflammatory MoMΦs results in increased production of proinflammatory cytokines, preventing tissue repair [25–28]. There are several reports of the placental membrane being able to reduce inflammation. It can suppress the expression of the potent proinflammatory cytokines, IL-1α, IL-1β, and TNF-α, and upregulates PGE2 and IL-10. Natural inhibitors of MMPs have also been found in the AM. Highmolecular-weight hyaluronic acid also exists in large quantities in the placental membranes and acts as a ligand for CD44 that is expressed on inflammatory cells and plays an important role in adhesion of inflammatory cells [29–32]. Placental membrane can also promote neovascularization and reduce scarring. They have been found to release angiogenic factors such as VEGF, increase capillary density, and promote endothelial cell migration and proliferation [33, 34]. Placental membranes also downregulates TGF-β and its receptor expression by fibroblasts, and in doing so, reduce the risk of fibrosis. Therefore, the placental membrane can modulate the healing of a wound by promoting tissue reconstruction rather than promoting scar tissue formation [35, 36]. Finally, chronic nonhealing wounds, including diabetic wounds, are commonly colonized by pathogens and the role of infection in diabetic wounds is being increasingly recognized as a potential barrier to healing. Amniotic membrane has antimicrobial properties and has been tested against clinical isolates of microbes commonly found in chronic wounds such as S. aureus, P. aeruginosa, E. faecium, K. pneumoniae, A. baumannii, and E. aerogenes. Antimicrobial peptides such as human-defensins such as HBD2 and HBD3 are secreted by placental membranes and directly contribute to its antimicrobial activity [37–39].

4. Experimental evidence of wound repair by placental membrane in animal models There are a few experimental animal models investigating the effects of applying placental membrane allografts on wounds. In an in vivo mouse model, a dehydrated amnion-chorion graft was tested in a skin flap model. A 5  5 mm square of the graft was surgically placed subcutaneously in 4-month-old wild-type mice. Negative controls were intact skin and sham-operated sites (surgical incision but no implant). At 3, 7, 14, and 28 days, the implant and overlying skin

Therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts

was harvested for FACS analysis. Results demonstrated that mesenchymal progenitor cells were being attracted to the site of implantation and were associated with soluble factors present in the tissue graft [9]. In another study, a dehydrated amnion-chorion graft was subcutaneously implanted under elevated skin. Three horizontal 6-mm incisions were created on the shaved dorsum of anesthetized mice. A subcutaneous pocket was bluntly dissected in the fascial plane underlying the panniculus carnosus and either a 5 mm  5 mm square of the graft or a 5 mm  5 mm square of control acellular fetal bovine dermal matrix of nonplacental origin was inserted. The third pocket did not receive an implant and acted as the sham surgical control. Wound tissue was harvested and processed for histology and flow cytometric analysis. Results demonstrated that the implanted graft recruited significantly more progenitor cells compared with controls and displayed in vivo stromal-derived factor 1 (SDF-1) expression with incorporation of CD34+ progenitor cells within the matrix. The authors concluded that the placental tissue graft effectively recruited circulating progenitor cells, likely because of SDF-1 expression. The recruited cells expressed markers of “stemness” and localized to sites of neovascularization, providing a partial mechanism for the efficacy of amniotic membrane in the treatment of chronic wounds [40]. A diabetic wound model was used to investigate the efficacy of a lyopreserved amniotic membrane in wound closure. A 7-mm excisional wounds in diabetic (db/db) mice was treated weekly with an amniotic membrane. Saline gel was used as a control. Wound appearance, size, wound tissue granulation, neovascularization, inflammation, redox state in wounds were assessed. Results demonstrated that diabetic wounds treated with the lyopreserved amniotic membrane closed faster (4 days faster on average) than control wounds. The faster closure correlated with a decrease in the expression of proinflammatory factors and oxidative stress, and an increase in angiogenesis and dermal thickness [41]. Placental tissues that were processed to retain cell viability were tested for viability of the cells when placed in a chronic wound environment. The tissue graft was stained using VivoTrack 680 Imaging Agent before applying on a chronic wound in a diabetic (db/db) mouse model. Cells were found to remain viable after 4 and 8 days in vivo after application to the wound [8]. The efficacy of wound closure between a viable lyopreserved and a viable cryopreserved amniotic membrane was compared in a 6-month old diabetic db/db mice chronic wound model. The mice were treated once intraperitoneally with a catalase inhibitor and the 7-mm wounds were created on the mouse. Immediately post injury, wounds were treated topically with an inhibitor of glutathione peroxidase. A second dose was administered 6–12 h postrecovery. Within 20 days, wounds became chronic. These chronic wounds were treated weekly with topical applications of either normgel, a lyopreserved amniotic membrane, and a cryopreserved amniotic membrane. Both the amniotic

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membranes resulted in wound closure in a diabetic mouse chronic wound model, while the control wound did not close demonstrating the equivalency between the two grafts [17]. Chorion immunogenicity was tested in vivo in a C567BL/6 mice subcutaneous pocket implantation model. Two 0.5-cm skin excisions were created on the dorsum of the animal. One-cm2 trophoblast-free chorionic membrane was implanted into the pockets. Subcutaneous pockets not receiving any tissue material served as the sham control. Trophoblast-free chorionic membrane was found to be nonimmunogenic, whereas chorionic membrane still containing the trophoblast layer elicited an immunogenic response [23]. An in vivo rabbit abdominal surgical adhesion model was used to evaluate the antifibrotic properties of lyopreserved chorionic membrane in New Zealand White rabbits. The rabbit abdomen was subjected to a 10-cm midline laparotomy. The surface of the cecum placed adjacent to the sidewall was abraded with sterile gauze until capillary hemorrhage was observed. The surface of the cecum facing the bowel was also abraded in a similar manner. Next, a 9-cm2 region of the peritoneum and abdominal transverse muscle was removed from the right lateral abdominal wall. The rabbit had two surgical injury sites, onto one of which a 25-cm2 piece of lyopreserved chorionic membrane was applied and sutured in place to cover the abdominal wall defect. The second surgical site had no barrier graft applied. The muscle wall was then sutured closed, followed by closure of the skin. Lyopreserved chorionic membrane was found to prevent formation of postsurgical adhesions in the same model [23]. Finally, the same rabbit abdominal surgical adhesion model was used to investigate the antiadhesion property of a cryopreserved umbilical tissue with viable cells. The cecum was abraded on two opposing sides, and the umbilical tissue was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Data were analyzed up to 67 days postsurgery and no adhesions were detectable on the umbilical tissue treated side and histological scores for adhesion, inflammation, and fibrosis were lower on the treated side as compared to the control side (Fig. 3).

5. Clinical evidence for efficacy of placental membranes in diabetic foot ulcer patients The first reported clinical use of placental membrane grafts was at Johns Hopkins over a century ago in 1910 as an aid for dermal wound healing [1]. In recent years, both clinical and research applications of these tissues have increased. These allografts have translated well to regenerative medicine applications by improving wound site integration due to their low immunogenicity, positive wound-healing characteristics, and minimal tissue inflammation and have allowed them to be applied to any tissue injury from head to

Therapeutic benefits of treating chronic diabetic wounds with placental membrane allografts

Fig. 3 Experimental evidence form treating chronic wounds with placental membrane allografts.

toe. However, most of the clinical evidence lies in the application in diabetic foot ulcers. Some of the major completed clinical trials on the efficacy of placental allografts in diabetic foot ulcers have been listed here. 1. Prospective, randomized, single-center clinical trial. Twenty-five patients with a diabetic foot ulcer of at least 4-week duration without infection having adequate arterial perfusion were treated with a dehydrated amnion-chorion allograft with standard of care or standard care alone. Wound size reduction in the first group was reported to be 97.1% on average, which was significantly higher than the control group after 4 weeks [42]. 2. Prospective, randomized, nonblinded, single-center clinical trial. Forty patients with noninfected ulcers of 4 weeks’ duration were included for the study. During the 12-week study period, 92.5% ulcers completely healed. Mean time to complete healing was 4.1  2.9 when applied biweekly versus 2.4  1.8 weeks when applied weekly. Complete healing occurred in 50% versus 90% by 4 weeks in the biweekly and weekly groups, respectively [43]. 3. Prospective, randomized, controlled, multicenter clinical trial. Sixty subjects were randomized into three groups (20 per group). Each group received a dehydrated amnion-chorion allograft or a bilayered living cell graft or standard wound care with collagen-alginate dressing. The proportion of patients in the placental membrane group achieving complete wound closure within 4 and 6 weeks was 85% and 95%, significantly higher than the other groups. Median time to healing was 13 days, which was also significantly faster [44]. The proportion of wounds achieving complete closure within the 12-week study period were 97%, which was also significantly higher than the other groups. Mean time to heal within 12 weeks was 23.6 days [45]. 4. Prospective, randomized, multicenter trial. Eighty patients treated with a dehydrated amnion-chorion allograft with standard of care or standard of care alone were investigated for achieving wound closure in nonhealing DFUs. At 12 weeks, 85% of the

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5.

6.

7.

8.

allograft-treated DFUs healed, compared with 33% treated with SOC alone. Mean time to heal within 12 weeks was 37 days, which was significantly faster than control [45]. Prospective, randomized, controlled multicenter clinical trial. Ninety-eight patients with a lower-extremity ulcer of at least 4 weeks’ duration were treated with a dehydrated human amnion/chorion membrane allograft or standard of care alone. Allograft-treated group had a significantly higher percentage of study ulcers completely healed in 12 weeks (81% vs 55%) and a significantly improved time to healing with the use of allograft [46] There is controversy, however, whether this study was able to contribute Level 1 evidence, based on study design, execution, and analysis [47]. Prospective, randomized, controlled, blinded, multicenter clinical trial. Ninety-seven patients with diabetic foot ulcer were treated with a cryopreserved amniotic membrane with viable cells along with standard of care alone. The proportion of patients who achieved complete wound closure was significantly higher in patients who received the amniotic allograft (62% vs 21%) and the median time to healing was 42 days [48]. This study was independently reported to be of a very low risk of bias [49]. In a follow-up open label extension phase of the study, 26 patients in the standard wound care arm whose DFUs did not close in the blinded phase chose to receive weekly applications of the cryopreserved amniotic membrane. 65.4% of the patients closed their wounds in a median of 34 days [50]. Prospective, randomized, controlled, single-blind trial. Sixty-two patients with diabetic foot ulcers were treated with a viable cryopreserved placental membrane or a human fibroblast-derived dermal substitute. No significant difference was found between the two treatments. However, preliminary findings showed that the amniotic allograft may have better outcomes for wounds 5 cm2. 81.3% of wounds in the amniotic allograft group reached complete closure as compared to 37.5% in the comparative group [51]. Prospective, multicenter, open-label, single-arm clinical trial. Twenty-seven patients with a complex DFU extending through the dermis with evidence of exposed muscle, tendon, fascia, bone and/or joint capsule were treated with a cryopreserved amniotic membrane with viable cells. For patients completing the protocol, the primary endpoint, 100% wound granulation by week 16, was met by 96.3% of patients in a mean of 6.8 weeks. Complete wound closure occurred in 59.3% (mean: 9.1 weeks) [52].

References [1] Davis II JS. Skin grafting at the Johns Hopkins Hospital. Ann Surg 1909;50(3):542–9. [2] Stern M. The grafting of preserved amniotic membranes to burned and ulcerated surfaces, substituting skin grafts. JAMA 1913;60(13):973–4. [3] Sabella N. Use of fetal membranes in skin grafting. Med Rec 1913;83:478–80.

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[4] Silini AR, et al. The long path of human placenta, and its derivatives, in regenerative medicine. Front Bioeng Biotechnol 2015;3:162. [5] Riboh JC, et al. Human amniotic membrane-derived products in sports medicine: basic science, early results, and potential clinical applications. Am J Sports Med 2016;44(9):2425–34. [6] Heckmann N, Auran R, Mirzayan R. Application of amniotic tissue in orthopedic surgery. Am J Orthop (Belle Mead NJ) 2016;45(7):E421–5. [7] Oottamasathien S, et al. Amniotic therapeutic biomaterials in urology: current and future applications. Transl Androl Urol 2017;6(5):943–50. [8] Johnson A, et al. Understanding the impact of preservation methods on the integrity and functionality of placental allografts. Ann Plast Surg 2017;79(2):203–13. [9] Koob TJ, et al. Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing. Int Wound J 2013;10(5):493–500. [10] Adds PJ, Hunt CJ, Dart JK. Amniotic membrane grafts, “fresh” or frozen? A clinical and in vitro comparison. Br J Ophthalmol 2001;85(8):905–7. [11] Shortt AJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials 2009;30(6):1056–65. [12] Hermans MH. Preservation methods of allografts and their (lack of ) influence on clinical results in partial thickness burns. Burns 2011;37(5):873–81. [13] Schulze U, et al. Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing. Histochem Cell Biol 2012;138(2):243–50. [14] Cooke M, et al. Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/chorion tissue. J Wound Care 2014;23(10):465–74. 476. [15] Thomasen H, et al. The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane. Curr Eye Res 2011;36(3):247–55. [16] Tan KE, Cooke M, Mandrycky C, Mahabole M, He H, O’Connell J, McDevitt TC, Tseng SCG. Structural and biological comparison of cryopreserved and fresh amniotic membrane tissues. J Biomater Tissue Eng 2014;4:379–88. [17] Dhall S, et al. Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage. PLoS One 2018;13(10):e0204060. [18] Thomasen H, et al. Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications. Graefes Arch Clin Exp Ophthalmol 2009;247(12):1691–700. [19] Rodriguez-Ares MT, et al. Effects of lyophilization on human amniotic membrane. Acta Ophthalmol 2009;87(4):396–403. [20] von Versen-Hoeynck F, et al. Sterilization and preservation influence the biophysical properties of human amnion grafts. Biologicals 2008;36(4):248–55. [21] DiDomenico LA, et al. Aseptically processed placental membrane improves healing of diabetic foot ulcerations: prospective, randomized clinical trial. Plast Reconstr Surg Glob Open 2016;4(10):e1095. [22] Paolin A, et al. Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and gamma-irradiated human amniotic membranes. Cell Tissue Bank 2016;17(3):399–406. [23] Jacob V, Johnson N, Lerch A, Jones B, Dhall S, Sathyamoorthy M, Danilkovitch A. Structural and functional equivalency between lyopreserved and cryopreserved chorions with viable cells. Adv Wound Care 2019; (Ahead of print). [24] Graham CH, et al. Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation. Biol Reprod 1992;46(4):561–72. [25] Baltzis D, Eleftheriadou I, Veves A. Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights. Adv Ther 2014;31(8):817–36. [26] Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med 2014;6(265):265sr6. [27] Mirza RE, et al. Sustained inflammasome activity in macrophages impairs wound healing in type 2 diabetic humans and mice. Diabetes 2014;63(3):1103–14. [28] Salazar JJ, Ennis WJ, Koh TJ. Diabetes medications: impact on inflammation and wound healing. J Diabetes Complications 2016;30(4):746–52. [29] Solomon A, et al. Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. Br J Ophthalmol 2001;85(4):444–9.

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[30] Hao Y, et al. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 2000;19(3):348–52. [31] Duan-Arnold Y, et al. Retention of endogenous viable cells enhances the anti-inflammatory activity of cryopreserved amnion. Adv Wound Care (New Rochelle) 2015;4(9):523–33. [32] Witherel CE, et al. Immunomodulatory effects of human cryopreserved viable amniotic membrane in a pro-inflammatory environment in vitro. Cell Mol Bioeng 2017;10(5):451–62. [33] Duan-Arnold Y, et al. Angiogenic potential of cryopreserved amniotic membrane is enhanced through retention of all tissue components in their native state. Adv Wound Care (New Rochelle) 2015; 4(9):513–22. [34] Zheng Y, et al. Amniotic epithelial cells accelerate diabetic wound healing by modulating inflammation and promoting neovascularization. Stem Cells Int 2018;2018:1082076. [35] Tseng SC, Li DQ, Ma X. Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J Cell Physiol 1999;179(3):325–35. [36] Lee SB, et al. Suppression of TGF-beta signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane. Curr Eye Res 2000;20(4):325–34. [37] Mao Y, Hoffman T, Johnson A, Duan-Arnold Y, Danilkovitch A, Kohn J. Human cryopreserved viable amniotic membrane inhibits the growth of bacteria associated with chronic wounds. J Diabetic Foot Complications 2016;8(2):23–30. [38] Mao Y, et al. Antimicrobial peptides secreted from human cryopreserved viable amniotic membrane contribute to its antibacterial activity. Sci Rep 2017;7(1):13722. [39] Mao Y, et al. The effect of cryopreserved human placental tissues on biofilm formation of woundassociated pathogens. J Funct Biomater 2018;9(1):3. [40] Maan ZN, et al. Cell recruitment by amnion chorion grafts promotes neovascularization. J Surg Res 2015;193(2):953–62. [41] Dhall S, et al. A viable lyopreserved amniotic membrane modulates diabetic wound microenvironment and accelerates wound closure. Adv Wound Care (New Rochelle) 2019;8(8):355–67. [42] Zelen CM, et al. A prospective randomised comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int Wound J 2013;10(5):502–7. [43] Zelen CM, Serena TE, Snyder RJ. A prospective, randomised comparative study of weekly versus biweekly application of dehydrated human amnion/chorion membrane allograft in the management of diabetic foot ulcers. Int Wound J 2014;11(2):122–8. [44] Zelen CM, et al. A prospective, randomised, controlled, multi-centre comparative effectiveness study of healing using dehydrated human amnion/chorion membrane allograft, bioengineered skin substitute or standard of care for treatment of chronic lower extremity diabetic ulcers. Int Wound J 2015; 12(6):724–32. [45] Zelen CM, et al. Treatment of chronic diabetic lower extremity ulcers with advanced therapies: a prospective, randomised, controlled, multi-centre comparative study examining clinical efficacy and cost. Int Wound J 2016;13(2):272–82. [46] Tettelbach W, et al. A confirmatory study on the efficacy of dehydrated human amnion/chorion membrane dHACM allograft in the management of diabetic foot ulcers: a prospective, multicentre, randomised, controlled study of 110 patients from 14 wound clinics. Int Wound J 2019;16(1):19–29. [47] Lindblad AS, Weaver LK. Tettelbach et al fail to meet the bar for Level 1 evidence in their report of dHACM allograft in the management of diabetic foot ulcers. Int Wound J 2019;16(2):582–3. [48] Lavery LA, et al. The efficacy and safety of Grafix((R)) for the treatment of chronic diabetic foot ulcers: results of a multi-centre, controlled, randomised, blinded, clinical trial. Int Wound J 2014;11 (5):554–60. [49] NICE. Diabetic foot problems: prevention and management. NICE clinical guideline 19, National Institute for Health and Care Excellence; 2016.

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[50] Lavery L, et al. Open-label extension phase of a chronic diabetic foot ulcer multicenter, controlled, randomized clinical trial using cryopreserved placental membrane. Wounds 2018;30(9):283–9. [51] Ananian CE, et al. A multicenter, randomized, single-blind trial comparing the efficacy of viable cryopreserved placental membrane to human fibroblast-derived dermal substitute for the treatment of chronic diabetic foot ulcers. Wound Repair Regen 2018;26(3):274–83. [52] Frykberg RG, et al. A prospective, multicentre, open-label, single-arm clinical trial for treatment of chronic complex diabetic foot wounds with exposed tendon and/or bone: positive clinical outcomes of viable cryopreserved human placental membrane. Int Wound J 2017;14(3):569–77.

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CHAPTER 17

Debridement and negative pressure wound therapy Said A. Atway, Nicholas V. DiMassa

Department of Orthopedics, The Ohio State University Wexner Medical Center, Columbus, OH, United States

1. Introduction Diabetes is a global epidemic with staggering effects on patients’ overall health as well as a major cost burden to healthcare systems. A 2017 report by the CDC found that 30.3 million Americans, or 9.4% of the overall United States population had diabetes. Roughly 25% of seniors over the age of 65 are living with diabetes in the United States, and 1.5 million Americans are diagnosed as new diabetics each year [1]. A 2017 article exploring the economic costs of diabetes in the United States reported a $327 billion dollar expense to the economy, with $237 billion in direct medical costs and $90 billion in reduced productivity [2]. The most common diabetic complication affecting the lower extremity is a diabetic foot ulcer, with approximately 12% of the diabetic population having a history of foot ulceration. The morbidity associated with the development of a diabetic foot ulcer is astounding. The New England Journal of Medicine in 2017 reported that half of all diabetic foot ulcers will become infected at some point, and approximately 20% of moderate-to-severe diabetic foot infections will lead to an amputation. Most sobering of all, the 5-year survival rate following any form of nontraumatic amputation in the diabetic population is around 50% [3]. Whether a diabetic foot ulcer or any other type of diabetic wound is present, close monitoring by a specialist with intimate woundcare knowledge and debridement experience is of utmost importance. With the need for appropriate wound care knowledge comes an industry with a multitude of options for wound healing, and it is important to understand the evidence-based treatment options. This chapter will focus on the role of wound debridement and negative pressure wound therapy (NPWT) on healing diabetic wounds, with specific applications for the diabetic foot highlighted.

2. Debridement Ubi pus, ibi evacua is a Latin adage, first appearing in the American Medical Journal in 1868, which can be roughly translated as “Where there is pus, there evacuate it.” The original definition and indication for debridement has evolved from incision and drainage of Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00017-4

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abscesses to the present clinical definition, which also includes the removal of contaminated or nonviable tissue that impedes the growth of normal tissue. Wound debridement involves the physical removal of devitalized tissue including skin, soft tissue, tendon, and bone. Devitalized tissue within a wound is a major inhibitory factor of the woundhealing process; it serves as a nidus for infection and microbial proliferation, and thereby retards wound healing [4]. Debridement of a wound is critical to promote healing by removing infection, biofilm, and senescent cells. By removing these inhibitory factors, endogenous healing can occur more rapidly while the effectiveness of other therapeutic modalities is also augmented. When a burden of bacteria and senescent cells are present within a chronic wound, the wound will remain in a chronic inflammatory phase. Debridement of the wound bed is capable of stimulating a host response to morph the physiology of a chronic wound to an acute wound that progresses through the natural stages of healing [5]. This brings into question timing of debridement when an abscess or overt infection causing sepsis may be more obvious; stimulatory debridement and/or removal of biofilms may be less obvious.

2.1 Medical management and vascular status Understanding the need for debridement and when to perform a debridement is key to wound healing, but an understanding of when debridement is contraindicated is equally important. Optimizing the patient’s overall medical management is a key to the overall wound-healing process. Aside from diabetes, numerous other comorbidities can impede wound healing, including chronic renal failure, cancer, immune-suppressing diseases, malnutrition, and nicotine use—but perhaps most detrimental is the presence of peripheral vascular disease. Meticulous surgical debridement cannot substitute for lack of tissue perfusion. Perfusion is essential to supplying the wound with oxygen, nutrients, and cells [6]. Hypoxia occurs in wounds with tissue perfusions less than 20 mmHG and can result in cell death, tissue necrosis, and bacterial proliferation. Involvement of a vascular surgeon to confirm or improve local blood flow when possible is imperative in diabetic patients with lower-extremity wounds. In the presence of a severe, active infection, surgical debridement needs to occur urgently. The timing of operative debridement in chronic wounds without active infection; however, should ideally take place after revascularization when the difference between nonviable and viable tissue becomes more evident [7]. Clinical acumen is just as important as surgical finesse and should be employed to delineate between viable and nonviable tissue and leave as much remaining viable tissue as possible—as this becomes the foundation for eventual healing. Specific to the diabetic foot and where an amputation may be the ultimate end-point debridement without compromising limb function and/or eventual wound closure or amputation flaps are important to consider but should not compromise appropriate evacuation of wound necrosis.

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2.2 Literature review Debridement of wounds has been a staple of the surgeon’s arsenal since the 18th century. While nearly every surgeon is keenly aware of the vital role of debridement in wound management, the evidence-based rationale has been slow to follow. There are few highquality independently published studies that evaluate the role of debridement in wound healing. Steed et al. conducted a randomized, prospective, double-blind multicenter study in 1996 that enrolled 118 patients with diabetic foot ulcers and looked at the role of rhPDGF in healing. Independent of their findings involving rhPDGF, they did also conclude that a lower percentage of wound healing occurred in the centers where debridement was performed less frequently [8]. Piaggesi et al. continued work on the topic in 1998 and randomized patients with diabetic foot ulcers into groups undergoing surgical debridement and excision of the ulcer vs conservative local wound care. While this study was low powered, it did conclude that the diabetic foot ulcers undergoing surgical debridement and excision were much less likely to recur, while those undergoing in-office debridement and local wound care were much more likely to have infectious complications while also having a lower healing rate at 6 months [9]. Saap and Falanga employed a unique “debridement performance index” in which they evaluated wounds pre- and postdebridement, scoring them on the need for debridement as well as on the adequacy of debridement performed. Their index did have predictive value for ultimate wound closure and can be easily adapted for clinical practice [10]. A study from the University of Wales College of Medicine in 2005 by Williams et al. prospectively evaluated the use of sharp debridement on chronic venous leg ulcers in the outpatient setting. They separated patients into a study group with wounds containing slough, nonviable tissue, and no granulation tissue vs a control group that also had minimal granulation tissue, but no slough or nonviable tissue. They performed outpatient sharp debridement in the study group. They analyzed mean surface area of the ulcers and found a 6-cm2 reduction in the study group vs 1-cm reduction in control group at 4 weeks. Similarly, at 20 weeks, they noted a 7.4-cm2 reduction in the control group vs an increase of 1.3 cm2 in the control group. The overall healing rate was 16% for the study group ulcers undergoing debridement vs 4.3% of the control ulcers. They concluded that outpatient debridement of chronic venous leg wounds was safe, well tolerated, and effective [11] (Figs. 1 and 2).

2.3 Types of debridement – Surgical or sharp excisional debridement is the most rapid and effective form of debridement. It may be accomplished as a bedside procedure if a patient is capable of tolerating or may also be performed in the operating room under general anesthesia. Local anesthesia may be used to facilitate bedside debridement. In cases where there is concern for bleeding or exposure of neurovascular structures, debridement should be

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Fig. 1 Diabetic heel ulceration with necrotic eschar cap and underlying necrosis to the level of bone.

performed in the operating room. Sterile surgical instrumentation is preferred over the use of disposable suture removal kits. The workhorse instruments of surgical debridement include scalpel blades for sharp removal of tissue and wound edge preparation, sharp curettes for the removal of nonviable and necrotic tissue clumped onto an underlying granular bed, various-sized rongeurs for grasping and removing nonviable soft tissue as well as nibbling away and culturing bone. Sagittal power saws can also be used to resect prominent portions of bone, especially cortical bone. Care must be taken when performing surgical debridement, as overaggressive debridement may lead to unnecessary removal of viable tissue. Color endpoints can be helpful in ensuring all necrotic tissue (gray/green/brown/black) is removed and guiding the surgeon in the differentiation of healthy tissues, including muscle (red), tendon and bone (white), and subcutaneous fat (yellow). – Mechanical debridement is one of the oldest and still most commonly used methods. Mechanical debridement is best characterized as the use of wet-to-dry gauze dressings

Debridement and negative pressure wound therapy

Fig. 2 Surgical excisional debridement of previous heel ulceration with the use of sharp instrumentation including rongeur. Debridement is performed until all nonviable tissue, including bone, has been excised.

that are applied to the wound bed, left to dry, and upon removal lift and nonselectively remove necrotic and infected tissue. It should be noted that mechanical debridement may be painful to the patient. The level of evidence is rated III to IV for this type of debridement. The cost for this form of treatment is minimal. Generally wet-to-dry dressings can be employed between interval debridements or definitive reconstruction in the acute care setting. Longer-term care facilities have more advanced wound care treatments available. Of note, hydrotherapy also falls under the category of mechanical debridement and involves soaking the wound in a whirlpool bath. This technique has been shown to be effective; however, care should be taken in patients with circulatory compromise. Also, there is innate potential for wound maceration as well as risk of contamination and infection. – Autolytic debridement tends to be the best-tolerated and most gentle form of wound debridement. Autolytic debridement relies on the body’s own enzymatic processes to

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prepare wound bed surfaces by removal of nonvitalized tissue and slough. The mechanism of action involves either occlusion or semiocclusion of wound fluids within the wound bed to hydrate, soften, and eventually liquefy necrotic tissue and eschar. Autolytic debridement tends to be facilitated by the use of special dressings, including hydrocolloids, hydrogels, and transparent films. Autolytic debridement is a slow process and is generally only suitable for stable, shallow wounds without signs of infection. Frequent wound monitoring is necessary with this treatment. – Enzymatic debridement with the use of a collagenase ointment has been shown to be effective in the debridement of chronic dermal ulcers as well as severe burns. The mechanism of action for this type of debridement relies on an enzyme that anchors to necrotic tissue within the wound bed and then hydrolyzes peptide bonds to break down the necrotic tissue. This is a form of selective debridement with little damage to healthy surrounding tissue. Similar to autolytic debridement, it is a slow process and may be more well suited for stable wounds or patients that are not surgical candidates. This type of product may work well in combination with advanced dressings changed frequently. Some patients do experience an inflammatory reaction with this form of treatment and expense of therapy may also be an issue. – Maggot therapy is an alternative option for debridement that is better supported in the international literature. Maggot therapy involves using the larvae of Phaenicia sericata on chronically infected wounds in patient’s that may be poor surgical candidates. The maggots function by releasing an enzyme, which functions to decrease bacterial burden and dissipate necrotic tissue into a nutrient-rich food source. Maggot therapy is contraindicated near exposed blood vessels, the eyes, the upper gastrointestinal tract, and the upper respiratory tract (Figs. 3 and 4).

2.4 Endpoint and biopsies Debridement of diabetic wounds must be thorough and complete until all nonviable tissue has been completely excised and only healthy, viable-appearing tissue remains in the wound bed. This is relatively straightforward when the wound is limited to soft-tissue involvement; however, in cases with bone involvement including osteomyelitis, the debridement endpoint becomes less clear. Often an appropriate debridement for bad diabetic foot infections will require amputation and determining the level of amputation needs to take into account and balance the priorities of leaving a functional foot while still completing a thorough debridement of all devitalized and infected tissue. Any residual osteomyelitis can carry the risk of reamputation as well as a longer course of postoperative antibiotics. At our institution, a standard approach to debridement of diabetic foot infections with bone involvement includes a meticulous debridement of the soft tissue with care taken not to excise excess viable tissue. When amputation is warranted, the level of resection is determined by clinical appearance of

Debridement and negative pressure wound therapy

Fig. 3 Dorsal diabetic foot ulceration with wound bed that has a mix of granular tissue 70%, fibrotic tissue 20%, and even some exposed tendon 10%.

the bone first, but also with consideration given to preoperative advanced imaging, vascular supply, and always keeping the biomechanics of the foot in mind. After amputation has been performed, a bone biopsy will be obtained from the resected infected portion of bone and sent for both pathologic examination as well as microbiologic culture and sensitivity. Next, the surgical team will irrigate the wound thoroughly with normal saline, don sterile gloves and re-establish a sterile field, and then obtain a new “clean” sample of bone from the proximal margin of the amputation with the use of sterile instruments. This sample will also be sent to microbiology for culture and sensitivity. We published a retrospective study of our data using this debridement technique and found that 57.1% of our patients undergoing either a transmetatarsal or partial ray resection procedure had residual osteomyelitis. This proved significant as 81.8% of cases with residual osteomyelitis had a poor outcome compared to only 25% of cases with a negative bone margin having a poor outcome [12]. Any remaining infected tissue, especially bone, after debridement warrants an

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Fig. 4 Healed diabetic foot amputation after appropriate debridement and skin grafting.

extended course of antibiotics consistent with the guidelines of the Infectious Diseases Society of America’s recommendations [13].

2.5 Applications for the diabetic foot In regard to the diabetic foot, special considerations have to be given to debridement. As the distal extremity, the diabetic foot is most prone to the devastating neurological and vascular complications of diabetes. Any deformities or gait abnormalities will result in callus formation and subcutaneous hemorrhage that quickly progresses to ulceration. The combination of neuropathy and vascular disease both permits and hastens this pathway. After a diabetic foot ulcer has formed, it is then extremely prone to secondary infection because of the compromised diabetic immune system. While all of the aforementioned debridement principles still pertain to diabetic foot ulcerations and infection, the biomechanics of the foot often dictate whether a limb may be salvageable or if a proximal amputation is warranted. Levels of amputations include toe, partial ray resections, transmetatarsal, Lisfranc (tarsometatarsal), Chopart (tarsal), and Syme (ankle).

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Partial resection of the calcaneus is also common. Functionality must be taken into account to attempt to maintain a limb that has relatively even distribution of weightbearing forces, while also assuring riddance of osteomyelitis, and capable vascular healing potential of the local tissue. Level of amputation is often a clinical judgment based on these factors; however, patient preference and factors also play a large role.

3. Negative-pressure wound therapy There may be no other recent product that has affected the treatment of wounds more than that of vacuum-assisted closure (VAC). The utilization of saline moist-to-dry gauze as the standard wound dressing without a definitive alternative has opened the wound dressing market to a myriad of options. Since its inception, Negative-Pressure Wound Therapy (NPWT) has allowed patients with wound infections and difficult-to-heal, large, and deep wounds with a viable option for closure as well as provided an option for limb salvage in patients with diabetic foot wounds. The main benefit of the continuous negative-pressure therapy is its ability to improve blood flow to the wound while drawing fluid from it. This improves the environment for wound healing either by secondary intention healing or in preparation for a secondary procedure. The wound VAC has benefits to wound healing in its ability to stimulate granular tissue by removing pressure to a wound, promoting secondary intention healing, and decreasing bacterial burden of wounds while controlling fluid levels at the wound site—making this a truly dynamic dressing. NPWT has proven to be especially effective in preventing limb loss among patients with diabetic foot wounds and infections [14]. For a device that has become so ingrained and integral to wound care in both the surgical and home health setting, its origin can be traced back fairly recently. In 1989, Mark Chariker and Katherine Jeter developed a technique utilizing standard surgical dressings and wall suction to create a “vacuum” that was noted to aid in wound healing. However, it wasn’t fully developed until 1997 when Dr. Michael Morykwas and Dr. Lewis Argenta studied the use of suction to polyurethane foam in wounds—bringing this technology to the wound care forefront. The usage of the product grew immensely with the development of a company called KCI where the “V.A.C” was developed and marketed. To be fair, NPWT had been available and utilized for years, but this is thought to be the point at which usage increased with physician acceptance and success in healing wounds truly appreciated. The polyurethane foam was critical to providing the missing link and ultimately leading to the increased success of the wound VAC systems we know today. In 1997, the wound VAC product we would recognize today with the polyurethane interface ushered in the new wave of NPWT usage. Then finally in 2001, given the increased use and demand of NPWT, the Centers for Medicare and Medicaid approved the technique for reimbursement further propelling usage of wound VAC systems [15].

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3.1 Background Negative-pressure wound therapy (NPWT), also called vacuum-assisted wound closure, refers to wound dressing systems that continuously or intermittently apply subatmospheric pressure to the surface of a wound. Therefore, the NPWT dressing has been described as a “dynamic dressing” in its ability to respond to changes at the wound and wound VAC interface. NPWT has become a popular treatment modality for the management of many acute and chronic wounds. NPWT provides a dynamic wound environment that allows for a clean, fluid-controlled wound bed. This is achieved by applying suction at the wound surface incorporated with various interfaces at the wound surface, including gauze but more commonly a polyurethane foam. The polyurethane interface remains the most commonly used and studied interface given its popularity with the KCI system as well as benefits that will be discussed later, although studies do exist that support gauze usage with results similar to that of foam dressings [16]. Indications for wound VAC usage have grown from the treatment of chronic wounds and now are being commonly employed in trauma surgery and plastic surgery, as well as in healing of diabetic foot wounds. Given the relatively recent advent of the product and the continually expanding uses, the literature has not quite yet caught up to support all of the clinical uses. Clinical evidence of its superiority over conventional wound-dressing techniques for all wound types has not been proven or supported very well. The available randomized trials have failed to provide enough evidence for usage in a single wound type, therefore making clinical applications to standardize usage challenging. This has not slowed usage but may limit understanding of when the product may be best suited for application and extent of utilization, but success and acceptance is generally accepted in the wound care community. The evidence supporting the use of NPWT in treatment of chronic nonhealing wounds exists primarily in the form of nonrandomized, controlled trials, prospective, and retrospective large and small case series, single-center studies and single-case studies. While these are beneficial in providing information and a guide for NPWT utilization, there are limits to understanding the ability to apply direction with regards to application. Despite its lack of evidence-based support, NPWT has become a staple in the wound physician and surgeon’s armament. This may speak more to the lack of research in the field of wound care as opposed to the support for utilization of NPWT. Therefore, it has even been proposed that NPWT be thought of as a noninvasive adjunctive therapy for diabetic foot ulcers to standard of care that includes debridement, local wound care, infection control, and off-loading [17].

3.2 Mechanism of action NPWT has evolved from the basic principles of wound healing and is thought to help augment several phases of healing, which further supports the description as an adjunctive

Debridement and negative pressure wound therapy

therapy. The initial phase of wound healing involves both hemostasis and inflammation. The inflammatory phase lasts approximately 4–6 days and is followed by the proliferative phase lasting for around 21 days. This phase is dominated by fibroblast activity and is characterized by the formation of granulation tissue, neoangiogenesis, and re-epithelialization. The final stage in wound healing is tissue remodeling and involves the renewal of collagen fibers and contraction of the wound through the activity of myofibroblasts. These steps are important to understand to allow for a true understanding of how NPWT affects wound healing. Local factors at the wound bed need to be controlled to allow for appropriate healing. The presence of infection, edema, exudates, and ischemia has been proven to inhibit the healing process. However, as discussed earlier once the wound has been debrided to an appropriate healthy wound bed using NPWT on the wound is believed to reduce these negative effects—specifically by reducing infection and controlling bioburden, increasing vascularity and angiogenesis, cell proliferation as well as controlling exudate build up from the wound NPWT can positively influence the wound. In turn, these effects provide an environment promoting granulation tissue and encouraging the wound edges to come together in the remodeling phase. This theory has been developed and studied in animal models mainly demonstrated in swine and rabbit models [15]. Orgill et al. described four primary effects of NPWT on wound healing [18]: • Macrodeformation—drawing the wound edges together leading to contraction, which was further supported by Lavery et al. in diabetic foot ulcers [19]. This can simply be thought of as wound shrinkage. • Stabilization of the wound environment—ensuring it is protected from outside microorganisms in a warm and moist environment. • Reduced edema—with removal of soft tissue exudates. • Microdeformation—leading to cellular proliferation on the wound surface. Multiple secondary effects were noted to result from this, including cell proliferation, increased blood flow and angiogenesis, reduction of inflammation, and ultimately granulations tissue formation with the possibility of a decrease in bacterial load (bioburden) at the wound bed. Furthermore, two main theories prevail regarding the mechanism of action of NPWT used in conjunction specifically with a reticulated open-cell foam. The first is based on the theory that tissue strain caused by NPWT has a stimulatory effect on cellular proliferation. This theory is supported by the fact that tissue has been shown to undergo a 5%– 20% strain when subjected to NPWT. This level of strain is hypothesized to proactively cause cell division and angiogenesis in a process analogous to Ilizarovian distraction [20]. This theory is supported by studies evaluating histologic sections of wound under the strain of the reticulated sponge, which supported the theory of cell division, growth factors and angiogenesis.

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The second theory is focused on the effects of NPWT on the mechanical evacuation of excessive interstitial fluid and edema. By actively removing fluid at the wound bed, it is believed that the local microcirculation is improved and secondary necrosis is reduced. This prevents the need for demarcation at the wound edges. This hypothesis is supported by studies that have shown reduced need for debridement at the time of “second look” for wounds treated with NPWT [20]. It is also supported by in vivo studies using a porcine model, which have demonstrated acceleration in capillary formation and increase in luminal area in wounds treated with NPWT [21]. The benefits of NPWT have further been studied in relation to the effects of subatmospheric pressure at a cellular level. Specifically, macrostrain and microstrain have been studied as two working theories to the benefit of negative pressure on the wound. Macrostrain is the theory of physically drawing wound edges together and is appreciated almost immediately. Macrostrain has been described as the physical change in size and shape of the wound at the cellular level with application of the reticulated open-cell foam. The mechanism in which macrostrain benefits the wound is at 30 mmHg when the reticulated open-cell foam (ROCF) has been shown to collapse—this closes in on the wound, which results in deformation of the size and shape of the wound as it begins to heal. The theory of Macrostrain in providing an improved wound environment remains similar to that of bone distraction and tissue expansion and is thought to be possible only with ROCF as discussed earlier comparing this to Ilizarovian distraction. A study by Zannis et al. compared ROCF under pressure to that of gauze and found decreased surface area of up to 50% with the ROCF in comparison to that of gauze [22]. Ultimately, this creates smaller amount of granulation tissue to need to develop to fill the wound. This provides a clinical benefit to both secondary intention healing as well as wound preparation for skin grafting. This theory is thought to support NPWT as a closure device. Microstrain creates microdeformation at a cellular level, which promotes healing by limiting inflammatory build up to the wound and changes at the cellular level of the wound. Working off the proven theory that cells under strain tend to divide, microstrain is thought to provide this same type of reaction. Cells under microstrain induce 5%–20% strain to cells, which is the level shown to promote proliferation in vitro [23]. These forces are further believed to provide an environment for increased microvasculature formation and angiogenesis. This theory supports NPWT as a stimulatory device. Decreased edema and promotion of perfusion supplies blood, nutrients and oxygen, and growth factors required for healing are all working theories that have been studied in several studies looking at negative pressure wound therapy. All of these theories are thought to lead to granulation tissue in the wound bed in preparation for final closure. This is the end goal of the adjunct treatment with the wound VAC—to fill the wound void and provide a granular base of tissue for skin grafting or end closure by secondary intention healing.

Debridement and negative pressure wound therapy

3.3 Description/function Although NPWT has been shown to provide a benefit to wound healing, it is a more complex wound device, which does require specific training and understanding before applying. This may be seen as a contraindication to use this product. However as usage has increased over the years utilization of the product has grown, and more clinicians have become proficient with its use. The advent of the negative-pressure wound therapy systems years ago lent itself to multiple options as the success of this device grew and demand increased. The most well-known device is that of the KCI system, which is still the most utilized negative pressure system providing the ROCF interface. This negativepressure therapy system is thought to be the first to utilize the polyurethane interface, and we will review its specific components and applications. Open-pore polyurethane sponges are utilized against the wound. These are cut and shaped to the wound to fit the wound specifically being able to adapt to the multitude of ulcer shapes and sizes. The compressibility of the material allows for even and equal tension on various types of wounds. The ability to cut and shape the foam allows the wound VAC to function for various wound shapes and depths; therefore allowing application to various wound types and allowing the VAC ability to further function as a dynamic dressing. The pore size is typically between 400–600 μm, which allows maximal tissue growth. However, smaller pore size is utilized as well in certain circumstances and is thought to limit tissue ingrowth and reduce pain with dressing changes. The smaller size is reserved for tunneling wounds, tendons, and bone, allowing granulation tissue over these structures. This material is thought to be more hydrophilic allowing for increased moisture to these structures, as it is more dense and may slow fluid penetration through this material [24]. A seal is necessary to go over the foam and the wound to create an effective vacuum effect while allowing a suction tube to be attached through a hole made within the seal over the foam. This seal is an important step that can sometimes provide a challenge to the clinician applying the device specifically when working with complex surfaces involving the foot. However, this is an important step in providing a continuous, consistent, and uniform negative-pressure system to the wound. It is key to confirm appropriate application of the seal without leaks when applying the wound VAC. The suction tube is connected to the source that generates the negative pressure to a set amount (typically 125 mmHg) and regimen (continuous vs cyclical). Studies utilizing porcine models have shown 125-mmHg to be the ideal setting for development of granulation tissue when compared to 25 mmHg and up to 500 mmHg [25]. The pressure is regulated by the lumen within the tubing and therapy unit monitoring system, which provides a feedback loop to the VAC device. This constant monitoring is controlled by the therapy unit. This continuous communication and control of the pressure translated to the wound is important in providing the appropriate environment for healing as proven by the benefits of the negative-pressure system at the cellular level as discussed

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previously. Intermittent suction has been shown to be more beneficial as it is thought that cells can adapt to the continuous suction. The theory is the times between suction will allow moments between cycles of rest and cell division [25]. A canister is attached to the source to collect the exudate and can be exchanged as needed based on fluid amounts being drawn from the wound (Figs. 5–7).

3.4 Applications for the diabetic foot As discussed earlier there are applications for NPWT that have been poorly studied, although anecdotal indications have been well accepted in the medical community as

Fig. 5 Diabetic foot wound with exposed bone and mix of granular and fibrotic tissue.

Fig. 6 After thorough debridement of wound, with application of skin graft and overlying wound VAC.

Debridement and negative pressure wound therapy

Fig. 7 Healed diabetic foot wound.

the wound VAC is utilized in various circumstances. For the purpose of this chapter we will focus on negative-pressure therapy, specifically, the wound VAC and ROCF, which have been associated with increased granulation tissue formation as it applies to the diabetic foot [26]. The benefits to the diabetic foot are especially important once the wound has been debrided to allow for a healthy wound bed and the healing process truly begins as the NPWT device will not compensate for a poorly prepared wound bed. However, given the limited ability to debride the foot due to sheer size and functional limitations, the wound VAC may provide an option for closure without requiring amputation either by primary intention healing or secondary intention or even secondary procedures. This idea coincides with studies by Morykwas et al. utilizing a porcine model comparing wound VAC to standard moist to dry dressing, which allowed for a 60% increase in granulation tissue formation [27]. This was further supported in a clinical study specific to diabetic foot ulcers by Blume et al., which found the rate in which granulation tissue is formed around a wound is greater in that of groups utilizing a wound VAC, with faster wound closure compared to standard care [28]. As noted earlier, standard of care remains

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a moistened wound environment; the wound VAC may provide for a more dynamic option for providing moisture allowing more rapid healing. NPWT allows for an increasing percentage of granulation tissue formation, which often leads to re-epithelialization or skin grafting when necessary. An additional clinical benefit of NPWT is by reducing the time in which the wound fills in with granulation tissue; leading to a reduced risk of reinfection. Moreover, quicker time to healing is a known predictive value to diabetic foot ulcer’s ability to heal [29]. Armstrong et al. established NPWT’s effect on amputation rates in studying 162 diabetic patients after operative wounds and minor amputations of diabetic feet and application of NPWT. They compared NPWT patients to that of standard moist-to-dry dressing and found that the median time to reach 76%–100% granulation tissue in the NPWT group was 42 days compared to 84 days for the standard care group. This is important when performing debridement or amputations when primary closure is not feasible [26]. While the ability of NPWT to establish and promote a healthy wound environment composed of granulation tissue is important, Armstrong et al. further defined the superiority of NPWT to standard of care by noting patients who received NPWT treatment were a quarter less likely to need reamputation. The result of this multicenter randomized control trial, which indicated that NPWT could effectively reduce the occurrence of amputation, is especially encouraging for limb preservationists and patients with challenging diabetic foot wounds.

4. Unique clinical usage and future applications While most of the discussion has focused on traditional application of NPWT, including its use in clinical trials as well as animal models, there is a new horizon becomingly increasingly utilized—continuous-instillation NPWT. In this technique, a continuous-instillation port is combined with that of the suction port at the wound interface to enhance healing. Various fluid options have been studied, including saline, insulin, dilute betadine, doxycycline, and polyhexanide biguinide, without strong evidence to support usage regularly [30, 31]. This new method has been studied in porcine models and applied in the clinic setting, with various solutions showing mixed results. This, however, provides a further complicating step in applying the device and can bring about its own unique set of complications and risks with malfunctioning or suction failure. There is promise in this technique providing a form of infection control and further supports the wound vac as a dynamic device that can respond to the many challenges of healing a wound even beyond the operating room suite. Negative-pressure therapy has also been used in conjunction with split-thickness skin graft procedures in place of a bolster dressing to immobilize the skin graft [32, 33]. The benefits are thought to be from the VAC’s ability to drain fluid, promote contact, and enhance vascularization of the graft [32, 33].

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5. Safety and conclusion While negative-pressure wound therapy has gained interest and success rapidly, the product is not without risk. Given the active nature of this product and dynamic dressing varying from that of standard of care, it is important to think critically of the potential side effects and safety associated with negative-pressure therapy. The main adverse events for NPWT include edema, infection/sepsis, foam retention, pain, and bleeding. Therefore when considering NPWT on wounds that are especially prone to these risks, caution should be exercised and basic wound care principles applied. Previous studies have shown NPWT to neither increase nor decrease the incidence of treatment related side effects compared with standard dressing. However, in February 2011 the US Food and Drug Administration (FDA) issued a safety communication update warning of serious complications associated with negative-pressure wound systems. Specifically, there was mention of bleeding and infection associated with usage of NPWT. In 2011, FDA cited 12 deaths and 174 injuries since 2007 related to NPWT [34]. Ren and Li reported sepsis burns treated with NPWT that lead to acute hemorrhage, but this was thought to be related to large exposed vessels and bleeding. With education and increased experience of practitioners applying this product, these risks can be avoided by recognizing exposed vasculature and poor or unprepared wound beds. It is important to understand not only when wound VAC application is appropriate but also to understand appropriate application. As negative-pressure therapy has become more commonplace with various home health agencies and clinicians becoming more comfortable with using the product, the rate of complications will hopefully continue to decrease. As we come to understand the effects of negative-pressure wound therapy on diabetic foot wounds in better detail, we will potentially have a better understanding of expectations for healing and a standardization for application and usage.

References [1] Centers for Disease Control and Prevention. National Diabetes Statistics report, 2017. Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and Human Services; 2017. [2] American Diabetes Association. Economic costs of diabetes in the U.S. in 2017. Diabetes Care 2018;41 (5):917–28. [3] Armstrong DG, Boulton AJM, Bus SA. Diabetic foot ulcers and their recurrence. N Engl J Med 2017;376(24):2367–75. [4] Anghel EL, DeFazio MV, Barker JC, Janis JE, Attinger CE. Current concepts in debridement: science and strategies. Plast Reconstr Surg 2016;138(3 Suppl):82S–93S. [5] Golinko MS, Joffe R, Maggi J, Cox D, Chandrasekaran EB, Tomic-Canic RM, Brem H. Operative debridement of diabetic foot ulcers. J Am Coll Surg 2008;207(6):e1–6. [6] Faris I, Duncan H. Skin perfusion pressure in the prediction of healing in diabetic patients with ulcers or gangrene of the foot. J Vasc Surg 1985;2(4):536–40. [7] Cornell RS, Meyr AJ, Steinberg JS, Attinger CE. D
ebridement of the noninfected wound. J Vasc Surg 2010;52(3 Suppl)31S–6S. [8] Steed DL. Debridement. Am J Surg 2004;187(5A)71S–4S.

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[9] Piaggesi A, Schipani E, Campi F, Romanelli M, Baccetti F, Arvia C, Navalesi R. Conservative surgical approach versus non-surgical management for diabetic neuropathic foot ulcers: a randomized trial. Diabet Med 1998;15(5):412–7. [10] Saap LJ, Falanga V. Debridement performance index and its correlation with complete closure of diabetic foot ulcers. Wound Repair Regen 2002;10(6):354–9. [11] Williams D, Enoch S, Miller D, Harris K, Price P, Harding KG. Effect of sharp debridement using curette on recalcitrant nonhealing venous leg ulcers: a concurrently controlled, prospective cohort study. Wound Repair Regen 2005;13(2):131–7. [12] Atway S, Nerone VS, Springer KD, Woodruff DM. Rate of residual osteomyelitis after partial foot amputation in diabetic patients: a standardized method for evaluating bone margins with intraoperative culture. J Foot Ankle Surg 2012;51(6):749–52. [13] Lipsky BA, Berendt AR, Cornia PB, Pile JC, Peters EJ, Armstrong DG, Deery HG, Embil JM, Joseph WS, Karchmer AW, Pinzur MS, Senneville E, Infectious Diseases Society of America. 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis 2012;54(12):e132–73. [14] Xie X, McGregor M, Dendukuri N. The clinical effectiveness of negative pressure wound therapy: a systematic review. J Wound Care 2010;19(11):490–5 [Review]. [15] Argenta LC, Morykwas MJ. Vacuum-assisted closure: a new method for wound control and treatment: clinical experience. Ann Plast Surg 1997;38(6):563–76. [16] Campbell PE, Smith GS, Smith JM. Retrospective clinical evaluation of gauze-based negative pressure wound therapy. Int Wound J 2008;5(2):280–6. [17] Peinemann F, Sauerland S. Negative-pressure wound therapy: systematic review of randomized controlled trials. Dtsch Arztebl Int 2011;108(22):381–9. [18] Saxena V, Hwang CW, Huang S, Eichbaum Q, Ingber D, Orgill DP. Vacuum-assisted closure: microdeformations of wounds and cell proliferation. Plast Reconstr Surg 2004;114:1086–96. [19] Lavery LA, Boulton AJ, Niezgoda JA, Sheehan P. A comparison of diabetic foot ulcer outcomes using negative pressure wound therapy versus historical standard of care. Int Wound J 2007;4(2):103–13. [20] Webb LX, Pape HC. Current thought regarding the mechanism of action of negative pressure wound therapy with reticulated open cell foam. J Orthop Trauma 2008;22(10 Suppl):S135–7. [21] Norbury K, Kieswetter K. Vacuum-assisted closure therapy attenuates the inflammatory response in a porcine acute wound healing model. Wounds 2007;19(4):97–106. [22] Zannis J, Angobaldo J, Marks M, DeFranzo A, David L, Molnar J, Argenta L. Comparison of fasciotomy wound closures using traditional dressing changes and the vacuum-assisted closure device. Ann Plast Surg 2009;62(4):407–9. [23] McNulty A, Spranger I, Courage J, Green J, Wilkes R, Rycerz A. The consistent delivery of negative pressure to wounds using reticulated, open cell foam and regulated pressure feedback. Wounds 2010;22 (5):114–20. [24] Heit YI, Dastouri P, Helm DL, Pietramaggiori G, Younan G, Erba P, M€ unster S, Orgill DP, Scherer SS. Foam pore size is a critical interface parameter of suction-based wound healing devices. Plast Reconstr Surg 2012;129(3):589–97. [25] Morykwas MJ, Falter BJ, Perce DJ, Argenta LC. Effects of varying levels of sub atmospheric pressure on the rate of granulation tissue formation in experimental wounds in swine. Ann Plast Surg 2001;47:547–51. [26] Armstrong DG, Lavery LA, Diabetic Foot Study Consortium. Negative pressure wound therapy after partial diabetic foot amputation: a multicentre, randomized, controlled trial. Lancet 2005;366 (9498):1704–10. [27] Morykwas MJ, Argenta LC, Shelton-Brown EI, McGuirt W. Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation. Ann Plast Surg 1997;38 (6):553–62. [28] Blume PA, Walters J, Payne W, Ayala J, Lantis J. Comparison of negative pressure wound therapy using vacuum-assisted closure with advanced moist wound therapy in the treatment of diabetic foot ulcers: a multicenter randomized controlled trial. Diabetes Care 2008;31(4):631–6. [29] Margolis DJ, Allen-Taylor L, Hoffstad O, Berlin JA. Diabetic neuropathic foot ulcers, the association of wound size, wound duration and wound grade on healing. Diabetes Care 2002;25(10):1835–9.

Debridement and negative pressure wound therapy

[30] Kim PJ, Attinger CE, Olawoye O, Crisi BD, Gabriel A, Galiano RD, Gupta S, Lantis Ii JC, Lavery L, Lipsky BA. Negative pressure wound therapy with instillation: review of evidence and recommendations. Wounds 2015;27:S2–S19. [31] Kim PJ, Attinger CE, Steinberg JS, Evans KK, Lehner B, Willy C, Lavery L, Wolvos T, Orgill D, Ennis W. Negative-pressure wound therapy with instillation: international consensus guidelines. Plast Reconstr Surg 2013;132:1569–79. [32] Petkar KS, Dhanraj P, Kingsly PM, Sreekar H, Lakshmanarao A, Lamba S, Shetty R, Zachariah JR. A prospective randomized controlled trial comparing negative pressure dressing and conventional dressing methods on split-thickness skin grafts in burned patients. Burns 2011;37:925–9. [33] Azzopardi EA, Boyce DE, Dickson WA, Azzopardi E, Laing JH, Whitaker IS, Shorkrollahi K. Application of topical negative pressure (vacuum-assisted closure) to split-thickness skin grafts: a structured evidence-based review. Ann Plast Surg 2013;70:23–9. [34] FDA Safety Communication. UPDATE on serious complications associated with negative pressure wound therapy systems. FDA Safety Communication; 2011. http://www.fda.gov/MedicalDevices/ Safety/AlertsandNotices/ucm244211.htm.

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CHAPTER 18

Protease technology in wound repair Ira M. Hermana, Priya Niranjanb, Komel Groverb a

Program in Cell, Molecular and Developmental Biology, Center for Innovations in Wound Healing Research, Tufts University School of Medicine, Boston, MA, United States b Swiss American, Carrollton, TX, United States

The scientific and clinical view of proteins, proteases, and enzymes has evolved over the last century from a mechanism to digest large proteins into smaller polypeptides and amino acids, to the use of highly specific proteases, which cleave target sequences with high affinities that give rise to innumerable downstream signaling cascades that modulate untold biological pathways governing human development, adult life, or pathogenesis. Ranging from regulating cell cycle progression, cell growth, and migration to angiogenic activation or blood coagulation, endogenous proteolytic processing of key substrates gives rise to specific cues controlling such diverse cellular and tissue phenomena that range from cell communication, cell death-instructing pathways, inflammatory cascades and adaptive immune responses, protein and mRNA processing, and postinjury tissue remodeling during the repair and regeneration during wound healing [1]. Consequently, the use of enzymes as “protease-inspired” wound therapy not only degrades tissue byproducts, triggers autophagy, and cleans up necrotic tissue; but also through the specific signaling intermediates arising, protease-based treatments and therapies have been demonstrated as having explicit and significant potential to temper inflammation, activate angiogenesis, while promoting the migratory and proliferative responses to injury and tissue repair while stimulating healing [2–5]. Protease therapy as an enzymatic debrider has been an effective adjunctive treatment for wound healing for decades [6]. In this review, we summarize the current state of existing protease technologies for managing inflammation and reparative responses along with their potential for managing other pathogenic processes, e.g., wound healing, fibrosis, and tumor progression [7].

1. Diabetes, wound prevalence, and health economics Diabetic wounds often have extended healing times with many evolving into chronic, nonhealing ulcers. Table 1 summarizes the prevalence and health economics of diabetesinduced nonhealing wounds in the United States [8–19]. Approximately 25% to 34% of diabetic patients develop DFU during their lifetime [14, 20]; these patients have a >200% higher mortality rate than patients with diabetes and no DFU [21]. Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00018-6

© 2020 Elsevier Inc. All rights reserved.

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Table 1 Prevalence of diabetic foot ulcers and estimated healthcare costs. Patient health status

Percentage

Chronic wound [8, 9]

5.6% of adults in 2018

Diabetes mellitus (DM) Type 1 DM Type 2 DM Diabetic foot ulcers (DFUs) occurrences annually [12, 13] Nonhealing DFU within 1 year [15, 16] Amputation due to diabetic ulcer in lower extremity (cost per episode) [17]

Prevalence in United States

5% 95% 1%–15%

14,200,000 8,200,000 medicare 30,000,000 1,500,000 28,500,000 [10] 300,000–4,500,000

23%–40%

552,000–960,000 107,675 amputations/year [18]

Costs

$28.1 billion to $96.8 billion for medicare $237 billion direct costs $327 billion total costs (direct and indirect) [11] $9–13 billion in 2011 [14] $26,881–$28,031/ patient/year [14] Minor amputation: $13,580/episode Multiple minor amputations: $31,835 Major amputation: $73,813/episode

DM, diabetes mellitus; DFU, diabetic foot ulcer.

Failure to heal the DFU is associated with high risk of amputation and limb loss [22]. Diabetic patients with DFUs using Medicare or private insurance had a 95- or 250-fold higher lower-limb amputation rate (3.8% and 5%, respectively) than a matched set of diabetic patients without a DFU (0.04% and 0.02%, respectively) [14]. Regardless of insurance type, patients with DFU utilized approximately twofold higher medical resources than the matched set of diabetic patients without a DFU [14]. Furthermore, hospitalized diabetic patients with DFU stayed an average of 8 days longer than diabetic patients without ulceration in the United Kingdom [23]. The 5-year mortality rate of DFU patients is 42% [21, 24]. The 5-year survival of patients with a history of DFU (58%) is less than the 5-year survival rate of patients diagnosed with any cancer (67.1%) or many common cancers such as prostate (98%), breast (89.9%), and colorectal carcinoma (64.4%) [25]. Thus, treatments that promote the healing of DFUs would not only benefit the patients but also reduce the medical costs to patients and society.

2. Risk factors for primary and recurrent DFUs Risk factors for development of DFUs or other nonhealing wounds are (i) neuropathy that dulls or eliminates protective sensation, (ii) uneven or excessive pressure due to foot deformities, (iii) external trauma, prior ulcers or amputations, (iv) infection, and (v) venous or arterial insufficiency due to chronic ischemia [14, 20]. In addition, the

Protease technology in wound repair

following factors are more common in patients with DFU than patients without DFU: smoking, older age, longer duration of diabetes (mean difference of 5.8 years), lower body mass index (99%) collagenase from Vibrio alginolyticus + hyaluronic acid (2% w/w)

Highest activity at pH 7 to 9. Cleaves Y-Gly bonds (Y ¼ neutral AA) in -Pro-Y-Gly-Pro

Development

Enzymatic debridement of acute wounds (e.g., burns, pressure ulcers (PrUs), and surgical dehiscence wounds). Enzymatic Tx of fibrotic tissue. Adjunctive therapy w/ chemotherapy or biologic agents for improving penetration Enzymatic debridement of chronic DFUs, and venous leg ulcers (VLUs)

[38–47]

Enzymatic debridement of acute and chronic DFUs, PrUs, and burns

[50–52]

EMA approved for enzymatic debridement of burns

Debridement of PrUs, venous, diabetic, and posttraumatic ulcers, gum and dental wounds

[53–59]

Development

Biofilm reduction, cancer

[60, 61]

Not commercially available in United States

Enzymatic debridement

[62, 63]

[48, 49]

Plant derived protease therapy

Actinidin

Kiwi pulp and extract from Actinidia

Bromelain, NexoBrid

Bromelain is mixture of endopeptidases from stem or fruit of pineapple (Ananas comosus)

Ficin

Isolated from fig

Papain-urea

Isolated from papaya (Carica papaya)

Cysteine protease, belongs to papainlike peptidase C1 family Mixture of thiol endopeptidase, w/ cellulase, glucosidase, phosphatase, peroxidase Nonspecific sulfhydryl protease Cysteine protease

CCO, clostridial collagenase ointment; Col, collagenase; EMA, European Medicines Agency; FDA, Food and Drug Administration; PrUs, pressure ulcers; Tx, treatment; Vcol, collagenase isolated from Vibrio alginolyticus; w/, with.

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has been shown to reduce proliferation of keratinocytes, essential for epithelialization [65]. Thus, distinct protease therapies may affect cells involved in wound healing differently. Note that papain-urea preparation would be contraindicated for adjunctive therapy with many antibody-based biologics and with most growth factor therapies. Bromelain preparation is prepared from the fruit and/or the stem of the pineapple and contains various endopeptidase with other enzymes and is discussed further in Section 4. Both the bromelain preparation and the papain-urea preparations are not commercially available in the United States [6].

5. Protease therapy for DFUs Table 3 summarizes multiple clinical trials that investigated treatment of DFUs with currently available protease therapy. Use of either of the two bacterial collagenase ointment preparations, the collagenase from Clostridium histolyticum (Clostridial collagenase ointment (CCO), also known as collagenase Santyl ointment, CSO) or Vibrio alginolyticus (Vcol), for 4, 6, 8, or 12 weeks daily significantly reduced the target ulcer area (TUA) in comparison to the baseline TUA [48, 66, 68-70, 72, 73]. The enzymatic debridement also reduced the time to closure in most studies in comparison to standard of care [66, 68, 72], in some cases reaching statistical significance [48]. Lantis II and Gordon [72] had compared CCO ointment with or without surgical debridement; they concluded that CCO was more effective for debridement with many DFUs when combined with a weekly surgical debridement. Furthermore, Motley et al calculated that CCO treatment of DFUs in a randomized controlled trial (RCT) was as cost effective as standard of care (SOC) [67]. Taken together, collagenase-driven protease therapy provides an enzymatic debridement for DFUs that aids in restarting and promoting the wound-healing process in patients with diabetes while giving rise to bioactive wound-healing peptides that convert nonhealing wounds into those capable of closure [4, 5, 65, 74]. As an emerging protease therapy, a Kiwifruit extract (KW), which contains the protease actinidin, was investigated in a randomized clinical trial of 37 patients with neuropathic DFUs with no visible infection [51]. Wound closure occurred significantly sooner in the KW group (11.2days) vs the control (SOC) group (17.8days; P < .0001) and the reduction of the mean ulcer size of KW group was significantly greater than that of the control group (P < .0001). Interestingly, compared to baseline values in respective groups, collagen formation had significantly increased in the KW group (P ¼ .0001) at the end of study (21days), but had significantly declined in the control group (P ¼ .02). Furthermore, the inflammation score of the experimental group had significantly decreased (P ¼ .001) during the study, but the inflammation score of the control group had increased (P ¼ .07). These data suggest that the actinidin-containing pulp and extract treatments had promoted fundamental processes involved in DFU healing (collagen formation, inflammation resolution) and supports further research for use in chronic DFU and other wounds [51].

Table 3 Use of protease therapy for treatment of diabetic foot ulcers (DFUs).a Citation

Treatments

Multicenter, openlabel RCT, NCT01408277 [66]

Clostridial Collagenase ointment (CCO) + serial sharp debridement (SShD) vs PI-directed control (Cntl) + SShD, 6 wks of Tx, followed-up for 6 wks

Economic results, multicenter, openlabel RCT, NCT01408277 [67]

Phase 4, prospective randomized open label study, 14 sites, NCT # NR [68]

#/Group, sex

Age

Wounds

Outcome

55: 28, 27 ≧ 18 yrs 41 M; Mean: 14 F 57.9  12 yrs T1DM or T2DM

Neuropathic DFU but nonischemic. Mean size: 1.9  1.4 cm2

CCO + SShD or PI-chosen SOC + SShD 12 wk

55: 28, 27 ≧ 18 74.5% M Mean: 57.9 yrs 25.5% F T1DM or T2DM

Neuropathic DFU, same RCT as above

CCO daily vs silver product chosen by PI, daily; both groups allowed sharp debridement, as needed

Safety: 102 ITT: 51, 51 PP: 44, 42

CCO reduced target ulcer area (TUA) by end of Tx (EOT) (mean: 1.92 ! 0.56 cm2) P < .001; Cntl reduced TUA by EOT from mean: 1.77 ! 1.17 cm2, NS; time to closure CCO 9 wks, Ctrl 11 wks, NS: # AE similar Based on faster closure time in CCO group, estimated cost per DFU for Cntl Tx ($2376) was higher than that for CCO ($2099) CCO group: significant mean reduction in TUA 62% at 6 wks; silver group (40% reduction); 6-wk comparison P ¼ .071; EOS P ¼ .065 Closure EOT: CCO 31.1  7.7 d vs 37.1  7.7 d, NS

≧ 18 yrs Mean: 57.0 yrs Adequate arterial perfusion

DFU, ≧ 6 wks UWD (7.7 cm2) (P < .05)

Retrospective deidentified EHR [109]

CCO vs medicinal honey (MH)

517 PrUs (USWR 2007–13) CCO, 446 pts; vs MH 341 pts; F 48%

Mean: 66.2 yrs

517 wounds/group, majority at PrU stage III (56.1% vs 55.3%)

RCT [49]

Vcol (collagenase from Vibrio alginolyticus) + hyaluronic acid (HA) vs placebo control (Cntl) ointment; daily, 15 days

113: 58, 55 HA-Vcol: 41% M, 59% F Cntl: 46% M, 54% F

Mean HA-Vcol 66.5 yrs Cntl: 65.1 yrs, NS

Duration >6 mon Total lesion area (TUA) between 5 to 30 cm2

After matched via propensity score 1:3 (UWD, n ¼ 8862), CCO treated wounds were significantly more likely to improve (20% vs about 15%) CCO significantly greater percentage (P < .01) reached 100% granulation by the end of therapy (EOT) 30% vs MH wounds 19%; CCO significantly higher % closure (45%) vs MH at 31% HA-Vcol Tx significantly increased rate of full debridement at day 15, and at other time points (days 7, 21, 30). Total lesion area was reduced during Tx, NS between groups

CCO, clostridial collagenase ointment; CSA, collagenase clostridiopeptidase A; EHR, electronic health records; EOS, end of study; EOT, end of therapy; F, female; M, male; MH, medicinal honey; Mon, months; NXB, NexoBrid; NICU, neonatal intensive care unit; NPWT, negative pressure wound therapy; NR, not reported; NS, not significantly different; PrU, pressure ulcer; Pts, patients; SA, surface area; SgD, surgical debridement; Signif, significant; SSD, silver sulfadiazine; TBSA, total body surface area; TUA, total ulcer area; Tx, treatment; USWR, US wound registry; Vcol, collagenase from Vibrio alginolyticus; VLU, venous leg ulcer; wks, weeks; yrs, years.

Protease technology in wound repair

analysis of CCO-treated burn patients, CCO treatment of most patients with burns and eschar required more CCO treatments (e.g., 4 weeks) before closure than those without eschar [42]. Collagenase treatment (CCO) of children with partial-thickness burn wounds in prospective and retrospective trials indicated that CCO treatment was associated with a shorter hospital stay, fewer transfusions, fewer surgical excisions than the surgical debridement group [101, 102]. According to a survey of attending nurses during the clinical trial, 10 of 12 nurses preferred CCO due to lower frequency of dressing changes and less trauma to the patient [113]. These data support the use of CCO for treatment of burn wounds. Enzymatic debridement by bromelain and mixture of proteases from pineapple stems, NexoBrid (NXB), is approved by European Medicines Agency (EMA) for treatment of burns and is supported by results from RCT [103] and case series of facial burns, upperextremity burns, and genital burns [54-56, 104] (Table 4). Quality of scar was not significantly different between groups in the RCT [103] but was improved in the case series at one center [54]. Together, these data raise the possibility that a subgroup of patients with more superficial wounds treated with bromelain may end up with more pliable skin and less scars than those treated with surgical debridement [54], but further studies are warranted. However, one case series report of four patients with diabetes and thermal burns documented limitations to the use of the bromelain debridement therapy for debridement of their wounds [114] (Table 4). Novel methods using chitosan nanofibers to deliver bromelain to burn wound beds are in development; the bromelain-loaded nanofibers had induced more rapid healing in a rat model of burns [53]. As an emerging protease therapy, Kiwifruit pulp and extract (KW) containing actinidin, was compared to treatment with vaseline sterile gauze or silver sulfadiazine (SOC) on second-degree burns in a rat model with a 21-day duration (n ¼ 20/group). KW showed a significantly higher rate of complete healing, a high grade of neovascularization, and more rapid reduction in wound surface area (all P < .001) [50]. In summary, protease therapy has shown benefit for the treatment of burns and the presence of eschar may necessitate an additional two or more weeks of protease therapy [42]. Several protease therapies, including Kiwifruit with actinidin [52], bromelain [58], and CCO [45, 115], have shown benefit in the treatment of PrUs. Patients with PrU treated with CCO protease therapy experienced benefits in efficacy, required fewer follow-up visits, and also on average had lower economic costs in comparison to autolytic debridement with medicinal honey [106, 107]. Protease therapy appears useful for treatment of posttraumatic ulcers since all patients (9/9) with posttraumatic ulcers were considered responders to treatment with bromelain-containing therapy [58]. In regards to treatment of surgical dehiscence, the type of protease therapy used may also affect efficacy. Whereas the three patients with postsurgical necrotic flaps treated with bromelain-based protease therapy were nonresponders [58], CCO treatment of surgical dehiscence supported the healing process in

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adults (n ¼ 6) [44] and led to full closure in infants (n ¼ 6) [40] (Table 4). The use of NPWT with successful CCO treatment of surgical dehiscence [44] suggests that adjunctive therapies and specialized wound dressings may augment the efficacy of protease therapy and warrants at least retrospective analysis of databases of administrative claims, electronic health records, and/or patient registries, providing real-world evidence (RWE). All together (Table 4), these data support the use of protease therapy for acute wounds such as burns, surgical dehiscence [40, 44], and pressure ulcers [45, 50]. The data also support continued development of these protease therapies for treatment of acute and chronic wounds, including venous leg ulcers. Chronic wounds generally are considered stalled in the inflammatory stage because they have (i) excessive levels of MMPs, (ii) high levels of inflammatory cytokines IL-1, TNF-α, and IL-6, (iii) minimal levels of TIMPs, and (iv) low levels of growth factors [e.g., TGF-β and vascular endothelial growth factor (VEGF)]. The chronic wound bed contains many activated inflammatory cells (e.g., mϕinf), which secrete TNF-α and IL1-β, stimulate MMP production, and suppress TIMPs. These mediators impair the healing process via excess inflammation and degradation of growth factors, receptors, and ECM components. Restarting and maintaining the wound healing process can be a challenging task. Limited success of a single therapeutic protein with targeted specificity may be due to insufficient alteration of the wound milieu, a duplicity of multiple alternative pathways, both mechanisms, or degradation of the exogenously applied growth factors. An alternative strategy to revert to a more normal wound microenvironment is to modulate the activity of proteins such as proinflammatory cytokines and MMPs. Since chronic wounds display have a high ratio of MMP to TIMP, most of the MMP molecules are not inhibited and some TIMP molecules may be hydrolyzed [116]. Many chronic venous leg ulcers (VLUs) responded to bromelain-based therapy [58], Vcol [49], or CCO [109], with some VLUs reaching 100% granulation (Table 4) [109]. However, the effects of protease therapy for treatment of arterial insufficiency ulcers remain unclear since 4 of 4 cases of arterial insufficiency ulcers treated with bromelain-based therapy were nonresponders [58]. Retrospective analysis of healthcare databases such as administrative claims, electronic health records, and/or patient registries may provide further insights on the effects of protease therapy for treatment of chronic arterial insufficiency ulcers [58].

9.2 Effects of plant proteases on inflammation We expect that beyond debridement, the plant-based protease technology will also affect key players during the inflammatory response stage and help balance the cytokine network by releasing TIMPs, allowing for the proresolution cytokines to promote

Protease technology in wound repair

phagocytosis and efferocytosis, which clean up those fragments, thereby promoting the restarting of wound healing. These proteases balance the activity of wound-associated proteins, and diminish the rate of tissue destruction, edema, fever, inflammation, pain, itching, and hyperpermeability of the wound endothelium. The select proteases used together in a specified composition degrade inflammation-related proteins, and diminish the intensity of inflammation in skin or wounds, which can hasten the rate of resolution to inflammation and decrease scarring to the wound.

9.3 Angiogenesis and antiinflammation effects of proteases on wound repair and regeneration Recent studies indicate that several plant proteases either individually or in combination have a positive effect in balancing the proteins in the wound microenvironment by having an immunomodulatory effect, enabling the microenvironment to promote cell migration and angiogenesis. The proteases were introduced in wound macrophages at different concentrations and their effect was evaluated on LPS-induced cytokine expression. It was identified that the proteases differentially regulated the LPS-induced expression of inflammatory mediators, such as IL-1β, IL-10, and TNF-α, and proangiogenic VEGF. These factors are critical in contributing to the physical rebuilding process in wound healing (manuscript in preparation).

9.4 Effect on antimicrobial peptides in skin Another potential mechanism of action for protease therapy involves their effects on antimicrobial peptides. Studies using immortalized HaCaT human keratinocyte treated with different concentrations of the proteases in the protease technology were followed by analysis of antimicrobial peptides, cell death, and differentiation markers. Antimicrobial peptides (AMPs) function as a chemical shield on the skin surface, may participate in wound healing [117], and appear to coordinate and activate various components of the adaptive and innate immune system. Many cell types in the skin, including sebocytes, eccrine glands, keratinocytes, and mast cells, produce AMPs [118]. In the skin, β-defensins and cathelicidins are the most well-characterized AMPs. AMP dysfunction plays a key role in the pathogenesis of several skin diseases, including atopic dermatitis and rosacea [36]. Therapies modulating production and activity of AMPs might provide new strategies in the management of inflammatory and infectious skin diseases. Filaggrin and involucrin are crucial epidermal proteins and function as differentiation markers that are important for the optimum health of the stratum corneum. The imbalance of filaggrin and involucrin is a major factor in the pathogenesis of diseased skin conditions [119, 120]. Studies revealed that the treatment of the keratinocyte cell line with proteases differentially regulated the expression of AMPs β-defensin-1, β-defensin-3, S100A9, and differentiation markers filaggrin and involucrin (manuscript in preparation).

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Whether protease therapy may modulate expression and/or activity of AMPs is an important question in not only wound healing but also as potential alternate targets for therapy of challenging dermatologic diseases [36, 120].

9.5 Emerging protease technologies for treatment of fibrosis In some cases, fibrosis can develop during wound closure and during injury. Protease therapy may be beneficial for reducing fibrotic tissue. The connective tissue disorder, Dupuytren’s contracture of the hand, involves fibrous tissue, mainly composed of excess collagen deposits in rope-like cords that have accumulated within the fascia under the dermis. These rope-like cords prevent the full extension of fingers, most commonly the little and ring finger in Dupuytren’s contracture. Peyronie’s disease shows fibrotic scar tissue in the tunica albuginea of the penis, which prevents normal extension during penile erection. Xiaflex, an injectable form of collagenase from C. histolyticum, is FDA approved in adults and is a common treatment of the two connective tissue disorders, Dupuytren’s contracture of the hand [121, 122], and Peyronie’s disease [123, 124]. In rodent models, papain loaded into propylene glycol liposomes and applied to fibrotic skin tissue significantly reduced the fibrosis compared to the combined treatment of conventional liposomes and free papain solution [125]. Hypertrophic scars can also develop after burns and trauma [126]. Hypertrophic scars involve dysregulation of many of the same components active during wound healing (ECM, fibroblasts, growth factors, inflammation and immune system, keratinocytes, myofibroblasts) [126] and currently have few treatment options beyond surgery [126]. Investigating the effects of protease therapy on hypertrophic scars may lead to further understanding of the role of the ECM and inflammatory components in the dysregulation of skin growth and potentially new treatment options.

9.6 Emerging technologies for treatment of biofilm Biofilm contributes to the stalled healing process in chronic wounds [127] and is a common occurrence in gum disease and oral surgical wounds [128]. Some plant proteases have been proven to be edible herbal products with analgesic and antiinflammatory properties and are now being used in the oral treatment of gum disease and oral surgical wounds [59]. The proteases actinidin, papain, and trypsin reduced a biofilm of Actinomyces in vitro. Tablets containing a mixture of these proteases reduced the biofilm on the tongue in elderly patients [129]. Ficin was able to digest 3-day-old biofilms produced by the common pathogens, Staphylococcus aureus and Staphylococcus epidermidis in 4 h in a dose-dependent manner, ranging from 40% to 50% disrupted at 10 μg/mL ficin to near-complete disruption at 1000 μg/mL [60]. Biofilm disruption by ficin augmented the sensitivity of biofilm-embedded S. aureus and S. epidermidis to ciproflaxin [60].

Protease technology in wound repair

Additional protease preparations may be useful in reducing dental biofilm [130]. Researchers are also evaluating the efficacy of these proteases to enhance healing after plastic surgery [131].

9.7 Emerging protease-inspired therapies and cancer diagnostics Extracellular matrix forms part of the stroma surrounding tumor nodules and can limit the penetration of large-molecular-weight therapeutics and diagnostics into the tumor mass. The tumor-associated ECM consists primarily of fibrous structural proteins, including densely packed collagen and elastin; fibrous adhesion proteins such as laminin and fibronectin; and various proteogylcans [132]. Protease technology and specific compositions of the different proteases may be used to manage the microenvironment in and around cancerous and precancerous cells. Tumor cells secrete enzymes, growth factors, and cytokines to establish a blood supply and evade the immune system. The proteases may be used to modulate the microenvironment of the cancerous and precancerous cells in a patient, thereby promoting a normal tissue environment and reducing the ability of these cells to establish a metastasis. Modulation of the tumor microenvironment may thwart the tumor’s use of MMPs and certain cytokines and growth factors such as VEGF, FGF basic, PDEGF, EphrinB2, and Ang2 [116]. If the tumor microenvironment returns to a normal tissue milieu, the cancerous and precancerous cells can succumb to the lack of nutrients and the immune system, thereby promoting improved health and healing without exposure to chemotherapy [116]. As alternative strategies, combined targeted delivery of protease technology with antitumor agents may increase their penetration into the tumor nodules and has the potential to improve efficacy [38, 41, 43, 46]. Similar strategies may also apply to delivery of antibody-based cancer diagnostics in vivo.

10. Conclusions In summary, protease therapy is the backbone of enzymatic debridement for chronic wounds. Currently, only one of the six described protease therapies, CSO, is FDA approved for enzymatic debridement of chronic wounds, including DFUs. It may also be beneficial for the treatment of stalled acute wounds, such as DFUs, surgical wounds, and burns. Mechanistic studies revealed that the protease therapy impacts inflammation, proliferation, epithelialization, and wound neovascularization (Fig. 4). For example, the transition of mϕ1 proinflammatory macrophages into a predominance of mϕ2 macrophages in the wound bed was promoted, which helped reignite the normal wound healing progression from the inflammatory phase to the proliferative phase. Keratinocyte proliferation, which is essential during the proliferative, epithelialization, and remodeling phases, has also been demonstrated. These functions appear to be mediated by bioactive peptides released from the CCO-digested human collagen. Whether many of the benefits

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Fig. 4 The wound-healing process of diabetic foot ulcers can be delayed or stalled by complications of diabetes, and restarted by the effects of protease therapy. ECM, extracellular matrix; EPC, endothelial progenitor cells; FGF, fibroblast growth factor; KGF, keratinocyte growth factor; MMPs, matrix metalloproteases; mϕ, macrophage; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; TGF, transforming growth factor; TNF, tumor necrosis factor; TIMPs, tissue inhibitors of matrix metalloproteases; VEGF, vascular endothelial growth factor. (Modified from Nguyen TT, Mobashery S, Chang M. Roles of matrix metalloproteinases in cutaneous wound healing. In: Alexandrescu V, editors. Wound healing new insights into ancient challenges. London, UK: InTechOpen; 2016. p. 37–71.)

of other protease therapies are mediated by bioactive peptides and macrophage transition remains to be elucidated (manuscript in preparation). Since protease therapy is efficient in degrading ECM and potentially tumor-associated ECM, new frontiers for protease therapy range from adjunctive therapy for chemotherapy and/or immunotherapy to improved penetration of diagnostic probes, delayed healing of acute wounds, and potential clearance of dental biofilms and fibrotic lesions.

Acknowledgments The authors thank K.L. Molnar-Kimber, PhD (Kimnar Group LLC, Worcester, PA), for her editorial assistance.

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[102] Ostlie DJ, Juang D, Aguayo P, Pettiford-Cunningham JP, Erkmann EA, Rash DE, Sharp SW, Sharp RJ, St Peter SD. Topical silver sulfadiazine vs collagenase ointment for the treatment of partial thickness burns in children: a prospective randomized trial. J Pediatr Surg 2012;47:1204–7, 22703794. [103] Rosenberg L, Krieger Y, Bogdanov-Berezovski A, Silberstein E, Shoham Y, Singer AJ. A novel rapid and selective enzymatic debridement agent for burn wound management: a multi-center RCT. Burns 2014;40:466–74, 24074719. [104] Cordts T, Horter J, Vogelpohl J, Kremer T, Kneser U, Hernekamp JF. Enzymatic debridement for the treatment of severely burned upper extremities—early single center experiences. BMC Dermatol 2016;16:8, 27342276. [105] Berner JE, Keckes D, Pywell M, Dheansa B. Limitations to the use of bromelain-based enzymatic debridement (NexoBrid((R))) for treating diabetic foot burns: a case series of disappointing results. Scars Burn Heal 2018;4:2059513118816534. [106] Mearns ES, Liang M, Limone BL, Gilligan AM, Miller JD, Schaum KD, Waycaster CR. Economic analysis and budget impact of clostridial collagenase ointment compared with medicinal honey for treatment of pressure ulcers in the US. Clinicoecon Outcomes Res 2017;9:485–94, 28860830. [107] Dreyfus J, Delhougne G, James R, Gayle J, Waycaster C. Clostridial collagenase ointment and medicinal honey utilization for pressure ulcers in US hospitals. J Med Econ 2018;21:390–7. [108] Gilligan A, Waycaster C, Carter MJ, Fife CE. Comparative effectiveness of clostridial collagenase ointment for the treatment of venous leg ulcers in outpatient care settings. Value Health 2015;18: A230. [109] Gilligan AM, Waycaster CR, Bizier R, Chu BC, Carter MJ, Fife CE. Comparative effectiveness of clostridial collagenase ointment to medicinal honey for treatment of pressure ulcers. Adv Wound Care 2017;6:125–34, 28451469. [110] Pham CH, Collier ZJ, Fang M, Howell A, Gillenwater TJ. The role of collagenase ointment in acute burns: a systematic review and meta-analysis. J Wound Care 2019;28:S9–S15. [111] Salmeron-Gonzalez E, Garcia-Vilarino E, Perez-Del-Caz MD, Sanchez-Garcia A, ValverdeNavarro AA. Instantaneous specific burn debridement with an enzymatic debriding agent: a new resource for the treatment of burns. Plast Surg Nurs 2019;39:18–21. [112] Zacharevskij E, Baranauskas G, Varkalys K, Rimdeika R, Kubilius D. Comparison of non-surgical methods for the treatment of deep partial thickness skin burns of the hand. Burns 2018;44:445–52. [113] Sharp NE, Aguayo P, Marx DJ, Polak EE, Rash DE, Peter SD, Ostlie DJ, Juang D. Nursing preference of topical silver sulfadiazine versus collagenase ointment for treatment of partial thickness burns in children: survey follow-up of a prospective randomized trial. J Trauma Nurs 2014;21:253–7. [114] Brennan MB, Hess TM, Bartle B, Cooper JM, Kang J, Huang ES, Smith M, Sohn MW, Crnich C. Diabetic foot ulcer severity predicts mortality among veterans with type 2 diabetes. J Diabetes Complicat 2017;31:556–61. [115] Milne CT, Ciccarelli A, Lassy M. A comparison of collagenase to hydrogel dressings in maintenance debridement and wound closure. Wounds 2012;24:317–22. [116] Kling WO, Parnell LKS, O’neill PJ. In US Patent and Trademark Office, editor. Patent application no. 14/788175. Protease compositions for the treatment of damaged tissue. US: Swiss American Products; 2015. [117] Mangoni ML, Mcdermott AM, Zasloff M. Antimicrobial peptides and wound healing: biological and therapeutic considerations. Exp Dermatol 2016;25:167–73. [118] Schauber J, Gallo RL. Antimicrobial peptides and the skin immune defense system. J Allergy Clin Immunol 2008;122:261–6. [119] Kim BE, Leung DY, Boguniewicz M, Howell MD. Loricrin and involucrin expression is downregulated by Th2 cytokines through STAT-6. Clin Immunol 2008;126:332–7. [120] Thyssen JP, Kezic S. Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis. J Allergy Clin Immunol 2014;134:792–9. [121] Arora R, Kaiser P, Kastenberger TJ, Schmiedle G, Erhart S, Gabl M. Injectable collagenase Clostridium histolyticum as a nonsurgical treatment for Dupuytren’s disease. Oper Orthop Traumatol 2016;28:30–7.

Protease technology in wound repair

[122] Rohit A, Peter A, Paul A, Anja B, Christian D, Renate D, Stefan G, Dietmar H, Johannes J, Peter K, Marco K, Martin L, Maximilian N, Christoph P, Gernot S, Gerald S, Tobias S, Matthias W, Armin Z, Markus G. Prospective observation of Clostridium histolyticum collagenase for the treatment of Dupuytren’s disease in 788 patients: the Austrian register. Arch Orthop Trauma Surg 2019;139:1315–21. [123] Sherer BA, Levine LA. Contemporary review of treatment options for Peyronie’s disease. Urology 2016;95:16–24. [124] Tsambarlis PN, Yong R, Levine LA. Limited success with Clostridium collagenase histolyticum following FDA approval for the treatment of Peyronie’s disease. Int J Impot Res 2019;31:15–9. [125] Sahu K, Kaurav M, Pandey RS. Protease loaded permeation enhancer liposomes for treatment of skin fibrosis arisen from second degree burn. Biomed Pharmacother 2017;94:747–57. [126] Zhang J, Li Y, Bai X, Li Y, Shi J, Hu D. Recent advances in hypertrophic scar. Histol Histopathol 2018;33:27–39. [127] Snyder RJ, Bohn G, Hanft J, Harkless L, Kim P, Lavery L, Schultz G, Wolcott R. Wound biofilm: current perspectives and strategies on biofilm disruption and treatments. Wounds 2017;29:S1–S17. [128] Lasserre JF, Brecx MC, Toma S. Oral microbes, biofilms and their role in periodontal and periimplant diseases. Materials 2018;11:E.1802. [129] Mugita N, Nambu T, Takahashi K, Wang PL, Komasa Y. Proteases, actinidin, papain and trypsin reduce oral biofilm on the tongue in elderly subjects and in vitro. Arch Oral Biol 2017;82:233–40. [130] Pleszczynska M, Wiater A, Bachanek T, Szczodrak J. Enzymes in therapy of biofilm-related oral diseases. Biotechnol Appl Biochem 2017;64:337–46. [131] Orsini RA, Plastic Surgery Educational Foundation Technology Assessment, C. Bromelain. Plast Reconstr Surg 2006;118:1640–4. [132] Choi IK, Strauss R, Richter M, Yun CO, Lieber A. Strategies to increase drug penetration in solid tumors. Front Oncol 2013;3:193.

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CHAPTER 19

Collagen in diabetic wound healing Motaz Abasa, Mohamed El Masryb, Haytham Elgharablyc a

Ross University School of Medicine, Bridgetown, Barbados Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States c Department of Thoracic and Cardiovascular Surgery, Cleveland Clinic, Cleveland, OH, United States b

1. Introduction Collagen, mostly produced by fibroblasts, is a unique triple helix protein molecule that is the most abundant protein found in connective tissue, blood vessels, and skin while also being functionally pivotal by means of its wide-ranging functions in all phases of wound healing. Each molecule of collagen consists of three polypeptide protein chains that are coiled into a robust triple helix structure. The crosslinking and self-aggregation of these molecules creates additional tensile strength and stability that is characteristic of collagen fibers, yet has the ability to undergo constant remodeling to refine cellular behavior and tissue function [1]. There are over 20 genetically distinct human collagens that have so far been identified, with type I comprising approximately 70% of collagen in the skin [2]. Collagen accounts for 70%–80% of the dry weight of the dermis and is the major constituent of the dermal extracellular matrix, a three-dimensional structure that also contains glycosaminoglycans, proteoglycans, laminin, fibronectin, elastin and cellular components [3]. Injury to skin causes breaks to the extracellular matrix protective layer, and the extracellular matrix must be remodeled properly for wound healing to progress. Collagen plays a key functional role in granulation tissue formation by regulating recruitment, proliferation, and migration of activated cell types on the mesh of extracellular matrix [4]. Connective tissue mostly heals by formation of collagenous scar tissue [5]. Acting as a connective tissue scaffold in the early phase of wound healing, type III collagen is first laid down, while type I increases in proportion as scar formation progresses to increase the tensile strength of the wound [6]. A number of cell-surface proteins are capable of binding collagen [7]. There are four main types of proteins that mediate cell-collagen interactions, including receptors that recognize the peptide sequences Pro-Hyp-Gly unit (like glycoprotein VI) [8], discoidin domain receptor 1 and 2, receptors of the integrin-type, and receptors with affinity for noncollagenous domains. Many proteins such as decorin and laminin with integrinrecognition sequences are able to bind both collagen and integrins, which promotes cell adhesion and proliferation [9]. One central feature of diabetic wounds is an inappropriate

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00019-8

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inflammatory response caused partly by an abnormal collagen content that disrupt the normal adhesion and proliferation of inflammatory cells.

2. Collagen in diabetic wounds Diabetic wound-healing dysfunction and ulcer formation is thought to begin with an inclined susceptibility for injury at baseline. Diabetic skin is characterized by inferior biomechanical properties with greater stiffness and less flexibility, features that are thought to represent greater propensity for injury. These properties have been suggested to be the result of disparities in collagen synthesis versus degradation [10]. Hyperglycemia causes abnormal collagen production by increasing crosslinking while inducing nonenzymatic glycosylation of collagen. This leads to formation of abnormal and rigid collagen strands that inside the wound would ultimately fail to maintain the granulation tissue and is broken down to produce elevated levels of advanced glycation end products (AGEs) [11]. It was reported from a clinical study that collagen deposition was decreased in type I diabetes mellitus, where it was normal in type II [11]. This study also showed that the comparable collagenase activity in type I and II diabetes suggested the decrease in collagen synthesis rather than increased activity of collagenase. These findings were consistent with different animal studies that addressed the decrease in collagen synthesis in animals with induced diabetes [12, 13]. Interestingly, blockade of the receptor for advanced glycation end products (RAGEs) has been shown to restore the normal wound healing properties of diabetic (db/db) mice [14]. Hyperglycemic animals have also been shown to have significantly higher levels of glycated collagen with higher levels of collagenase activity [15]. In addition, diabetic (db/db) mice were also noted to have a prolonged inflammatory phase [16] with constant expression of the inflammatory cytokines macrophage chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) [17]. Once an open wound occurs, the surrounding skin normally offers a base structure for the fragile granulation tissue. However, the surrounding biochemically altered skin in diabetes is less suitable for this purpose, while trauma to the area is more likely to disturb the developing wound matrix due to the prolonged inflammation and proteolysis that occurs in diabetic wounds. Diabetic wounds like all wounds begin as acute wounds with a fibrin clot, but instead of advancing through the four normal phases of healing they are trapped in a prolonged inflammatory phase. It has been proposed that this extended phase of inflammation causes increased levels of proteases such as matrix metalloproteinase (MMP) collagenases, elastase, plasmin and thrombin, which devastate components of the ECM and damage the growth factors and their receptors that are necessary for healing [18]. The optimal conditions required for normal wound healing are thus disturbed in diabetic wounds, leading to their chronicity with possible complications such as infection, chronic foot ulcers, and amputation. This mainly stems not from a lack of adequate healing processes

Collagen in diabetic wound healing

Fig. 1 Impaired wound healing in diabetes.

but from hyperactivity of some of these processes that results in an out-of-phase, nonprogressive, persistent inflammatory state in the wound [19]. Collagen stimulates granulation tissue formation by regulating recruitment, proliferation, and migration of activated cell types on the mesh of extracellular matrix (Fig. 1). The abnormal collagen content of diabetic wounds leads to defective leucocyte chemotaxis and phagocytosis, which prolong inflammation, subsequently delaying proliferation and the formation of granulation tissue, further disturbing collagen deposition [20]. An additional impact of modified leucocytes levels is an increased vulnerability to infection, which also overstimulates the inflammatory response. The coexistence of infection and defective leukocytes likely perpetuates an inflammatory cycle, which delays the progression to proliferative phase of healing while the presence of infection also directly increases collagen breakdown. Significant collagen breakdown products maintain a state of excessive local bioburden in the wound and increases the vulnerability of the wound to microbial colonization, which further increases the bioburden of the wound and inflammation [21]. The local ischemia associated with peripheral vascular disease in diabetes also causes variations in oxygen tension in tissues, which modulates fibroblast proliferation and in turn collagen production. This causes a reduction of the structural support needed for capillary angiogenesis as well as producing excessive local inflammation [22]. Normal wound healing occurs through a balance of extracellular matrix degradation and formation, while nonhealing diabetic wounds sustain a chronic inflammatory state that lacks extracellular matrix formation. Matrix metalloproteinases (MMPs), capable of degrading all components of the extracellular matrix, play a central role in wound healing by removing damaged extracellular matrix during the inflammatory phase, degradation of the capillary basement membrane to facilitate angiogenesis and cell migration

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during the proliferative phase, and contraction of tissue in the remodeling phase [23]. Wounds require a certain level of these enzymes but can be very damaging at high concentrations leading to excessive degradation and impaired wound healing and chronicity, a characteristic problem seen in diabetic patients. MMP-1, MMP-8, MMP-13, and MMP-18 are the collagenase subset of MMPs known to directly interact with collagen. On the other hand, tissue inhibitors of metalloproteinase (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) counteract the action of MMPs and thus regulate their action, with an imbalance of these matrix proteins and their inhibitors causing degradation of the matrix collagen and act as a predictor of chronicity [24]. Diabetic wounds are known to be in a state of persistent inflammation. Proinflammatory cytokines such as IL1-α, IL1-β, IL-2, IL-17, C-reactive protein, insulin-like growth factor-1, and TGF-α stimulate the production of neutrophil gelatinase-associated lipocalin (NGAL), a potent activator of MMPs. The overactivation of MMPs through this process was found to occur in nonhealing venous ulcers and is a contributing factor to delayed wound healing [25]. Further studies noted the important role of the collagenases MMP-1 and MMP-8 in normal wound healing while observing their overexpression in patients with nonhealing chronic venous ulcers compared to patients with healing ulcers [26]. Many authors have agreed and linked the association of MMP/NGAL to the pathophysiology of chronic wounds during the inflammatory and proliferative phases where collagenases are found to be significantly high, causing repeated migration of PMNs and macrophages to the wound site. The overexpression of these proteins is thought to prolong the inflammatory phase due to excessive collagen breakdown and neutrophil presence that results in delayed wound healing [27–29]. Fibroblasts produce and arrange the ECM by constructing a collagen-rich matrix while communicating with each other and other cell types. In human studies, collagen-producing fibroblasts extracted from healthy human skin cultured in high glucose levels were shown to have slower proliferation rates [30]. Similarly, fibroblasts cultured from diabetic foot ulcers demonstrated impaired proliferation [31]. The state of reduced deposition of collagen in diabetic wounds leads to decreased mechanical strength of these wounds, while healing was found to be almost normalized with insulin treatment in several studies [32–34].

3. Diabetic-wound-infection-induced collagen dysfunction Diabetic wound infection has a significant role in chronic extracellular matrix degradation. Increased level of proteases, lack of growth factors, and increased inflammatory cytokines were evident by analysis of wound fluids [35, 36]. The rate of clinically infected diabetic foot ulcers (DFU) ranged from 58%–61% [37, 38]. Commonly DFU is infected with many gram-positive pathogens, such as Staphylococcus aureus, Enterococcus faecalis, and Streptococcus equi, via binding to collagen by utilizing collagen-binding adhesins of the

Collagen in diabetic wound healing

Bacterial colonization

Collagen triple helix

Elastase MMPs

Matrix degradation Fig. 2 Collagen dysfunction in diabetic wounds.

microbial surface component recognizing adhesive matrix molecules family [39–41] (Fig. 2). The collagen-binding microbial surface component recognizing adhesive matrix molecules on S. aureus is called CWA (cell-wall-anchored protein) and is the prototype member of this family. CWA participates in the infectious process of pathogenic S. aureus and is shown to be a virulence factor in many different animal models of staphylococcal infections, including arthritis, endocarditis, osteomyelitis [39, 42–44], indicating the general advantage of bacteria-collagen binding in the pathogenesis of delayed wound healing. Thus, one of the main challenges for collagen-based wound dressings is the high affinity of some bacteria for collagen.

4. Applications of collagen in wound healing Advantages of the application of collagen in wound dressings include its inherent biodegradability and nontoxic nature, with exogenous collagen being more biocompatible than other natural polymers while being weakly antigenic. One of the earlier introductions of collagen dressings was in the form of collagen sponges. Collagen-based sponges are insoluble forms of animal-derived collagen that are used in diabetic wounds due to their capability of absorbing large amounts of tissue exudate while adhering to the wound bed, maintaining a moist wound environment, and protecting against mechanical trauma and bacterial infection [45]. The collagen within the sponge promotes cellular motility and allows inflammatory cells to actively invade the porous scaffolding of the sponge [46]. When there are cells bound to an extracellular matrix, in this case an implanted collagen sponge, an increase in the production of new collagen is seen [47]. Furthermore, the collagen sponge undergoes degradation by tissue collagenases into peptide fragments and amino acids and replaced by native type I collagen produced by fibroblasts.

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Other formulations of collagen-based wound dressings that have been introduced include dressings with or without active cellular components, as well as those containing silver, alginate, protease inhibitors, hydrolyzed collagen, bone marrow, or amniotic membrane. Wound dressings containing collagen are thought to create a biological scaffold matrix that supports the regulation of extracellular components by encouraging deposition and organization of newly formed collagen through their chemotactic properties on wound fibroblast. Experimental data also suggest several roles of collagen wound dressings that address some of the anomalous diabetic wound processes such as to inhibit MMPs, induce fibroblast production, increase bioavailability of fibronectin, maintain chemical and thermostatic microenvironment of the wound, and preserve leukocytes, fibroblasts, and epithelial cells [48–51]. These biomaterials can also provide moisture or absorption, depending on the delivery system, and are usually formulated with bovine, avian, or porcine collagen. On the other hand, oxidized regenerated cellulose, a plantbased material, has been merged with collagen, which allows for the capability of binding to and protecting growth factors by inactivating matrix metalloproteinases in the wound [52]. Collagen-based dressings have the propensity for the stimulation of wound healing with the ability of absorption of wound drainage to decrease the wound’s bioburden. Such added function is achievable by adding alginate to the product, for example, although pure collagen dressings maintain to some degree an absorptive quality of their own. Pure collagen or denatured collagen dressings may also contain silver as another absorptive component to decrease the bioburden in the dressing. On the other hand, plant or marine-based cellulose derivatives are thought to lead to less giant cell-type reactions in the wound bed due to the absence of nonbiodegradable materials [53]. Studies from preclinical swine models of excisional and ischemic wounds utilizing a bovinederived modified collagen gel dressing reported increase in the length of rete ridges, which suggest improved biomechanical properties of the healing wound tissue. Other findings include accelerated neutrophil and macrophage recruitment to the wound site, induction of monocyte chemotactic protein-1 expression, upregulation of transforming growth factor-β, vascular endothelial growth factor, von Willebrand’s factor, higher abundance of fibroblasts and endothelial cells with increased blood flow to the wound area, enhanced proliferation of wound-site endothelial cells, higher abundance of collagen, and increased collagen type I:III ratio [54, 55]. Another study applying a murine model utilizing a modified collagen gel dressing reported attenuated proinflammatory and promoted antiinflammatory macrophage polarization, increased antiinflammatory IL-10, IL-4, and proangiogenic VEGF production, bolstered macrophage engulfment, and induced miR-21 expression [56]. In a different murine study utilizing equine pericardial collagen-based matrix dressing showed the inhibitory effect of the host primed dressing on the biofilm formation of both S. aureus and Pseudomonas aeruginosa by inhibiting their metabolic activity [57].

Collagen in diabetic wound healing

Overall, collagen-containing wound dressings demonstrate some benefit in the treatment of diabetic wounds. However, there is currently not sufficient evidence pointing to the superiority of a particular collagen biological source, combination, or method of delivery. Due to its low antigenicity and inherent biocompatibility with most endogenous tissue, natural collagen has continued to be a subject of interest in the field of chronic wound research. Another advantage of wound dressings based on collagen is their practicality and easy remodeling due to their simple structure, relative uniformity, and abundant availability. Furthermore, they have a distinctive practical and economic advantage compared to growth-factor and cell-based treatment of diabetic wounds and have been formulated in a number of different ways. A major advantage of collagen-based wound care dressings is breaking down the persistent inflammatory response. Clinical application of collagen-based dressings helps to reduce excess proteolysis and restore the physiological conditions required for wound healing. Given the vital role of collagen in wound optimization, collagen has continued to be considered for nonhealing wounds as a component of wound care products.

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[13] Grotendorst GR, Martin GR, Pencev D, Sodek J, Harvey AK. Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats. J Clin Invest 1985;76:2323–9. [14] Goova MT, Li J, Kislinger T, Qu W, Lu Y, Bucciarelli LG, Nowygrod S, Wolf BM, Caliste X, Yan SF, Stern DM, Schmidt AM. Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice. Am J Pathol 2001;159:513–25. [15] Hennessey PJ, Ford EG, Black CT, Andrassy RJ. Wound collagenase activity correlates directly with collagen glycosylation in diabetic rats. J Pediatr Surg 1990;25:75–8. [16] Khanna S, Biswas S, Shang Y, Collard E, Azad A, Kauh C, Bhasker V, Gordillo GM, Sen CK, Roy S. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One 2010;5:e9539. [17] Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol 2000;115:245–53. [18] Mast BA, Schultz GS. Interactions of cytokines, growth factors, and proteases in acute and chronic wounds. Wound Repair Regen 1996;4:411–20. [19] Trengove NJ, Stacey MC, MacAuley S, Bennett N, Gibson J, Burslem F, Murphy G, Schultz G. Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 1999;7:442–52. [20] Acosta JB, del Barco DG, Vera DC, Savigne W, Lopez-Saura P, Guillen Nieto G, Schultz GS. The pro-inflammatory environment in recalcitrant diabetic foot wounds. Int Wound J 2008;5:530–9. [21] White R, Cutting K. Critical colonisation of chronic wounds: microbial mechanisms. Wounds UK 2008;4:70–8. [22] Hunt TK, Pai MP. The effect of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg Gynecol Obstet 1972;135:561–7. [23] Ayuk SM, Abrahamse H, Houreld NN. The role of matrix metalloproteinases in diabetic wound healing in relation to photobiomodulation. J Diabetes Res 2016;2016:2897656. [24] Muller M, Trocme C, Lardy B, Morel F, Halimi S, Benhamou PY. Matrix metalloproteinases and diabetic foot ulcers: the ratio of MMP-1 to TIMP-1 is a predictor of wound healing. Diabet Med 2008;25:419–26. [25] Serra R, Grande R, Buffone G, Molinari V, Perri P, Perri A, Amato B, Colosimo M, de Franciscis S. Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines. Int Wound J 2016;13:53–8. [26] Amato B, Coretti G, Compagna R, Amato M, Buffone G, Gigliotti D, Grande R, Serra R, de Franciscis S. Role of matrix metalloproteinases in non-healing venous ulcers. Int Wound J 2015;12:641–5. [27] Busceti M, Grande R, Amato B. Pulmonary embolism, metalloproteinases and neutrophil gelatinase associated lipocalin. Acta Phlebol 2013;14:115–21. [28] de Franciscis S, Mastroroberto P, Gallelli L, Buffone G, Montemurro R, Serra R. Increased plasma levels of metalloproteinase-9 and neutrophil gelatinase-associated lipocalin in a rare case of multiple artery aneurysm. Ann Vasc Surg 2013;27. 1185.e5-7. [29] Serra R, Buffone G, Molinari V, Montemurro R, Perri P, Stillitano DM, Amato B, de Franciscis S. Low molecular weight heparin improves healing of chronic venous ulcers especially in the elderly. Int Wound J 2015;12:150–3. [30] Hehenberger K, Hansson A. High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C-inhibitors. Cell Biochem Funct 1997;15:197–201. [31] Hehenberger K, Kratz G, Hansson A, Brismar K. Fibroblasts derived from human chronic diabetic wounds have a decreased proliferation rate, which is recovered by the addition of heparin. J Dermatol Sci 1998;16:144–51. [32] Gottrup F, Andreassen TT. Healing of incisional wounds in stomach and duodenum: the influence of experimental diabetes. J Surg Res 1981;31:61–8. [33] Tengrup I, Hallmans G, Agren MS. Granulation tissue formation and metabolism of zinc and copper in alloxan-diabetic rats. Scand J Plast Reconstr Surg Hand Surg 1988;22:41–5. [34] Verhofstad MH, Hendriks T. Complete prevention of impaired anastomotic healing in diabetic rats requires preoperative blood glucose control. Br J Surg 1996;83:1717–21.

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[35] Holmes C, Wrobel JS, Maceachern MP, Boles BR. Collagen-based wound dressings for the treatment of diabetes-related foot ulcers: a systematic review. Diabetes Metab Syndr Obes 2013;6:17–29. [36] Mustoe TA, O’Shaughnessy K, Kloeters O. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 2006;117:35S–41S. [37] Lavery LA, Armstrong DG, Murdoch DP, Peters EJ, Lipsky BA. Validation of the infectious diseases society of America’s diabetic foot infection classification system. Clin Infect Dis 2007;44:562–5. [38] Prompers L, Huijberts M, Apelqvist J, Jude E, Piaggesi A, Bakker K, Edmonds M, Holstein P, Jirkovska A, Mauricio D, Ragnarson Tennvall G, Reike H, Spraul M, Uccioli L, Urbancic V, Van Acker K, van Baal J, van Merode F, Schaper N. High prevalence of ischaemia, infection and serious comorbidity in patients with diabetic foot disease in Europe. Baseline results from the Eurodiale study. Diabetologia 2007;50:18–25. [39] Patti JM, Boles JO, Hook M. Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus. Biochemistry 1993;32:11428–35. [40] Lannergard J, Frykberg L, Guss B. CNE, a collagen-binding protein of Streptococcus equi. FEMS Microbiol Lett 2003;222:69–74. [41] Ma W, Ma H, Fogerty FJ, Mosher DF. Bivalent ligation of the collagen-binding modules of fibronectin by SFS, a non-anchored bacterial protein of Streptococcus equi. J Biol Chem 2015;290:4866–76. [42] Patti JM, Allen BL, McGavin MJ, Hook M. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 1994;48:585–617. [43] Hienz SA, Schennings T, Heimdahl A, Flock JI. Collagen binding of Staphylococcus aureus is a virulence factor in experimental endocarditis. J Infect Dis 1996;174:83–8. [44] Elasri MO, Thomas JR, Skinner RA, Blevins JS, Beenken KE, Nelson CL, Smeltzer MS. Staphylococcus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis. Bone 2002;30:275–80. [45] Yannas IV. Biologically-active analogs of the extracellular-matrix—artificial skin and nerves. Angew Chem Int Ed 1990;29:20–35. [46] Chvapil M, Chvapil TA, Owen JA. Reaction of various skin wounds in the rat to collagen sponge dressing. J Surg Res 1986;41:410–8. [47] Postlethwaite AE, Seyer JM, Kang AH. Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci USA 1978;75:871–5. [48] Brett D. A review of collagen and collagen-based wound dressings. Wounds 2008;20:347–56. [49] Burton JL, Etherington DJ, Peachey RD. Collagen sponge for leg ulcers. Br J Dermatol 1978;99:681–5. [50] Doillon CJ, Silver FH, Olson RM, Kamath CY, Berg RA. Fibroblast and epidermal cell-type I collagen interactions: cell culture and human studies. Scanning Microsc 1988;2:985–92. [51] Palmieri B. Heterologous collagen in wound healing: a clinical study. Int J Tissue React 1992;14 (Suppl):21–5. [52] Cullen B, Smith R, McCulloch E, Silcock D, Morrison L. Mechanism of action of PROMOGRAN, a protease modulating matrix, for the treatment of diabetic foot ulcers. Wound Repair Regen 2002;10:16–25. [53] Fleck CA, Simman R. Modern collagen wound dressings: function and purpose. J Am Coll Certif Wound Spec 2010;2:50–4. [54] Elgharably H, Ganesh K, Dickerson J, Khanna S, Abas M, Ghatak PD, Dixit S, Bergdall V, Roy S, Sen CK. A modified collagen gel dressing promotes angiogenesis in a preclinical swine model of chronic ischemic wounds. Wound Repair Regen 2014;22:720–9. [55] Elgharably H, Roy S, Khanna S, Abas M, Dasghatak P, Das A, Mohammed K, Sen CK. A modified collagen gel enhances healing outcome in a preclinical swine model of excisional wounds. Wound Repair Regen 2013;21:473–81. [56] Das A, Abas M, Biswas N, Banerjee P, Ghosh N, Rawat A, Khanna S, Roy S, Sen CK. A modified collagen dressing induces transition of inflammatory to reparative phenotype of wound macrophages. Sci Rep 2019;9:14293. [57] El Masry MS, Chaffee S, Das Ghatak P, Mathew-Steiner SS, Das A, Higuita-Castro N, Roy S, Anani RA, Sen CK. Stabilized collagen matrix dressing improves wound macrophage function and epithelialization. FASEB J 2019;33:2144–55.

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CHAPTER 20

Nutrition and diabetic wound healing Amit Kumar Madeshiyaa,∗, Nandini Ghosha,∗, Nirupam Biswasa,∗, Abhishek Sena, Debasis Bagchib, Jennifer Mohnackya, Sashwati Roya, Amitava Dasa a

Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States b Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX, United States

1. Introduction Optimal nutrition is pivotal in maintaining physiological and immunological homeostasis. It is clinically established that an individual consuming a diet deficient in one or more of its components is more susceptible to diseases [1]. Therefore, not only is good nutrition vital to evading pathological symptoms, it plays an equally important role in recovering from morbidities. Wound care is a significant aspect of the clinical setting, and it is directly affected by nutrition, which is often disregarded [2–4]. Simply put, a wound (ranging from a paper cut to a foot ulcer) is perceived as a stress stimulus by the human body, which then inadvertently reacts by mounting an immunological response. During the three phases of wound healing—inflammation, tissue formation and tissue remodeling—it is essential for our body to replenish its energy reserves and build new tissue (anabolism). Amino acids, fats, carbohydrates, and minerals drive this anabolic process of wound repair to completion. A deficiency thereof slackens wound healing and, if persistent, leads to chronic wound infections. In the United States, as of 2015, more than 30.3 million people (9.4% of the US population) were diabetic [5]. Diabetes results in the body’s inability to produce or utilize insulin, which allows the body to turn glucose into energy. When the body is incapable of metabolizing glucose, it can lead to high blood glucose levels. While the goals for treating diabetic complications have developed significantly over time, the primary objective is to control glucose levels to forestall diabetic complications. Nowadays, complementary medicines are used extensively worldwide and amount to global sales greater than US$60 billion [6]. At present, more than 100 plant species known for various chemical classes of compounds are in consideration for diabetes care [7, 8]. Diabetic wounds are one of the serious complications associated with type 2 diabetes mellitus (T2DM) [9, 10] characterized by persistent inflammation and impaired wound healing [11–16]. Like all other pathophysiological processes, diabetic wound healing is also governed by the nutritional ∗

Contributed equally.

Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00020-4

© 2020 Elsevier Inc. All rights reserved.

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status of the body and the affected tissue. This chapter is dedicated to explaining the nutritional requirements and interventions in the treatment of diabetic wound complications.

2. Dysregulated wound inflammation during diabetes Inflammation is a pathophysiological response of living mammalian tissues to injury or infection. Inflammation is defined as “a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, postischemic, toxic or autoimmune injury” [17]. Inflammation is characterized by a change in the blood flow, increased cellular metabolism, dilatation of blood vessels, release of soluble mediators, fluid leakage and infiltration of various cells [18]. During the initiation of inflammation, resident tissue cells release a number of mediators such as chemokines, cytokines, lipid mediators and bioactive amines [19]. Inflammation response, after its scheduled role, is actively terminated and resolved [20, 21]. Failure of termination of inflammation could result in a chronic inflammation [22]. The beginning of the reparative phase and the termination of the inflammatory phase is termed as resolution of inflammation [11–14]. The desired outcome of the acute inflammation is successful resolution and repair of the damaged tissue. Persistence of inflammation might be detrimental, causing loss of organ function [23]. Low-grade chronic inflammation is the cause of a wide range of disorders such as chronic obstructive pulmonary disease, atherosclerosis, obesity, cancer, asthma, inflammatory bowel disease, neurodegenerative disease, multiple sclerosis, and rheumatoid arthritis [24]. Chronic nonresolving wounds are the result of dysregulated inflammatory response, which is becoming a major social and economic burden [25, 26].

3. Nutrition in diabetic wound healing Chronic wounds are commonly seen in individuals without adequate nutrition. Under pathological conditions such as metabolic syndrome (MetSyn) and diabetes, acute wounds become chronic. It has been suggested that the systemic and tissue-specific inflammatory conditions resulting from excessive adipose tissue may result in obesityrelated comorbidities, such as T2DM and CVD [27, 28]. Along with other complications, impaired wound healing is a major problem in T2DM patients [29]. Inadequate consumption and metabolism of nutrients may have a negative impact on the immune system, which may result in a chronic inflammatory status. A number of clinical studies have revealed that an imbalance of proteins, carbohydrates, fats, vitamins, and minerals may impair wound healing outcomes [3] (Fig. 1). An imbalance in nutritional status is exacerbated in the case of diabetic patients where wound healing is compromised. This phenomenon is attributed to delayed and chronic inflammation and is commonly coupled with inadequate angiogenesis [30, 31]. One in five such diabetic

Nutrition and diabetic wound healing

Fig. 1 Nutritional factors affecting wound healing.

patients are likely to suffer from foot ulcers, of which nearly half develop infections needing an amputation [32]. Returning to the process of wound healing in normal and diabetic patients alike, the initial stages demand greater amounts of building blocks; notably, amino acids, carbohydrates, fats, and vitamins A and D must be supplemented through a structured diet. Therefore, when the body is provided with suboptimal amino acids through food, it resorts to its own lean body mass (LBM) as a source—a process termed autocannibalism. A prolonged case of dependence on LBM leads to protein energy malnutrition (PEM). Carbohydrates such as glucose are an important source of energy in wound healing and otherwise. Their mode of action stimulates insulin production in the body, but since this mechanism is impaired in diabetic patients, their body is unable to wield this source of energy, naturally leading to infectious complications. The delayed wound healing is coupled with hindrance in basophil, neutrophil, eosinophil, and mast cell activity in hyperglycemic/diabetic conditions. Therefore, in diabetic patients, fats serve as the second source of energy, in lieu of carbohydrates, while also functioning as a platform for fat soluble vitamin A (retinoic acid), which stimulates epithelial cells and fibroblast growth and has antiinflammatory effects for wound closure, irrespective of a diabetic condition [33, 34]. Vitamin D has been shown to regulate immune cell proliferation, skin differentiation, and angiogenesis [35].

3.1 Carbohydrates Carbohydrates, along with fats, are the primary energy source in the body and are also required for the different phases of wound healing, especially for collagen synthesis [36]. However, the role of carbohydrates, especially glucose, is complex. Under hyperglycemic conditions characterized by increased blood glucose levels, wound inflammation is prolonged and wound healing is impaired [11–13, 20]. One of the important causes for this impairment is the formation of advanced glycation end products in the hyperglycemic wound milieu [12]. On the other hand, fibroblast proliferation is reported to increase

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with glucose treatment [37]. Recent studies show that carbohydrate-rich supplements help in management of diabetic wound outcomes [7, 38–40].

3.2 Fats and fatty acids Like carbohydrates, fats provide energy and building blocks for tissues [41]. Fats have a critical role in synthesis of cell membrane, phospholipids, and intracellular matrix [41]. Fatty acids constitute the cell membrane and act as substrates for the eicosanoid pathway, which governs the inflammatory processes [36]. Supplementing with omega-3 fatty acids and low trans fats helps attenuate inflammation [42]. Application of omega-3 fatty acids has been reported to improve wound healing [43]. Interestingly, it has been recently reported that eicosapentaenoic and docosahexaenoic polyunsaturated fatty acids may increase proinflammatory cytokine production at wound sites [44], thereby provoking the need for more clinical studies.

3.3 Proteins and amino acids Providing the principal building blocks for tissues, cell renewal, and injury-induced repair, proteins affect all the wound healing phases through their roles in nucleotide synthesis, collagen tissue formation, the immune system, growth of the epidermis, and keratinization [41, 45]. Protein malnutrition for an extended period of time results in thin and wrinkled skin and dampening of immune responses [46]. The risk of amputations is higher in diabetic patients suffering from protein malnutrition [47, 48]. Among amino acids, arginine has several effects on the immune function. Macrophage biology, which determines the fate of the inflammatory process, is governed by arginine metabolism. The proinflammatory M1 macrophages express nitric oxide synthase, which is responsible for metabolizing arginine to nitric oxide (NO). NO can be metabolized to reactive nitrogen species and citrulline, which may be reused for NO synthesis via the citrulline-NO cycle. On the other hand, antiinflammatory M2 macrophages express the enzyme arginase that hydrolyzes arginine to ornithine and urea, thereby limiting the availability of arginine for NO synthesis. The ornithine thus formed is critical in synthesis for polyamine and proline, which are required for cellular proliferation, collagen synthesis, and tissue repair [49]. Though glutamine was found to confer protection against inflammation injury through heat shock proteins by providing cellular protection during inflammation, injury, and stress [50, 51], glutamine supplementation has not been shown to be beneficial in wound healing. Cysteine and methionine play critical roles in connective tissue and collagen synthesis.

3.4 Vitamins Vitamins improve wound healing by facilitating the transition between the stages of wound healing and by promoting collagen synthesis [52]. Vitamin C acts as a cofactor

Nutrition and diabetic wound healing

in hydroxylation of proline and lysine residues in procollagen, which is critical for conferring the strength and stability of collagen fibers. In addition, ascorbic acid acts as an antioxidant and bolsters neutrophil function. Thus, vitamin C deficiency may impair wound healing outcomes, primarily due to its critical role in collagen formation and posttranslational modification. On the other hand, vitamin A increases the inflammatory response in wounds through increased macrophage infiltration, greater lysosomal membrane lability, and its activation and stimulation of collagen synthesis. Vitamin A increases the influx of monocytes and macrophages at the wound site during the early inflammatory phase and facilitates epithelial cell differentiation [34]. Interestingly, it also reverses corticosteroid-induced inhibition of cutaneous wound healing [53]. Vitamin D supplementation was found to improve wound healing in streptazotocin induced diabetic mice through a NF-κB mediated pathway [54]. Although vitamin E supplementation has been reported to induce antimicrobial activity and improve the efficacy of daptomycin in murine wounds infected with methicillin-resistant Staphylococcus aureus [55], the literature from robust studies of the effect of vitamin E on wound healing is scanty [56]. Recent studies have revealed that inadequate vitamin B can impede wound healing and is associated with disorders related to skin manifestations [41].

3.5 Minerals Minerals have pivotal roles in optimal wound healing, as they modulate preliminary enzymes in the early inflammatory phases. Copper functions as a cofactor for enzymes essential for remodeling, possesses angiogenic properties, and induces the expression of vascular endothelial growth factor (VEGF) [57, 58]. Another micronutrient, zinc, also serves as a cofactor for the key wound healing enzymes and transcription factors in addition to promoting reepithelialization and generating of new tissues [59, 60]. Magnesium is a critical cofactor for enzymes associated with the synthesis of both protein and collagenase. Iron has a critical role in oxidative stress and the hydroxylation of the amino acids lysine and proline [61]. Iron deficiency has been reported to stall the inflammation in a proinflammatory environment and thereby impair wound healing [62]. In addition, iron deficiency has been directly correlated to chronic inflammatory diseases such as rheumatoid arthritis and lupus erythematosus [63].

4. Other nutritional interventions Apart from the various macronutrients and micronutrients described previously, several nutritional supplements have been reported to accelerate wound healing [64]. Polyphenols like resveratrol, which are obtained from berries, peanuts, and red wine, have been shown to play pivotal roles in diabetic foot ulcers (DFUs) [65]. Resveratrol activates sirtuins (SIRTs), thereby decreasing the proinflammatory TNF-α. The polyphenolic compound curcumin, obtained from turmeric, is effective in inhibiting the transcription

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factor NF-κB, thereby dampening inflammation [66]. Production of nitric oxide via iNOS and COX-2 are attenuated by the flavonoids naringenin and apigenin. Lipopolysaccharide-induced release of proinflammatory mediators from BV2 microglia is inhibited by naringenin through NF-κB and the mitogen-activated protein kinase (MAPK) signaling pathway [67]. Additionally, apigenin, a natural product belonging to the flavone class, is reported to decrease apoptosis of endothelial cells by limiting the activity of caspase 3 [68].

5. Papaya: A natural remedy for diabetic wounds Papaya (Carica papaya Linn), belonging to the family Caricaceae and known in some areas as paw-paw, has nutritional value [69]. The different parts of the papaya plant, including leaves, latex, seeds, and fruit, all have medicinal significance. The latex of unripe papaya fruit contains the enzymes papain and chymopapain [70]. A wide array of research studies show that treatment with papaya preparations facilitates wound-healing responses [7, 38–40, 69, 71–73] (Table 1). The different parts of the papaya plant have been extensively used in traditional systems of medicine for curing several ailments [78]. Papaya is known to have wound-healing properties that may be due to the presence of protease enzymes. Papaya dressing is effective and safe for use in wound bed preparation of postoperative wound gape patients [75]. Wounds treated with aqueous extract of papaya fruit promoted epithelialization and increased granulation tissue weight and hydroxyproline content. Further, treatments with aqueous extract of papaya demonstrated antimicrobial activity and promoted significant wound healing in streptozotocin-induced diabetic rat excision wound models [74]. Papaya extract improved wound repair by recruitment of fibroblasts, limiting leukocyte infiltration by early transient expressions of TGF-β1 and VEGFA at the wound site. In addition, healing outcomes were accelerated by the addition of Se2+ [76]. Treatment with papaya in the presence of selenium for 10 days dampened inflammation-related oxidative damage in excisional wounds via cyclooxygenase specific inhibition, arginine metabolism, and upregulation of antioxidant enzymes [77]. Comparatively, the aqueous extracts of green papaya epicarp (GPE) were reported more efficient in wound healing than the ripe papaya epicarp (RPE) when administered orally [69]. The use of papaya pulp in second- and third-degree burns was found to be a very effective debriding agent and helpful in the healthy granulation of burn tissue [79]. Recently several studies observed the significant impact of standardized fermented papaya preparation (FPP) on wound healing in humans as well as adult obese diabetic (db/db) mice [7, 38–40]. FPP is a carbohydrate-rich nutritional supplement. In FPP, glucose coexists with fructose and maltose in addition to multiple other sugars and alcohols such as inositol. In a murine study, 8-week oral supplementation of 0.2 g/kg body FPP reduced the total blood sugar level, effectively corrected wound closure, and improved

Nutrition and diabetic wound healing

Table 1 Wound healing using papaya and its products. References

Model

Key findings

Mikhal’chik et al. [71]

Rat

Nayak et al. [74]

Rat

Anuar et al. [69]

Mice

Gurung and Skalko-Basnet [72] Collard and Roy [7]

Mice Mice

Dickerson et al. [40]

Human

Murthy et al. [75]

Human

Dickerson et al. [39]

Human

Nafiu and Rahman [76]

Rat

Nafiu and Rahman [77]

Rat

Figueiredo Azevedo et al. [73] Das et al. [38]

Mice

Improved wound healing outcome and reduced inflammation in rats upon treatment with the phytopreparation from papaya Aqueous extract of C. papaya exhibited antimicrobial activity and promoted wound healing in diabetic rats Green papaya epicarp (GPE) extract was reported more efficient in wound healing than the ripe papaya epicarp (RPE) when administrated orally Papaya latex formulation in the Carbopol gel is effective in burn treatment Diabetic-wound outcomes benefit from fermented papaya preparation (FPP) supplementation by modulating woundsite macrophages and angiogenic response FPP corrected respiratory burst in T2DM PBMC via an Sp-1-dependent pathway Papaya dressing is effective and safe as compared to hydrogen peroxide dressing for wound bed preparation in postoperative wound gape patients FPP improves diabetic wound outcome by correcting inducible “respiratory burst” in T2DM patients Papaya extract improved wound repair by recruitment of fibroblasts, limiting leukocyte infiltration by early transient expressions of TGF-β1 and VEGFA at the wound site Treatment of papaya extract lowered inflammation associated oxidative damage in excision wounds via cyclooxygenase inhibition, arginine metabolism, and upregulation of antioxidant enzymes 3% papain gel improved cutaneous wound healing in mice

Human

FPP bolstered PMA-induced respiratory burst in wound-site macrophages of diabetic patients. Wound closure in FPPsupplemented patients showed improvement

the respiratory burst by inducing the production of reactive oxygen species like NO in the viable macrophages isolated from the wound site. Additionally, FPP supplementation resulted in an increased abundance of CD68+ and CD31+ at the wound site, suggesting increased infiltration of monocytes/macrophages and an improved proangiogenic response. However, interestingly, the consumption of a carbohydrate-rich FPP mixture, including significant amounts of glucose, did not increase HbA1c [7, 38]. These findings of FPP were validated in healthy human volunteers as well as diabetic patients. The production of reactive oxygen species by T2DM peripheral blood mononuclear cells

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(PBMC) was comparatively decreased to that of the PBMC from non-DM donors when stimulated with phorbol 12-myristate 13-acetate. The compromised respiratory burst was improved upon the treatment with FPP in T2DM PBMC through a p47phoxRac2-Sp1 pathway [40]. In addition, FPP supplementation improved inducible “respiratory burst” in T2D PBMC, probably due to increased availability of ATP and NADPH resulting from increased mitochondrial respiration [39]. Finally, patients supplemented with FPP and on standard of care (SoC) for wound management had significantly higher ROS production in wound-site immune cells and improved wound closure compared with those patients receiving SoC alone [38]. In diabetic rats, aqueous extract Carica papaya Linn leaves (AECPL) were effective in controlling blood glucose levels and improving the lipid profile [80]. In addition, papaya extract exhibited antioxidant potential in diabetic rats [81]. In vitro, FPP treatment improved platelet function by enhancing Na(+)/K(+)-ATPase activity. FPP also enhanced membrane fluidity and ameliorated antioxidant functionality by increasing TAC and SOD activity. In T2DM patients, FPP decreased conjugated diene levels [82].

6. Conclusion Nutritional status plays a major role in determining the fate of wound healing and its complications, especially in diabetes. A wide array of macronutrients, micronutrients, and food supplements are instrumental in orchestrating a wound repair. However, several questions pertaining to the effect of supplementation of these nutrients on wound care remain unanswered and this warrants more randomized clinical trials.

References [1] Krehl WA. The role of nutrition in maintaining health and preventing disease. Health Values 1983; 7(2):9–13. [2] Stechmiller JK. Understanding the role of nutrition and wound healing. Nutr Clin Pract 2010; 25(1):61–8. [3] Dryden SV, Shoemaker WG, Kim JH. Wound management and nutrition for optimal wound healing. Atlas Oral Maxillofac Surg Clin North Am 2013;21(1):37–47. [4] Irvin TT. Effects of malnutrition and hyperalimentation on wound healing. Surg Gynecol Obstet 1978;146(1):33–7. [5] https://www.cdc.gov/media/releases/2017/p0718-diabetes-report.html. [6] Tanaka MM, Kendal JR, Laland KN. From traditional medicine to witchcraft: why medical treatments are not always efficacious. PLoS One 2009;4(4):e5192. [7] Collard E, Roy S. Improved function of diabetic wound-site macrophages and accelerated wound closure in response to oral supplementation of a fermented papaya preparation. Antioxid Redox Signal 2010;13(5):599–606. [8] Samad A, et al. Status of herbal medicines in the treatment of diabetes: a review. Curr Diabetes Rev 2009;5(2):102–11. [9] Sen CK, et al. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen 2009;17(6):763–71. [10] Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;366(9498):1736–43.

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[11] Das A, et al. Engulfment of apoptotic cells by macrophages: a role of microRNA-21 in the resolution of wound inflammation. J Immunol 2014;192(3):1120–9. [12] Das A, et al. Correction of MFG-E8 resolves inflammation and promotes cutaneous wound healing in diabetes. J Immunol 2016;196(12):5089–100. [13] Das A, Roy S. Resolution of inflammation. In: Roy S, Bagchi D, Raychaudhuri S, editors. Chronic inflammation: molecular pathophysiology, nutritional and therapeutic interventions. Boca Raton, FL: CRC Press; 2013. p. 119–28. [14] Das A, et al. Monocyte and macrophage plasticity in tissue repair and regeneration. Am J Pathol 2015;185(10):2596–606. [15] Pierce GF. Inflammation in nonhealing diabetic wounds: the space-time continuum does matter. Am J Pathol 2001;159(2):399–403. [16] Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J Clin Investig 2007;117(5):1219–22. [17] Nathan C. Points of control in inflammation. Nature 2002;420(6917):846–52. [18] Ferrero-Miliani L, et al. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol 2007;147(2):227–35. [19] Lawrence T, Fong C. The resolution of inflammation: anti-inflammatory roles for NF-kappaB. Int J Biochem Cell Biol 2010;42(4):519–23. [20] Khanna S, et al. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One 2010;5(3):e9539. [21] Roy S. Resolution of inflammation in wound healing: significance of dead cell clearance. Adv Wound Care 2010;1:253–8. [22] Lawrence T, Gilroy DW. Chronic inflammation: a failure of resolution? Int J Exp Pathol 2007; 88(2):85–94. [23] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2005;6(12):1191–7. [24] Nathan C, Ding A. Nonresolving inflammation. Cell 2010;140(6):871–82. [25] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341(10):738–46. [26] Crovetti G, et al. Platelet gel for healing cutaneous chronic wounds. Transfus Apher Sci 2004; 30(2):145–51. [27] Emanuela F, et al. Inflammation as a link between obesity and metabolic syndrome. J Nutr Metab 2012;2012:476380. [28] Romeo GR, Lee J, Shoelson SE. Metabolic syndrome, insulin resistance, and roles of inflammation— mechanisms and therapeutic targets. Arterioscler Thromb Vasc Biol 2012;32(8):1771–6. [29] Pradhan L, et al. Inflammation and neuropeptides: the connection in diabetic wound healing. Expert Rev Mol Med 2009;11:e2. [30] da Costa Pinto FA, Malucelli BE. Inflammatory infiltrate, VEGF and FGF-2 contents during corneal angiogenesis in STZ-diabetic rats. Angiogenesis 2002;5(1–2):67–74. [31] Chesnoy S, Lee PY, Huang L. Intradermal injection of transforming growth factor-beta1 gene enhances wound healing in genetically diabetic mice. Pharm Res 2003;20(3):345–50. [32] Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA 2005; 293(2):217–28. [33] Molnar JA, Underdown MJ, Clark WA. Nutrition and chronic wounds. Adv Wound Care 2014; 3(11):663–81. [34] Levenson SM, et al. Supplemental vitamin A prevents the acute radiation-induced defect in wound healing. Ann Surg 1984;200(4):494–512. [35] Bouillon R, et al. Skeletal and extraskeletal actions of vitamin D: current evidence and outstanding questions. Endocr Rev 2019;40(4):1109–51. [36] Arnold M, Barbul A. Nutrition and wound healing. Plast Reconstr Surg 2006;117(7 Suppl):42S–58S. [37] Han J, Hughes MA, Cherry GW. Effect of glucose concentration on the growth of normal human dermal fibroblasts in vitro. J Wound Care 2004;13(4):150–3. [38] Das A, et al. May dietary supplementation augment respiratory burst in wound-site inflammatory cells? Antioxid Redox Signal 2018;28(5):401–5.

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[39] Dickerson R, et al. Does oral supplementation of a fermented papaya preparation correct respiratory burst function of innate immune cells in type 2 diabetes mellitus patients? Antioxid Redox Signal 2015;22(4):339–45. [40] Dickerson R, et al. Correction of aberrant NADPH oxidase activity in blood-derived mononuclear cells from type II diabetes mellitus patients by a naturally fermented papaya preparation. Antioxid Redox Signal 2012;17(3):485–91. [41] Brown KL, Phillips TJ. Nutrition and wound healing. Clin Dermatol 2010;28(4):432–9. [42] Giugliano D, Ceriello A, Esposito K. The effects of diet on inflammation: emphasis on the metabolic syndrome. J Am Coll Cardiol 2006;48(4):677–85. [43] Shingel KI, et al. Solid emulsion gel as a vehicle for delivery of polyunsaturated fatty acids: implications for tissue repair, dermal angiogenesis and wound healing. J Tissue Eng Regen Med 2008;2(7):383–93. [44] McDaniel JC, et al. Omega-3 fatty acids effect on wound healing. Wound Repair Regen 2008; 16(3):337–45. [45] Munro HN. Report of a conference on protein and amino acid needs for growth and development. Am J Clin Nutr 1974;27(1):55–8. [46] Hackam DJ, Ford HR. Cellular, biochemical, and clinical aspects of wound healing. Surg Infect 2002; 3(Suppl. 1):S23–35. [47] Kay SP, Moreland JR, Schmitter E. Nutritional status and wound healing in lower extremity amputations. Clin Orthop Relat Res 1987;217:253–6. [48] Eneroth M, et al. Nutritional supplementation for diabetic foot ulcers: the first RCT. J Wound Care 2004;13(6):230–4. [49] Rath M, et al. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front Immunol 2014;5:532. [50] Wischmeyer PE, et al. Glutamine induces heat shock protein and protects against endotoxin shock in the rat. J Appl Physiol (1985) 2001;90(6):2403–10. [51] Wischmeyer PE. Glutamine and heat shock protein expression. Nutrition 2002;18(3):225–8. [52] MacKay D, Miller AL. Nutritional support for wound healing. Altern Med Rev 2003;8(4):359–77. [53] Hunt TK, et al. Effect of vitamin A on reversing the inhibitory effect of cortisone on healing of open wounds in animals and man. Ann Surg 1969;170(4):633–41. [54] Yuan Y, Das SK, Li M. Vitamin D ameliorates impaired wound healing in streptozotocin-induced diabetic mice by suppressing NF-kappaB-mediated inflammatory genes. Biosci Rep 2018;38(2). [55] Pierpaoli E, et al. Vitamin E supplementation in old mice induces antimicrobial activity and improves the efficacy of daptomycin in an animal model of wounds infected with methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2011;66(9):2184–5. [56] Hobson R. Vitamin E and wound healing: an evidence-based review. Int Wound J 2016;13(3):331–5. [57] Xie H, Kang YJ. Role of copper in angiogenesis and its medicinal implications. Curr Med Chem 2009;16(10):1304–14. [58] Sen CK, et al. Copper-induced vascular endothelial growth factor expression and wound healing. Am J Physiol Heart Circ Physiol 2002;282(5):H1821–7. [59] Agren MS, Chvapil M, Franzen L. Enhancement of re-epithelialization with topical zinc oxide in porcine partial-thickness wounds. J Surg Res 1991;50(2):101–5. [60] Agren MS. Studies on zinc in wound healing. Acta Derm Venereol Suppl 1990;154:1–36. [61] Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res 2010;89(3):219–29. [62] Sindrilaru A, et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest 2011;121(3):985–97. [63] Wright JA, Richards T, Srai SK. The role of iron in the skin and cutaneous wound healing. Front Pharmacol 2014;5:156. [64] Chaffee S, et al. Diabetic wound inflammation. In: Bagchi D, Nair S, editors. Nutritional and therapeutic interventions for diabetes and metabolic syndrome. Elsevier; 2018. [65] Bashmakov YK, et al. Resveratrol promotes foot ulcer size reduction in type 2 diabetes patients. ISRN Endocrinol 2014;2014:816307. [66] Lim R, et al. Dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta, foetal membranes and myometrium. Mol Hum Reprod 2013;19(7):451–62.

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[67] Park HY, Kim GY, Choi YH. Naringenin attenuates the release of pro-inflammatory mediators from lipopolysaccharide-stimulated BV2 microglia by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Int J Mol Med 2012;30(1):204–10. [68] Duarte S, et al. Apigenin protects endothelial cells from lipopolysaccharide (LPS)-induced inflammation by decreasing caspase-3 activation and modulating mitochondrial function. Int J Mol Sci 2013; 14(9):17664–79. [69] Anuar NS, et al. Effect of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food Chem Toxicol 2008;46(7):2384–9. [70] Yogiraj V, et al. Carica papaya Linn: an overview. Int J Herb Med 2014;2(5):8. [71] Mikhal’chik EV, et al. Wound-healing effect of papaya-based preparation in experimental thermal trauma. Bull Exp Biol Med 2004;137(6):560–2. [72] Gurung S, Skalko-Basnet N. Wound healing properties of Carica papaya latex: in vivo evaluation in mice burn model. J Ethnopharmacol 2009;121(2):338–41. [73] Figueiredo Azevedo F, et al. Evaluating the effect of 3% papain gel application in cutaneous wound healing in mice. Wounds 2017;29(4):96–101. [74] Nayak SB, Pinto Pereira L, Maharaj D. Wound healing activity of Carica papaya L. in experimentally induced diabetic rats. Indian J Exp Biol 2007;45(8):739–43. [75] Murthy MB, Murthy BK, Bhave S. Comparison of safety and efficacy of papaya dressing with hydrogen peroxide solution on wound bed preparation in patients with wound gape. Indian J Pharm 2012; 44(6):784–7. [76] Nafiu AB, Rahman MT. Selenium added unripe carica papaya pulp extracts enhance wound repair through TGF-beta1 and VEGF-a signalling pathway. BMC Complement Altern Med 2015;15:369. [77] Nafiu AB, Rahman MT. Anti-inflammatory and antioxidant properties of unripe papaya extract in an excision wound model. Pharm Biol 2015;53(5):662–71. [78] https://www.drugs.com/npc/papaya.html. [79] Jayarajan R, Narayanan P, Adenwalla H. Papaya pulp for enzymatic wound debridement in burns. Indian J Burns 2016;24(1):24–8. [80] Maniyar Y, Bhixavatimath P. Antihyperglycemic and hypolipidemic activities of aqueous extract of Carica papaya Linn. leaves in alloxan-induced diabetic rats. J Ayurveda Integr Med 2012;3(2):70–4. [81] Juarez-Rojop IE, et al. Hypoglycemic effect of Carica papaya leaves in streptozotocin-induced diabetic rats. BMC Complement Altern Med 2012;12:236. [82] Raffaelli F, et al. In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes. Nutr Metab Cardiovasc Dis 2015;25(2):224–9.

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CHAPTER 21

Nanotechnology in diabetic wound healing Mark Azeltinea, Andrew Clarkb, Carlos Zgheiba, Subhadip Ghatakb a

Laboratory for Fetal and Regenerative Biology, Department of Surgery, University of Colorado Denver–Anschutz Medical Campus and Children’s Hospital Colorado, Aurora, CO, United States b Department of Surgery, IU Health Comprehensive Wound Center, Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, United States

1. Diabetic wounds—An overview Cuts and scrapes are an inevitable part of life. How those wounds heal, though, can range widely from person to person. Most will begin by a combination of clotting and remodeling proteins closely followed by a concert of inflammatory cytokines. Eventually, that culminates in a scab that gradually reduces in size until full-tissue repair is achieved. What differs, however, lies at the junction of personal genetics, physiology, wound type, and treatment strategies. The National Diabetes Statistics report for 2017 (the most recent report currently available) estimates that there were 30.3 million people living with diabetes in the United States for the year 2015 [1]. New diagnoses for adult patients were estimated to be 1.5 million people in that same year. Despite current interventions and treatments, many patients living with diabetes will experience wounds typically concomitant with common comorbidities such as neuropathy and vascular disease. Peripheral neuropathy and poor circulation are main drivers for the development of ulcers in the diabetic population. A review on the incidence rate of foot ulcers details that between an estimated 3.1%–11.8% of the global diabetic population have had a foot ulcer [2]. The subsequent lack of angiogenesis and possibility of infection then significantly increase and vary the time from the initial wound development to the time of wound closure. Hyperglycemia is the defining factor of diabetes and its effects on wound healing are far reaching, from chronic inflammation, defective angiogenesis to impaired barrier function that often times result in wound recurrence. Skin remodeling involves the removal of damaged, degraded, or otherwise nonpractical tissues to make way for new tissue. Matrix metalloproteases (MMP) are enzymes that catalyze the breakdown of the extracellular matrix (ECM) for the remodeling and are expressed in greater quantities in diabetic patients than in patients without a metabolic disorder [3]. Wound repair requires that the rate of collagen synthesis to exceed the rate of collagen degradation by MMP [4]. Failure to Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00021-6

© 2020 Elsevier Inc. All rights reserved.

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achieve this results in inappropriate synthesis of the ECM that results in chronic wound. High glucose also promotes the formation of reactive oxygen species (ROS) production initially by macrophages but also mitochondria, aortic smooth muscle cells, and endothelial cells, which can promote cardiovascular disease such as atherosclerosis [5, 6]. Increased angiopoietin-2 (ang-2) gene expression in endothelial kidney cells and diabetic mice was induced by a high glucose environment. When tumor necrosis factor-α (TNF-α) is factored into these models, increased levels of intracellular- and extracellular-vascular adhesion molecules (ICAMS; VCAMS) ensued [7]. Inflammation is a natural part of wound healing. Neutrophils and monocytes migrate to the wound and secrete many growth factors and proinflammatory cytokines at the onset of wound healing. Some circulating monocytes will then settle to become tissue-resident macrophages, while the chemokines, cytokines, and growth factors continue to direct cellular migration and function. At an injury site, efficient clearance of apoptotic cells by wound macrophages or efferocytosis is a prerequisite for the timely resolution of inflammation (Fig. 1). Previous work has demonstrated the significance of miR-21 in the regulation of efferocytosis-mediated suppression of innate immune response, a key process implicated in resolving inflammation following injury [8]. Apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. It has been demonstrated that milk fat globule epidermal growth factor-factor 8 (MFG-E8), a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, is critical in scavenging the apoptotic cells from affected tissue [9]. Such controlled processes normally induce cellular changes that promote progression of wound healing and mitigate the chances of infection [10]; however, the pathophysiology of diabetes prevents these changes from happening in a controlled manner. Due in part to the dysfunctional macrophage population and lack of leukocyte chemotaxis, chronic inflammation leads to cellular changes that are counterproductive and even damaging [11]. Chronic expression of proinflammatory cytokines has been shown to reduce vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)β1, while also implicated in increasing the expression of MMP [12]. Complicating matters is the risk of infection. When a microbe infects a diabetic wound, it can quickly lead to severe tissue damage resulting in greatly increased risk of limb amputation. The lack of blood flow to a diabetic wound limits the amount of oxygen, minerals, and nutrients needed by cells in the area of a wound to stimulate the growth of new blood vessels. Microvascular damage occurs when ROS expression is initially increased by macrophages, thereby inducing five main mechanisms by which hyperglycemia induces vascular damage: increased polyol pathway flux; increased hexosamine pathway flux; increased intracellular advanced glycation end-products (AGE) formation; increased expression of AGE receptors (RAGEs); and increased protein kinase C (PKC) activity. Furthermore, a direct correlation between hyperglycemia and a lack of angiogenesis and tissue granulation is a major contributor to the chronicity of wounds in diabetic patients

Fig. 1 Stages of typical wound healing. (https://pubs.acs.org/doi/10.1021/acscentsci.6b00371 © 2017 American Chemical Society. Reprint requests should be directed to ACS.)

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[13]. Although insulin-dependent mechanisms are null, insulin-independent mechanisms of glucose transport into cells remain active. Some metabolites of the nonenzymatic breakdown of glucose, like methylglyoxal, modify p300 to where it can no longer associate with hypoxia-inducible factor-1 (HIF-1) and prevents transcriptional activation by HIF-1 [14]. Consequently, VEGF expression is not induced in this manner, thereby contributing to the lack of angiogenesis. The implications of nanotechnology applications in medicine pose as promising treatment modalities for diabetic foot ulcers. Nanotechnology is described as materials that range from 1 to 1000 nm in size that exhibit exceptional surface-to-size ratios. The uses of nanotechnology include nanoparticles and nanotubes that can be altered in physical and chemical properties such as size, charge, hydrophobicity, or conjugated material [15]. Current strategies to prevent the development of a foot ulcer include frequent visual examinations of the feet to ensure wounds do not go unnoticed due to neuropathy, footwear such as socks and shoes designed to mitigate areas of pressure on the foot when standing, maintaining a healthy diet low in sugar, and frequently monitoring blood glucose concentrations.

2. Applications of nanotechnology in wound healing and management 2.1 Nanosensors One of the current shortfalls in monitoring glucose levels is found in how monitoring of glucose is performed. Discrete time points are selected by the patient to measure blood glucose concentration and insulin is administered as needed as part of the open loop system. The problem therein is that glucose concentrations in the blood fluctuate continuously and discrete sampling misses the data required to treat those fluctuating concentrations most effectively. To solve that problem, biosensors were generated that use glucose oxidase (GOx) to catalyze the reaction of glucose to gluconic acid and H2O2, through which a nearby electrode could measure oxidation of the H2O2 molecule as a means to quantify the concentration of glucose in the blood [16]. Nanotechnology improves upon this concept through a few different strategies. One such strategy is by using nanotubes or nanowires to increase the effectiveness of electrodes [17]. Of the most popular strategies are those that use nanoparticles as electron conductors meant to shuttle electrons between GOx and the electrode. Carbon nanotubes (CNT) are a popular choice in this application due to their exceptional surface area and ability to conduct electrons. They are often used in conjunction with a variety of other nanomaterials such as nanoparticles and biological molecules such as GOx. A major drawback of systems involving immobilized GOx is the biodegradability of the GOx enzyme. Excellent stability can be granted to give longevity to the sensor. For example, stability granted by the chitosan for the GOx film coated on a gold electrode was able to maintain 80% activity after 20 days under 60°C [18]. Nevertheless, such a sensor will inevitably need to

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be replaced. There is also the possibility for such a system to react to the electrochemical changes in blood as a result of pharmaceuticals like acetaminophen or diet. To overcome this, hollow nanoparticles were synthesized from a conductive polymer with GOxcontaining polyaniline nanotube electrode which could operate at an electrochemical potential that is different from the electrochemical potentials that may arise from pharmaceuticals, diet, or metabolism [19]. Nonenzymatic sensors seek to eliminate the drawback of biodegradability from glucose sensors. The most popular choice for such a sensor is one that involves the reduction of the electrode and oxidation of glucose directly of which the efficiency and longevity depend on the material used in development (Fig. 2). Not all sensors that have been studied focus on oxidation-reduction reactions, though. Other sensors seek to identify changes in pH. The catabolism of glucose by GOx decreases pH through the eventual freeing of hydrogen ions. As endogenous GOx breaks down available glucose, the change in pH located immediately near the field effect transistor (FET) can be then be detected and those data can then feedback information about the glucose concentration through that relationship [20]. CNT were also functionalized for use in detecting pH changes by affixing them to a silicon chip together

Fig. 2 The developmental progress of nanosensors for glucose concentrations. (A) First-generation glucose sensors, (B) second-generation sensors, (C) single nanomaterial sensors, and (D) nanocomposite sensors. (https://doi.org/10.1016/j.molmed.2010.08.002 © 2010 Elsevier Ltd.)

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with GOx. Lastly, some sensors function via fluorescence [21]. Sensors that elicit a fluorescent signal in response to glucose do not have electrodes like the other sensors based on electrochemical changes. A major benefit of these sensors is that they do not need to be implanted as deeply into the patient as sensors with electrodes, thereby mitigating the probability that the patient develops an infection as a result of implantation [22].

2.2 Nanoparticles Emergence of nanoparticle-mediated drug-delivery systems has significantly improved the delivery of small-molecule drugs that could dramatically improve the quality of life for those living with diabetes. Being a chronic disease associated with several other comorbidities, nanotechnology-based approaches hold more promise in terms of providing site-specific delivery of drugs with higher bioavailability and reduced dosage regimen unlike conventional dosage forms. The possibility of administering peptide drugs like glucagon-like peptides orally by encapsulation into nanoparticles has been a matter of intense research. Additionally, nanoparticles allow the opportunity of further modifications in encapsulation into microparticles, polyethylene glycol (PEG)-PEGylation-, or functionalization with ligands for active targeting. In addition to delivering drugs that are of direct relevance to treat diabetes, nanoparticles can also be used to treat other comorbid conditions such as nonhealing wounds. Topical application of oligonucleotide-functionalized lipid nanoparticles [23] or cell-targeted lyophilized lipid nanoparticles [24] using FDA-cleared material are promising as topical therapeutic agents in the management of diabetic wounds. The size and shape of nanoparticles allow for high surface area upon which reactions can occur with the surface of the particle and functionalized biomolecules may be attached. Many have antibacterial properties themselves or associated antibacterial molecules that contribute to mitigating infection and hastening wound healing. They can disperse within a 3-D medium (Fig. 3), making them particularly well suited for eliminating the harmful biofilms that certain pathogenic bacteria synthesize as a survival mechanism [25]. A myriad of coatings or functional groups have been used in conjunction with nanoparticles to achieve accelerated wound healing in otherwise chronic wounds. For example, gold nanoparticles were coated with epigallocatechin gallate (EGCG) and a-lipoic acid, thereby combining the antioxidant effects of gold while promoting angiogenesis and reducing the activity of macrophages [26]. Poly(lacticco-glycolic acid) (PLGA) nanoparticles have also been coated with molecules such as VEGF [27], curcumin [28], and antibiotics [29], thereby fantastically demonstrating the versatility of nanoparticles as a delivery vehicle. Nanoparticles have also been used in combination with other nanomaterials, such as nanofibers, to create nanocomposite wound dressings that contain a variety of factors that promote proper wound healing and prevent infection [30–32].

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Fig. 3 SEM image of silver nanoparticles suspended in ethanol and coated onto silicon wafers. (https:// doi.org/10.1016/j.jcis.2014.11.074 © 2015 Elsevier Ltd.)

Depending on the nanoparticle composition, there are several different methods of fabrication. Polymer-based nanoparticles often use self-assembly processes such as desolvation [33–35]. Desolvation dissolves the nanoparticle material in a solvent and then adds an antisolvent (miscible in the solvent but unable to dissolve the nanoparticle material). This antisolvent causes formation and precipitation of the nanoparticles. Another selfassembly method of polymers and lipids is through emulsion-based methods [36–38]. Emulsion causes self-assembly of nanoparticles due to hydrophobic interactions of the nanoparticle material when placed in an aqueous solution. The size of the nanoparticle can be controlled by how much energy is added to the solution, usually homogenization or sonication. Depending on the use of single or double emulsions, the resulting nanoparticle can be solid or a shell [39, 40]. Emulsions are capable of simultaneously loading drugs during the formation of the nanoparticle [41]. Metallic nanoparticles (e.g., gold, silver) can be made through reduction of metallic compounds, such as AgNO3 or HAuCl4 [42, 43]. Metallic nanoparticles can also be made from laser ablation of a metallic sheet [44, 45]. Living cells can also be used to make metallic and lipid nanoparticles [46–48].

2.3 Nanofibers Nanofibers are an attractive technology for wound healing because they can provide both a means for hemostasis and a scaffold upon which cell regeneration may occur [49]. The basic principle for nanofibers is to create wound dressings that mimic the form and function of the ECM. Naturally occurring molecules found in the skin’s ECM are a common choice for nanofibers. Examples of these include collagen, gelatin, fibrin, silk fibroin,

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Fig. 4 A diagram displaying the basics of nanofiber synthesis. (https://doi.org/10.1016/j.biotechadv. 2010.01.004 © 2010 Elsevier Ltd.)

hyaluronic acid, chitosan, cellulose, and dextrans among others [50–53]. Other popular nanomaterials of nonbiological origin, such as polylactic acid, polycaprolactone, polyurethane, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, and PLGA, have also been investigated [54–61]. Benefits inherently related to nanofiber networks include the permeability of the 3-D structure of the applied dressing and their ability to be a local drug-delivery system [62–65]. On top of the hemostatic potential of nanofibers, nanomats, and nanogels, loading the network with growth factors or antiinflammatory and antimicrobial molecules poise the technology to be tailored to treat a variety of wound types and related concomitant ailments. The most common method of producing nanofibers is through electrospinning, which requires a high-voltage supplier, micropump, syringe, and electrode metal collector. A polymer solution is extruded from a syringe under high voltage and forms a Taylor cone (Fig. 4). A jet of the polymer comes out of the Taylor cone and collects on the electrode metal collector [66]. The polymer solution can contain multiple polymers to make coreshell nanofibers or drugs that will be incorporated in the nanofibers [67–69].

2.4 Nanotransfection Nanotransfection or rather tissue nanotransfection (TNT), as it is commonly known, is an electroporation-based technique capable of topical gene and drug cargo delivery or transfection at the nanoscale. Furthermore, TNT is a scaffold-less tissue engineering (TE) technique that can be considered cell-only or tissue inducing depending on cellular- or tissue-level applications. The transfection method makes use of custom-fabricated

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nanochannel arrays to deliver cargo to tissues topically. This technology was fabricated using cleanroom photolithography techniques and deep reactive ion etching (DRIE) of silicon wafers to create nanochannels with backside etching of a reservoir for loading desired therapeutics. This postage-stamp-sized chip is then connected to an electrical source capable of delivering an electrical field to drive the factors from the reservoir into the nanochannels, and onto the contacted tissue within few milliseconds. Delivering nucleic acids to cells to direct transcriptional and translational changes is feasible with the help of nanotechnology. Short interfering RNA (siRNA) and microRNAs (miRNA) are small noncoding RNA molecules between 20 and 25 base pairs in length that regulate transcription and translation through binding to their complementary base pairs on mRNA molecules. Specifically, siRNAs contain structural motifs like hairpin loops or pseudoknots that physically block translation. Alternatively, miRNAs bind to complementary base pairs in the 30 untranslated region of an mRNA molecule. Ultimately, both of these molecules mark target mRNA molecules for degradation while preventing translation to the corresponding protein. The list of siRNAs and miRNAs with potential clinical applications continues to grow well into the thousands. While simply injecting siRNA or miRNA will induce transcriptional and translational changes, they are also rapidly degraded. Cells of the innate immune system use toll-like receptor (TLR)3, TLR7/8, and TLR9 to identify viral infections by double-stranded RNA, single-stranded RNA and DNA, respectively, in order to detect possible viral infections. The extracellular milieu is not a safe place for unprotected RNAs as many will be degraded. Nanoparticles can help deliver these RNA molecules to targeted cells such that the efficacy of the treatment is increased.

3. Strategies of nanotechnology applications 3.1 Hemostasis Hemostasis is the body’s first step in wound healing once wounding has occurred. Matrix proteins will aggregate at the wound site to begin forming the scaffold to build upon for wound closure, while the ensuing growth factors instigate the cascade of events that comprise the early steps of wound healing. Current technology used in hemostatic efforts and subsequent wound dressings include gauze and tulle, though these materials are not optimal since they absorb blood and exudate creating the need for frequent changing (which may be painful and damaging upon removal) and prevent the use and effects of the molecules within that exudate. A range of hemostatic nanotechnology continues to be developed with interest by combat forces for the purposes of hemostasis on the battlefield. Nonetheless, those applications can trickle down to serve civilian populations with excellent results. To date, a wide variety of materials have been used to synthesize the fibers, mats, sponges, sheets, foams, gels, and nanoparticles used to achieve rapid and effective

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hemostasis [52, 70–74]. A major benefit of these topical applications is their ability to be used as a primary hemostatic agent and secondary vehicle carrier for antibiotics/antimicrobials, antiinflammatories, and cell regenerative compounds allowing for continued use as a wound dressing.

3.2 Cell proliferation and skin remodeling Certain cell types are positively productive in the process of wound healing when they behave according to their respective biological functions. As already mentioned, the inability to form an ECM is a major barrier to wound healing in diabetic wounds. Therefore, a bioactive and biocompatible artificial ECM provides a means to overcome this problem. The ideal wound environment is one which has the appropriate amount of moisture and is free of environmental contaminants and pathogens. Similarly, the properties of the ideal wound dressing include acting as a barrier to maintain the optimal amount of moisture around the wound; prevent environmental contaminants from entering the wound; conform well to the wound area and are comfortable to wear; protect the wound from further trauma; are nontoxic, nonallergic, and nonimmunogenic; allow for the bidirectional transmission of gasses while only allowing liquid exudate to leave but preventing external liquids in the environment from entering; require minimal changes; have a long shelf-life; and are affordable [75]. Some of the most promising results come from nanofibers, specifically those manufactured through electrospinning [75, 76]. There are two main types of wound dressings with nanotechnological properties: biological and synthetic. Biological proteins, such as collagen, gelatin, and fibrin, have all been used as the core material for nanofibers; however, rapid degradation of these proteins can occur. To prevent this, crosslinking of fibers increases their in vivo stability. Other biologically derived materials include polysaccharide polymers. Chitosan, an alkaline polysaccharide polymer, has been extensively researched in medical nanotechnology (Fig. 5). Nanosilk derived from the electrospinning of silk fibroin isolated from the cocoons of Bombyx mori silkworms improves biomechanical properties of the skin. It accomplishes this by acting as a durable yet breathable barrier to protect the wound and promote healing [77]. Properties of this nanosilk include exceptional biocompatibility, permeability, and the ability to strengthen skin well beyond its normal limit. With just a 7% solution of the nanosilk, human diabetic skin samples were able to withstand an additional 23% of applied load in tensile strength testing resulting in 16% more distance in millimeters before the skin succumb to the load and broke apart when compared to a PBS control treatment.

Nanotechnology in diabetic wound healing

Fig. 5 Scanning electron micrograph of electrospun nanofibers produced using three different manufacturing techniques. (A), (C) and (E) represent the corresponding cross-sections of (B), (D) and (F), respectively. (https://doi.org/10.1016/j.nano.2014.11.007 © 2014 Elsevier Ltd.)

3.3 Antiinflammatories The inflammatory process is far too dysregulated in a diabetic wound to ignore, but correcting it is a challenge that has yet to be well explored. As already discussed, macrophages are a major producer of cytokines such as IL-1β and TNF-α in chronic wounds. Those cells are being targeted in efforts to reduce the amount of inflammation present to promote wound healing. Turmeric comes from a type of ginger (Curcuma longa), which has for millennia had its roots ground into a powder used as a spice for food that is well known for its antiinflammatory properties. Curcumin, a lipophilic molecule, is responsible for this action. Lipophilic molecules, however, struggle with pharmacokinetics as they can be absorbed into multiple tissue compartments, thereby removing them from systemic circulation and reducing the systemic concentration to an unappreciable level. As such, studies on the oral administration of curcumin noted that large daily intakes

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of curcumin (3.6 g) correlated to a decrease in preinflammatory markers [78]. Liposomal delivery systems loaded with antiinflammatory molecules such as curcumin have been developed as a vehicle aimed at delivering their antiinflammatory cargo to macrophages [79]. Curcumin has the added bonus of scavenging ROS and suppressing NF-κB, which makes it well suited for study in the treatment of chronic wounds associated with diabetes. The results from a study regarding the use of topical delivery of liposomal curcumin to treat vaginal inflammation provides confidence to its possible effectiveness for the treatment of skin wounds [80]. Antiinflammatory molecules can also be conjugated to nanoparticles. One such case was explored using PLGA nanoparticles for the oral administration of rolipram, an antiinflammatory drug with many associated adverse effects due in part to the high concentrations clinically administered to combat inflammatory bowel disease [81]. When rolipram was conjugated to the PLGA nanoparticles or delivered as an oral solution of the drug only (not conjugated to the nanoparticle) for 5 days, the clinical activity scores improved following 24–48 of treatment. Drug treatment was stopped at day 10 for both the rolipram solution and rolipram-PLGA nanoparticle and clinical activity scores for the solution group decreased back toward pretreatment levels. Many studies to date have examined the effectiveness at using nanotransfection to downregulate TNF-α production. One such study utilized a chitosan-based nanoparticle with an TNF-α siRNA conjugated to a chitosan-based nanoparticle for mitigating the inflammation experienced by arthritis patients [82]. Yet another study took advantage of the lower pH present in areas of chronic inflammation to determine if it could be used to release TNF-α siRNA from the nanoparticle at the site of chronic inflammation [61]. To do so, researchers prepared a PLGA nanoparticle by coating them in stearoylhydrazone-polyethylene glycol 2000 (PHC), an acid-sensitive emulsifying reagent, and the TNF-α siRNA. They found that their molecule targeted an LPS-induced inflamed murine foot following intravenous injection. Furthermore, macrophages pretreated with the acid-sensitive nanoparticle-siRNA complex was quickly endocytosed and TNF-α expression remained at concentrations similar to control cells treated with PBS only in vitro. Cerium oxide nanoparticles (CNP) function with the intrinsic property of scavenging ROS and can be conjugated to miRNAs aimed at correcting the wound healing process [83]. Zgheib et al. conjugated miRNA-146a to CNP (CNP-miR146a) and delivered them intradermally to wounds on diabetic mice and pigs to observe how it affects the woundhealing process and how it functioned in reducing inflammation in other inflammatory diseases. They showed that CNP-miR146a improved diabetic wound healing in both animal models. There was a significant decrease in hematopoietic cells, identified by the presence of CD45, in the vicinity of the wound following treatment. The data give confidence that CNP-miR146a decreases the immune response on the cellular level, subsequently reducing the inflammation present in the milieu of the wound area (Fig. 6).

Nanotechnology in diabetic wound healing

Fig. 6 Accelerated wound healing with varying concentrations of miRNA-146a conjugated to cerumide nanoparticles in diabetic murine wounds. (https://doi.org/10.1016/j.jamcollsurg.2018.09.017 © 2018 American College of Surgeons. Published by Elsevier, Inc.)

3.4 Antimicrobial activity A worry with all wounds is the possibility of developing an infection. Many opportunistic pathogens will readily replicate and cause further damage to the tissue surrounding a wound. It is therefore necessary to prevent infection or, in the case that a microbe has already infected the wound, to decrease the time of infection to the shortest feasible duration. Metal nanoparticles are one method of controlling the risk or growth of microbial infection in the wound. The exact mechanism by which this works varies according to the specific metal used. Some materials have antibacterial properties while other particles function by physically damaging membranes or by using conjugated molecules to the surface of the nanoparticle. Silver has been used as an antibiotic in the form of silver nitrate for decades due to its antibacterial effectiveness since it was commercialized as silver sulfadiazine cream for the prevention of infections in recovering burn patients. Nanoparticle preparations with silver have demonstrated the expected antibacterial properties are retained and are bacteristatic against Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa. Bactericidal effects were also observed with silver nanoparticles with the proposed mechanism of action of silver ions being released from the surface of the silver nanoparticle or interactions of proteins and DNA with intracellular nanoparticles.

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Copper also contains the intrinsic antibacterial properties that silver exhibits, though possibly requiring a smaller comparative dose against both E. coli and Bacillus subtilus. Zinc oxide (ZnO) has been shown to be effective at treating infections caused by gram-positive and gram-negative bacteria [84, 85]. Susceptible bacteria include grampositive species like B. subtilis and Staphylococcus aureus and gram-negative bacteria such as E. coli and Pseudomonas fluorescens. It is important to note, however, that some studies have demonstrated that the effectiveness of ZnO nanoparticles may be photodependent to function utilizing varying degrees of ultraviolet light for the treatment of water [86]. Aluminum oxide nanoparticles have also been investigated for antibiotic properties and were effective at reducing the bacterial populations of B. subtilis, E. coli, and P. fluorescens. Nonmetal molecules have also been used widely as antimicrobials. Nitric oxide (NO) has been previously documented as effective in treating bacterial infections. Silicon dioxide nanoparticles have minimal data currently available on them. Of what has been studied, SiO2 nanoparticles appeared to be bactericidal against B. subtilis, E. coli, and P. fluorescens cells in suspension. Chitosan also has displayed some antibacterial activity, although that property is driven further by incorporating antibacterial molecules such as ZnO to create a nanocomposite wound dressing [57]. Silk-fibroin-based nanosilk, a tissue scaffold, can also be combined with antibacterial molecules to enhance its characteristics to perform multiple functions with promising results [87] (Fig. 7).

4. Concerns regarding nanotechnology in medicine Anytime a novel product becomes available, people start to wonder whether there are any contraindications for its use. Many exist within the realm of nanotechnology regarding the safety of such products. Previously, the ideal properties for wound dressings were described. Some of those properties are shared among all materials aimed at being physically applied to areas of trauma with the goal of stimulating wound healing. Biomaterials may carry the risk of being allergenic/immunogenic, cytotoxic, and environmentally damaging. Each of these risks is well studied prior to application, especially in the case of products intended for use on the human body. Cytotoxicity from the use of nanomaterials is of interest to many research groups. As such, it is explored alongside the proposed beneficial effects of the nanomaterials. Since nanoparticle research has focused heavily on metals (i.e., silver and gold), it is of particular concern about how those metals may degrade within the body and how those particles may distribute before they are excreted [88]. The clearance of nanoparticles from the body occurs primarily through filtration by the liver but some very small particles (90%) after 24-h incubation [192]. Multiple lamellipodia and cell-cell interactions are shown, demonstrating that the developed films can potentially be used as a flexible platform for wound dressings (Fig. 8).

4. Conclusion and future directions Any injury in tissue will affect the local environment and will cause systemic effects as well. The host’s response to wounds involves various processes of tissue healing that are triggered by tissue injury and encompasses four continuous phases, including coagulation and hemostasis, inflammation, proliferation, and wound remodeling with scar tissue deposition. Proper wound management effectively can influence healing time and also reduce potential complications. In this chapter, we have reviewed different nanotechnology-based drugdelivery systems in the area of skin wound healing. We explain the pros and cons for each nanocarrier, which has been used in wound healing recently. It is important to note that any outcome of these delivery systems and devices depends on the nanocarrier formulation, doses and methods of application, which has also been explained. Beneficial effects of using hybrid/ various nanocarriers for wound-healing applications have been reported; however, the molecular mechanisms/signaling pathway of such nanomaterials are not clearly understood yet. A better understanding of the signaling pathway will elucidate the actions of nanocarriers on the wounds and will help to establish novel promising nanotechnology-based woundhealing therapy. Understanding the cellular response and analysis of cell signaling pathways involved in wound healing with varying physicochemical features of nanomaterials may open up new routes for novel nanotherapeutics. Because nanomaterials are highly active at the cellular level due to their size compared to its bulk, the toxicity of nanocarriers must be taken into consideration in every case before its usage in wound care products. Traditional wound therapy approaches (i.e., traditional dressings) are intended to provide wound cover, bleeding arrest, fluid adsorption, moistening or/and drying, and in some cases infection protection and dead tissue removal. However, nanotechnologybased approaches due to their unique properties open a new array of wound-healing products. Nanomaterials can modify each phase of wound healing as they possess antibacterial, antiinflammatory, and antioxidant activities, along with proangiogenic and proliferative properties that usually are given to them by loaded drugs. At the same time,

Drug-delivery nanocarriers for skin wound-healing applications

Fig. 8 Schematic of the formation of hydrogel nanotubes (HNTs) via self-rolling of polymer nanosheets at 40°C with ice helices entrapped in their hollow channels and their application in diabetic wound healing [193] (A); illustration of the synthesis of antibacterial ssDNA-AgNPs@GO and their application to wound healing [194] (B).

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single or multiple therapeutic agents can be delivered to their desired target sights through these nanomaterials. At cellular level, nanomaterials are able to correct the expression level of some crucial proteins and signal molecules to enhance wound healing as it has been explained in some of the examples in this chapter. Therefore, nanocarriers may become sophisticated enough in the near future to overcome most of the challenges regarding wound healing. Distinguishing the functions of conventional and novel biomaterials/nanomaterials in wound-healing therapy may lead to successes in managing complicated wounds, such as infected, chronic, and ischemic ulcers by a combination of nanocarriers hybrid/heterogeneous nanomaterials. Development of novel biocompatible and biodegradable nanocarriers, which are able to correct all phases of wound healing, can be a future goal for researchers working in this area.

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CHAPTER 23

Volatile organic compounds: Potential biomarkers for improved diagnosis and monitoring of diabetic wounds Ali Daneshkhaha, Amanda P. Siegela,b, Mangilal Agarwala,c a

Integrated Nanosystems Development Institute (INDI), Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States b Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States c Department of Mechanical and Energy Engineering, Indiana University–Purdue University Indianapolis (IUPUI), Indianapolis, IN, United States

1. Introduction Through history, scents have been used by humans for the detection of diseases, including complications associated with wounds. Identification of gangrene by smelling wounds and diabetes ketoacidosis by smelling the breath has been widely practiced for generations [1, 2]. Animals demonstrate a superior ability over humans in detecting disease through olfaction. Recently, researchers and scientists have identified a wide variety of diseases using an animal’s sense of smell [3–6]. For example, trained African giant rats (Cricetomys gambianus) can identify Mycobacterium tuberculosis in mucus from the lungs [7, 8]. Trained canines have successfully detected lung cancer [3], and breast cancer [9] from human breath, and prostate cancer [10, 11] from human urine headspace with sensitivity and specificity superior to current diagnostic tools. Furthermore, canines can detect transient health conditions such as hypoglycemia [12] and have shown promising results in the detection of melanoma [13]. Canines can detect a cell-cultured virus [14] with high sensitivity and specificity. Studies have shown that canines can detect the microbial activities and infections from bacteria such as Clostridium difficile [15] with sensitivity and specificity of 83% and 98%. Studies from another group have further validated the ability of canines in detection of toxin gene-positive Clostridium difficile [16]. These studies show the importance of VOCs and how animals can be used as a “point-of-care” diagnostic tool for the detection of different diseases through smell generally. More specifically, smells have great potential for identifying and analyzing wounds, and thus, streamlining wound healing. In fact, malodorous wounds are such a common problem in wound care, clinicians have developed an “Odour assessment scoring tool,” [17] which scores wounds from no odor to strong odor (odor is evident when entering Wound Healing, Tissue Repair, and Regeneration in Diabetes https://doi.org/10.1016/B978-0-12-816413-6.00023-X

© 2020 Elsevier Inc. All rights reserved.

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the room with the dressing intact). As research in other diseases demonstrates, however, even wounds that do not smell strongly enough for scoring on the odor assessment tool contain VOCs that can be analyzed and quantified for disease monitoring. Poor blood circulation [18, 19], plasma glucose fluctuation, and changes in metabolic pathways in patients with DM impact the wound healing and also the smells produced by the wound. Patients with DM are at higher risk of wound-related complications [20]. It appears that healing of diabetic wounds are slower than in nondiabetic wounds [21] and diabetic patients are at risk of kidney injury during their diabetic foot osteomyelitis treatment [22]. This requires further attention and research concerning the identification of VOCs associated with wounds in patients with DM. This chapter will start by describing the different origins of VOCs in wounds, continuing with specific biosamples from wounds to analyze for VOCs and quantitative methods for analyzing such samples. Next, specific VOCs that may be useful for wound monitoring are discussed in connection with different microbial sources, including research on whether biofilms display different VOC profiles than bacteria that have not yet formed biofilms. Additionally, the potential role of VOCs for prediction and monitoring of foot wounds in patients with diabetes is discussed. Finally, the chapter will conclude with progress toward the use of nanosensors and eNoses as a noninvasive mobile real-time woundmonitoring machine.

2. How wounds produce VOCs Microorganism activity has been shown to produce VOCs that can be identified through the analysis of cultured bacterial colonies. The VOCs produced by bacterial microorganisms and their impact on different organs can lead to the formation of a unique VOC profile or VOC “fingerprinting.” As mentioned earlier, animals such as canines and rats can identify a unique VOC signature (panel of VOCs) linked to the disease. The main sources for the production of VOCs associated with a wound are (1) infection, (2) slough (dead tissue) and exudate [23]. Devitalized tissue is a suitable place for growth and formation of colonies of bacteria leading to infection [23]. The odor produced by the infection can be analyzed to evaluate the infection and may be caused by gram-positive, gram-negative, polymicrobial, aerobic, or anaerobic bacteria. Generally speaking, certain microorganisms have such distinct odors and are often present in such high concentrations that they can be recognized by health care professionals trained in wound healing. Another source of wound-related VOCs is the necrotic tissue itself. Capillary occlusion caused by arterial ulcers, pressure ulcers, and diabetic foot ulcers leads to the formation of dead tissue, which produces certain VOCs [23]. Accumulation of fluids such as fibrin and white blood cells leaking, and liquefied dead tissue (by bacteria activity) lead to the formation of exudate [23, 24].

Volatile organic compounds: Potential biomarkers for improved diagnosis and monitoring of diabetic wounds

Table 1 Gram stain and dependency on oxygen for the microorganism linked to a diabetic wound. Bacteria

Gram +/2

Aerobic/ anaerobic

Reported as present in diabetic wounds

S. aureus (methicillin resistant) S. epidermidis E. faecalis S. pyogenes E. coli

Positive

Aerobic

Reveles et al. [25]

Positive Positive Positive Negative

Arago´n-Sa´nchez et al. [26] Shettigar et al. [27] Shanmugam et al. [28] Anisha et al. [29]

P. aeruginosa

Negative

Aerobic Anaerobic Anaerobic Aerobic/ anaerobic Aerobic/ anaerobic

Shankar et al. [30]

In addition, wounds cause changes in metabolic requirements that may also contribute endogenous VOCs that may be diagnostic. It is hypothesized that VOCs produced by the bacteria can be used for monitoring and evaluating wound infection status, including diabetic foot wound. Bacteria such as Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), E. coli, Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), and Streptococcus pyogenes (S. pyogenes) are found in diabetic wound infections and their properties are shown in Table 1.

3. Role of VOCs in diabetes wound formation Damage to the blood vessels supplying oxygen and nutrients to the nerves can lead to foot wounds in people with DM. Low blood flow and high glucose plasma levels impair the blood vessels elasticity and make diabetic foot ulcers prevalent among patients with DM; in fact, approximately 15% of the patients with DM experience a wound on the bottom of their foot. Unfortunately, 3.1%–11.8% of diabetic foot wounds lead to foot ulcers and more than half of the foot ulcers form an infection [31]. The foot wound infection causes swelling, pain, thickened skin (near the wound), fever (in advanced stages), and foul smell [32, 33]. Limitations due to processing time for microbial analysis of samples, collected by superficial surface-swabbing technique or invasive deep-tissue biopsies [34–36], have led researchers to investigate techniques for rapid analysis of wound-related VOCs [32]. Diabetic foot infections can be caused by aerobic gram-positive bacteria such as S. aureus (smells like decomposition), and their genus Staphylococcie (with a “fruity” smell) or due to aerobic gram-negative bacteria such as P. aeruginosa (with “grape juice” smell) and E. coli (with a foul smell) [32, 37]. VOCs can be produced by bacteria, metabolic changes and decaying tissue may provide information for analyzing the status of the wound.

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4. Quantitative collection and processing of VOCs from wounds While previously it has been the practice to analyze wound dressings, and old wound discharge or exudate for diagnosing bacterial infections associated with wounds, these samples are a better description of what state a wound was in previously, and not the current status of a wound. The best practice is to clean and debride a wound thoroughly prior to collecting samples [34]. Once the wound has been cleaned, theoretically, the air surrounding the wound will contain significant VOCs for disease detection. While this may suffice for canines, other animals, or experimental sensors in development, for current analytical techniques a better practice is to either collect discharge/exudate into a syringe or onto a swab or dressing [33], or if the wound is not oozing, a sterilized polydimethylsilicone membrane can also efficiently collect volatile compounds (“skin-patch method”) [38]. These materials can be stored in headspace vials for analysis. In this case, the collection materials should be precleaned, which involves heating the pad or blotting paper to over 40°C for 48 h and storing the materials away from volatile chemicals (authors’ unpublished results). The heating drives out VOCs that may have been trapped on the pad or blotting paper during packaging.

5. Analytical and statistical methods for analyzing VOCs Advances in the field of electrical engineering and electronics, nanotechnology, and data science have led scientists and researchers to develop analytical techniques to identify and quantify VOC biomarkers without prior knowledge of their existence in the biosamples. Analytical methods such as GC-MS (gas chromatography-mass spectroscopy analysis), GC-ToF-MS (ToF, time of flight), SIFT-MS (selected ion flow tube-mass spectrometry), IMR-MS, SESI-MS (secondary electrospray ionization-mass spectrometry), PTR-MS (proton transfer reaction-mass spectroscopy), IMS (ion mobility spectroscopy), GC-IMS, FID, GC-FID, eNose, GC-eNose. These methods have presented a high sensitivity for identifying the target VOCs used for identifying the infection [39–43]. Analytical tools such as GC-eNose and SIFT-eNose may show high sensitivity and selectivity for detecting diseases; however, they are not suitable for identification of biomarkers, as the eNose’s nonlinear response does not lend itself to deconvolution of the panel of VOCs supported by a standard library. Direct detection techniques such as IMRMS, SESI-MS, and SIFT-MS present fast, analytical methods for the detection of VOCs. However, these instruments are less sensitive and are more suitable for targeted analysis where the researcher is identifying a known compound [39]. GC-IMS presents a simple, fast, and sensitive analytical method that can be utilized and used at the site [39]. This method has a limited quantitation dynamic range and is less suitable for the detection of unknown analytes than GC-MS. GC-MS and GC-QTOF-MS have been considered as the gold standard of analytical methods for screening of unknown compounds and

Volatile organic compounds: Potential biomarkers for improved diagnosis and monitoring of diabetic wounds

identification of potential biomarkers. The analytes are separated due to their boiling point, polarity, and their affinity to the column, while different MS detectors identify and quantify the VOCs. Powerful and reliable reference libraries and databases are available for tentatively identifying VOCs for the instrument, which makes it more popular among researchers. Over 3500 VOCs have been reported to be observed by different researchers [44]. Different statistical methods and techniques have been utilized for identification of biomarkers linked to a targeting disease. Univariate statistical analysis has been used to identify potential biomarkers. The following have been used to identify VOC biomarkers: direct correlation with disease [45], the distance between the mean of two classes [46], student t-test [47], Wilcoxon rank-sum [48], AUC of ROC curve [49], Kruskal-Wallis tests [41], analysis of variance (ANOVA) [50], and Monte Carlo simulations [51]. Volcano plot and clustergram are also widely used to identify the potential biomarkers and show their discriminatory powers. Accumulating evidence shows that a disease can be identified with higher sensitivity and specificity by using a VOC biomarker profile (panel of VOCs) rather than a single biomarker [41]. Different pattern recognition tools and classification algorithms (supervised or unsupervised) such as neural network [52], genetic algorithm [43], linear regression [53], linear discriminant analysis (LDA) [54], support vector machines (SVM) [55], principal component analysis (PCA) [54], partial least squares discriminant analysis (PLSDA), discriminant function analysis (DFA) [52] can be used to identify the panel of VOCs linked to the targeting disease [54, 56, 57].

6. Bacterial VOCs 6.1 In vitro Initially, researchers looked for VOCs produced by bacteria in culture media. In vitro analysis of VOCs with the help of culture media enables researchers to produce a high concentration of VOCs linked to the bacteria. This allows them to target only the VOCs produced by the bacteria and isolate them from the VOCs produced by other metabolic processes or changes in tissues. This could lead to targeting a more selective and specific set of biomarkers as the VOCs produced by the changes in metabolic pathways or tissue decaying can be observed in other situations. It should be noted that different researchers may report several similar VOCs but also differing VOC profiles produced by the same bacteria [58]. This inconsistency can be due to the effect of culture medium, SPME fiber material, and GC column on the VOC profile identified by each researcher [59]. In the following, the VOCs produced and identified due to the activity of each wound-related bacteria are presented and discussed. Filipiak et al. [60] investigated the VOCs produced or elevated by S. aureus and P. aeruginosa and determined the character of the VOCs for each bacterium. They identified that aldehydes

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and acids could distinguish the two bacteria from one another selectively. Different concentration of aldehydes (3-methylbutanal, 2-methylpropanal, acetaldehyde, and (Z)-2-methyl-2-butenal) and acids (acetic acid, and isovaleric acid) were released by S. aureus, while P. aeruginosa did not produce any aldehyde or acid [60]. Lawal et al. [61] cultured P. aeruginosa in artificial sputum medium for 24 h and proposed consumption of aldehydes such as benzaldehyde, 2-methylbutanal, furfural, hexanal, 2-ethylhexenal, 2-methylpropanal, 2,2-dimethylpropanal, 2-ethyl-trans-2-butenal by the activity of the bacteria. This means that the increase in the concentration of aldehydes could be linked to the process of wound healing. Alcohols such as ethanol, 2-butanol, 3-methyl-1-butanol were produced with different concentrations in both bacteria. S. aureus, released ketones such as acetoin (hydroxybutanone), 2,3butanedione, and acetol (hydroxacetone), while P. aeruginosa released entirely different ketones such as 2-butanone, methyl isobutyl ketone, 2-heptanone, 2-pentanone, 4-heptanone, 3-octanone, and 2-nonanone [60]. Medium-chain hydrocarbons (C9– C12) such as 1-undecane were an imperative VOC for detection of P. aeruginosa, while S. aureus released mostly short-chain hydrocarbons (C3–C4) such as 2-methylpropene and E-2-butene [60]. Hydrogen cyanide (HCN) is another important compound that has been reported to be produced by P. aeruginosa by different groups and can be considered as a biomarker of the bacteria [43, 62, 63]. Earlier studies have also identified an aromatic ketone, 2-aminoacetophenone, as an important biomarker of P. aeruginosa [64, 65]. Filipiak et al. [60] investigated the VOCs produced by P. aeruginosa and S. aureus and suggested the esters (except methyl methacrylate) are found in low concentrations and at a late time point. More recently, a study by Siripatrawan [66]) investigated the VOCs produced by E. coli and found that ethanol, 2-nonanone, 2-heptanone, dimethyl disulfide, pentyl-cyclopropane, and indole are produced with the bacteria. Ethanol and indole were also richly observed by another research group who used MS-MS analysis for the identification of VOCs linked to E. coli [67]. In another study Thorn et al. [68] also reported that E. coli produces ethanol, indole, and dimethyl disulfide. Their study showed that E. coli can further produce 1-butanol, 1-pentanol, acetone, acetic acid, butanoic acid, phenylacetic acid, indole, acetoin, ethyl acetate, formaldehyde, hydrogen sulfide, isoprene, methyl mercaptan, and trimethylamine. Thorn et al. further identified that E. faecalis produces 1-butanol, ethanol, formaldehyde, hydrogen sulfide, methyl mercaptan at a higher concentration (>100 ppb) while producing 1-pentanol, ethyl butanoate, phenylacetic acid, trimethylamine, ethyl butanoate, pyrrole, and 2-aminoacetophenone at a lower concentration (