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TherapeuticDressings

andWoundHealingApplications

ADVANCESINPHARMACEUTICALTECHNOLOGY

AWileyBookSeries

SeriesEditors:

DennisDouroumis, UniversityofGreenwich,UK

AlfredFahr, Friedrich–SchillerUniversityofJena,Germany

JürgenSiepmann, UniversityofLille,France

MartinSnowden, UniversityofGreenwich,UK

VladimirTorchilin, NortheasternUniversity,USA

TitlesintheSeries

Hot-MeltExtrusion:PharmaceuticalApplications

EditedbyDionysiosDouroumis

DrugDeliveryStrategiesforPoorlyWater-SolubleDrugs

EditedbyDionysiosDouroumisandAlfredFahr

ComputationalPharmaceutics:ApplicationofMolecularModelinginDrugDelivery

EditedbyDefangOuyangandSeanC.Smith

PulmonaryDrugDelivery:AdvancesandChallenges

EditedbyAliNokhodchiandGaryP.Martin

NovelDeliverySystemsforTransdermalandIntradermalDrugDelivery

EditedbyRyanDonnellyandRajSingh

DrugDeliverySystemsforTuberculosisPreventionandTreatment

EditedbyAnthonyJ.Hickey

ContinuousManufacturingofPharmaceuticals

EditedbyPeterKleinebudde,JohannesKhinast,andJukkaRantanen

PharmaceuticalQualitybyDesign

EditedbyWalkiriaSSchlindweinandMarkGibson

InVitro DrugReleaseTestingofSpecialDosageForms

EditedbyNikolettaFotakiandSandraKlein

ForthcomingTitles:

CharacterizationofMicro-andNanosystems

EditedbyLeenaPeltonen

ProcessAnalyticsforPharmaceuticals

EditedbyJukkaRantanen,ClareStrachan,andThomasDeBeer

MucosalDrugDelivery

EditedbyReneHolm

BasicBiopharmaceutics

EditedbyHannahBatchelor

TherapeuticDressings andWoundHealing Applications

Editedby

JOSHUABOATENG

SchoolofScience,UniversityofGreenwichMedway, ChathamMaritime,UK

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LibraryofCongressCataloging-in-PublicationDataappliedfor

HardbackISBN:9781119433262

Coverdesign:Wiley

Coverimages:©KaterynaKon/Shutterstock, ©Designua/Shutterstock,©Umpaporn/Shutterstock, ©molekuul_be/Shutterstock

Setin10/12pt,TimesLTStdbySPiGlobal,Chennai,India

10987654321

ListofContributors xiii

SeriesPreface xvii

Preface xix

1ChronicWoundHealing:MolecularandBiochemicalBasis1 SophiaTateandKeithHarding

1.1Introduction1

1.2AcuteWoundHealing1

1.3CategoriesofChronicWound3

1.3.1PressureUlcers3

1.3.2VenousStasisUlcers4

1.3.3IschaemicUlcers4

1.3.4DiabeticFootUlcers4

1.4HowaChronicWoundDevelops:IntrinsicComponents4

1.4.1CellPhenotype5

1.4.2ImmuneCellsandInflammatoryMediators6

1.4.3ReactiveOxygenSpecies8

1.4.4GrowthFactors8

1.4.5TheRoleofMatrixMetalloproteinases12

1.5HowaChronicWoundDevelops:ExtrinsicFactors13

1.5.1Infection13

1.5.2Nutrition13

1.5.3TobaccoSmoking14

1.5.4HypoxiaandIschaemia–ReperfusionInjury15

1.6ConcludingRemarks15 References16

2ClinicalPerspectivesforTreatingChronicWounds21 BarunMajumder,KirstieLane,DianeBeck,SandeepSinghandDuniya Majumder

2.1Background21

2.2AetiologyofDiabeticFootUlcers22

2.3StandardofCareforTreatmentofDiabeticFootUlcers22

2.4CommonlyUsedWoundDressingsforDiabeticFootUlcersandTheir MechanismofAction22

2.5AbsorbentandSuperabsorbentDressings23

2.6Alginates23

2.7Films23

2.8Foams24

2.9Honeys24

2.10Hydrogels25

2.11TheRoleofaSplitThicknessSkinGraftinDiabeticFootUlcers25

2.12NegativePressureWoundTherapy25

2.13LarvalTherapy27

2.14ClinicalCaseStudiesfromMultidisciplinaryDiabeticFootClinic27

2.14.1NeuropathicWound27

2.14.2IschaemicWound29

2.14.3Neuro-IschaemicWound31

2.14.4Osteomyelitis33

2.14.5Charcot’sFoot35

2.14.6NecrotisingFasciitisinaPatientwithDiabetes36

2.15Summary39 Acknowledgements39 References39

3Prediction,Prevention,Assessment,andManagementofSkinTearsin theAgingPopulation43 KimberlyLeBlancandKarenCampbell

3.1Introduction43

3.2SkinTearPrevalenceandIncidence44

3.3PredictingSkinTears45

3.4Prevention47

3.5ISTAPRiskReductionProgram49

3.5.1GeneralHealth49

3.5.2Mobility50

3.5.3Skin51

3.6Assessment52

3.7Management54

3.8Treatment54

3.9Conclusion55 References55

4ImportanceofDebridingandWoundCleansingAgentsinWound Healing59

GwendolynCazander,BiancaK.denOttelander,SandraKamga, MartijnC.H.A.Doomen,TimH.C.DamenandAnneMarieE.vanWell

4.1WhatisDebridement?59

4.2TheHistoryofDebridement59

4.3WhyUndertakeDebridement?60

4.4DebridementTechniquesandWoundCleansingAgents62

4.4.1MechanicalDebridement62

4.4.2BiologicalDebridement72

4.4.3EnzymaticDebridement74

4.4.4AutolyticDebridement77

4.4.5WoundCleansing79

4.4.6OtherDebridementTherapies80

4.5WhatistheFutureofDebridement?81 References82

5TreatmentofMixedInfectionsinWounds91

AsifAhmedandJoshuaBoateng

5.1Introduction91

5.1.1WoundHealingProcess92

5.1.2TypesofChronicWounds92

5.2PrevalenceofMixedInfections94

5.2.1Bacterial–FungalInteractions95

5.2.2Bacterial–BacterialInteractions98

5.2.3HostResponsestoMixedInfectionsandDrugResistance99

5.3ManagementofMixedInfectedWounds100

5.3.1ClinicalandMicrobiologicalDiagnosis101

5.3.2DebridementandCleansing101

5.3.3AntimicrobialTherapies102

5.3.4HyperbaricOxygenTherapy104

5.3.5PhageTherapy104

5.4SummaryandFuturePerspectives104 References105

6TreatmentofBiofilmsinInfectedWounds115

PhilipDebrah,AwoAfiKwapongandMansaFredua-Agyeman

6.1Introduction115

6.2WhyandHowBiofilmsForm116

6.3WoundBiofilms118

6.3.1WoundHealing119

6.4BiofilmsandWounds119

6.4.1SimulationofBiofilmsinWounds120

6.5TreatmentofBiofilmsinWounds126

6.5.1BiofilmEradication126

6.5.2CurrentTreatmentProtocols128

6.6ClinicalExamples128 6.7Summary128 References130

7Freeze-DriedWafersforWoundHealing137 Shiow-FernNg

7.1Introduction137

7.2WaferasaModernWoundDressing138

7.3Freeze-DryingProcess139

7.4WaferPreparation140

7.5WaferAssessments141

7.5.1Morphology142

7.5.2SwellingIndex144

7.5.3MechanicalProperties145

7.5.4InVitroDrugRelease145

7.5.5CellViability146

7.6WaferBiopolymers146

7.6.1Alginate147

7.6.2Chitosan148

7.6.3Carboxymethylcellulose149

7.7Conclusion150 References150

8SilverandSilverNanoparticle-BasedAntimicrobialDressings157 JoshuaBoatengandOvidioCatanzano

8.1Introduction157

8.1.1BriefHistoryofSilverasanAntibiotic159

8.1.2MechanismofAction160

8.1.3BacterialResistancetoSilver164

8.2SilverDressingsinWoundHealing167

8.2.1Silver-BasedAntimicrobialDressings169

8.2.2SilverNanoparticle-BasedAntimicrobialDressings170

8.3Cost-EffectivenessofSilverDressings175

8.4ConcludingRemarks176 References177

9HydrogelDressings185 GaliyaS.Irmukhametova,GrigoriyA.MunandVitaliyV.Khutoryanskiy 9.1Introduction185

9.1.1ClassificationbyOriginofMaterialsUsedtoPrepare Hydrogels186

9.1.2ClassificationbyCompositionandStructureofHydrogels186

9.1.3ClassificationbytheTypeofCross-Linking187

9.1.4ClassificationBasedontheShapeandDimensionsof Hydrogels187

9.1.5ClassificationBasedontheChargeofMacromolecules FormingHydrogels187

9.1.6ClassificationBasedonFunctionalPropertiesofthe Hydrogels187

9.2MechanismofHydrogelSwelling187

9.2.1SwellingofTemperature-SensitiveHydrogelsandTheir ApplicationinWoundHealing189

9.2.2SwellingofLight-SensitiveHydrogels190

9.2.3SwellingofElectro-SensitiveHydrogels191

9.3ApplicationofHydrogelsasWoundDressings191

9.4IndustrialMethodsfortheSynthesisofHydrogelsforWound Dressings193

9.4.1PolymerizationMethods193

9.4.2Cross-LinkingofPolymers195

9.5AntimicrobialHydrogelswithSpecialAdditives198

9.6Conclusion200 Acknowledgments201 References201

10GeneTherapyfortheTreatmentofChronicWounds209 MarcosGarcia-Fuentes

10.1Introduction209

10.2PharmacodynamicsofGeneTherapyinChronicWounds210

10.2.1SignallingSupplementation210

10.2.2PathwayInhibition211

10.3AdministrationRoutesandMethods212

10.3.1SystemicDelivery212

10.3.2TopicalDelivery212

10.3.3IntralesionalDelivery213

10.4GeneDeliverySystems213

10.4.1PhysicalMethods214

10.4.2ViralVectors215

10.4.3ChemicalDeliverySystems217

10.4.4Gene-ActivatedMatrices220

10.5ClinicalEvaluation221

10.6Conclusion226 Acknowledgements226 References227

11HoneyinWoundHealing235 EmiMaruhashi

11.1TheHistoryofHoney235 11.2Composition236

11.3HoneyResearch236

11.4MedicalGradeHoney237 11.5ModesofAction238

11.6ApplicationsandSpecificWoundTypes242 11.7PracticalConsiderations246 11.8NovelConceptsandConclusions247 References248

12RegenerationUsingTissueEngineeredSkinStrategies255 LucíliaP.daSilva,MarianaT.CerqueiraandAlexandraP.Marques

12.1Introduction255

12.2SkinPhysiologyandWounding256

12.3SkinTissueEngineering258

12.4EvolvingSkinTissueEngineeringStrategies259

12.4.1BalancingtheInflammatoryPhase261

12.4.2EnhancementofRe-Epithelialization263

12.4.3TargetofDermalMatrixSynthesisandRemodeling269

12.4.4Re-EstablishmentoftheVascularNetwork270

12.4.5InnervationShaping280

12.4.6AppendagesandPigmentation281

12.5Conclusion282 References283

13LocalDeliveryofGrowthFactorsUsingWoundDressings291 OvidioCatanzanoandJoshuaBoateng

13.1WoundDressingsasDeliveryPlatformsforGrowthFactors291

13.2GrowthFactorsInvolvedintheWoundHealingProcess292

13.3LocalDeliveryofGrowthFactorsUsingWoundDressings296

13.4IntegrationofPlatelet-RichPlasmainWoundDressings299

13.5EnhancingLocalGrowthFactorExpressionUsing GeneTherapy300

13.6WoundDeliveryofGrowthFactorsfromLivingSystems302

13.7RegulatoryConsiderations305

13.8ConclusionsandFuturePerspectives306 References307

14ElectrospinningTechnologiesinWoundDressingApplications315 GiuseppinaSandri,SilviaRossi,MariaCristinaBonferoni,CarlaCaramella andFrancaFerrari

14.1Introduction315

14.2BasicConceptandElectrospinningSet-Up316

14.3ParametersAffectingtheElectrospinningProcess318 14.4ProcessParameters319

14.4.1ElectricFieldStrength319

14.4.2FlowRate319

14.4.3Needle-to-CollectorDistance320

14.4.4CollectorandNeedleTypes320 14.5SolutionParameters321

14.5.1MolecularWeightandPolymerConcentration321

14.5.2SurfaceTension322

14.5.3Conductivity/SurfaceChargeDensity322

14.5.4EnvironmentalParameters322

14.6BiomedicalApplicationsofNanofibrousMembranes323

14.6.1WoundDressingsandWoundHealing323

14.6.2ElectrospunDressings325

14.7ChemicophysicalandBiopharmaceuticalCharacterizations325

14.8Dressing/ScaffoldParametersAffectingCellFunctions327

14.9MaterialsforFabricatingNanofibers328

14.9.1Biopolymers328

14.10ConcludingRemarks333 References333

15ThePlaceofBiomaterialsinWoundHealing337 AnnalisaBianchera,OvidioCatanzano,JoshuaBoatengandLisaElviri

15.1IntroductiontoBiomaterialsforWoundHealing337

15.1.1DefinitionofBiomaterials337

15.1.2FunctionalRequirementsofWoundRepairBiomaterials338

15.1.3ClassificationofBiomaterialsCommonlyUsedinWound Healing338

15.2SyntheticBiomaterialsforWoundHealing339

15.2.1PolyurethanesandtheirDerivatives340

15.2.2Polyl-LacticAcid340

15.2.3Poly(EthyleneGlycol)341

15.2.4Polycaprolactone341

15.2.5Poly(GlycolicAcid)andPoly(Lactic-co-GlycolicAcid)342

15.3NaturalBiomaterialsforWoundHealing343

15.3.1Polysaccharide-BasedBiomaterials343

15.3.2Protein-BasedBiomaterials348

15.4ApplicationofBiomaterialsinWoundHealing350

15.4.1TraditionalandImpregnatedDressings350

15.4.2Hydrogels352

15.4.3FilmDressings353

15.4.4FoamDressings354

15.4.5Nanofiber-BasedDressings355

15.4.6Three-DimensionalPrintedDressings356

15.5NewTrendsinBiomaterialsforWoundHealing357

15.5.1ExtracellularMatrix-DerivedBiomaterials357

15.5.2TissueEngineeredSkinSubstitutes357

15.6ConclusionsandFuturePerspectives358 References359

16WoundDressingsandPressureUlcers367 MichaelClark

16.1Overview367 16.2IntroductiontoPressureUlcers367

16.3TheImpactofPressureUlcers369 16.4ManagingPressureUlcers370

16.5WoundDressingsinPressureUlcerTreatment371

16.6PressureUlcerPreventionandWoundDressings377

16.6.1PressureUlcersattheNose378

16.6.2PressureUlcersattheHeel378

16.6.3PressureUlcersattheSacrum378

16.7Conclusions380 References380

173DPrintedScaffoldsforWoundHealingandTissueRegeneration385 AtabakGhanizadehTabriz,DennisDouroumisandJoshuaBoateng

17.1Introduction385 17.23DPrinting386 17.3Laser-BasedBioprinting387 17.4Jet-BasedPrinting389

17.5Extrusion-BasedPrinting391

17.6HybridPrinting393

17.7Conclusions395 References395

ListofContributors

AsifAhmed, SchoolofScience,FacultyofEngineeringandScience,Universityof GreenwichMedway,ChathamMaritime,UK

DianeBeck, AshfordandStPeter’sHospitalsNHSFoundationTrust,Chertsey,UK

AnnalisaBianchera, InterdepartmentalCentreBiopharmanet-TEC,UniversityofParma, Italy

JoshuaBoateng, SchoolofScience,FacultyofEngineeringandScience,Universityof GreenwichMedway,ChathamMaritime,UK

MariaCristinaBonferoni, DepartmentofDrugSciences,UniversityofPavia,Italy

KarenCampbell, WesternUniversity,London,ON,Canada

CarlaCaramella, DepartmentofDrugSciences,UniversityofPavia,Italy

OvidioCatanzano, DepartmentofLifeSciences,UniversityofTrieste,Italy

GwendolynCazander, WoundExpertiseCenter(WEC),Ikazia,Rotterdam, TheNetherlands

MarianaT.Cerqueira, 3B’sResearchGroup,I3Bs–ResearchInstituteonBiomaterials, BiodegradablesandBiomimetics,UniversityofMinho,HeadquartersoftheEuropeanInstituteofExcellenceonTissueEngineeringandRegenerativeMedicine,Barco, Guimarães; andICVS/3B’s–PTGovernmentAssociateLaboratory,Braga/Guimarães,Portugal

MichaelClark, BirminghamCityUniversity,Birmingham;andWelshWoundInnovation Centre,Ynysmaerdy,UK

TimH.C.Damen, WoundExpertiseCenter(WEC),Ikazia,Rotterdam,TheNetherlands

LucíliaP.daSilva, 3B’sResearchGroup,I3Bs–ResearchInstituteonBiomaterials, BiodegradablesandBiomimetics,UniversityofMinho,HeadquartersoftheEuropeanInstituteofExcellenceonTissueEngineeringandRegenerativeMedicine,Barco,Guimarães; andICVS/3B’s–PTGovernmentAssociateLaboratory,Braga/Guimarães,Portugal

PhilipDebrah, DepartmentofPharmaceuticsandMicrobiology,SchoolofPharmacy,UniversityofGhana,Accra,Ghana

BiancaK.denOttelander, WoundExpertiseCenter(WEC),Ikazia,Rotterdam, TheNetherlands

MartijnC.H.A.Doomen, WoundExpertiseCenter(WEC),Ikazia,Rotterdam, TheNetherlands

DennisDouroumis, SchoolofScience,FacultyofEngineeringandScience,Universityof Greenwich,ChathamMaritime,UK

LisaElviri, FoodandDrugDepartment,UniversityofParma,Italy

FrancaFerrari, DepartmentofDrugSciences,UniversityofPavia,Italy

MansaFredua-Agyeman, DepartmentofPharmaceuticsandMicrobiology,Schoolof Pharmacy,UniversityofGhana,Accra,Ghana

MarcosGarcia-Fuentes, CenterforResearchinMolecularMedicineandChronic Diseases(CIMUS),UniversidaddeSantiagodeCompostela,Spain

AtabakGhanizadehTabriz, SchoolofScience,FacultyofEngineeringandScience, UniversityofGreenwich,ChathamMaritime,UK

KeithHarding, DivisionofPopulationMedicine,CardiffUniversitySchoolofMedicine, Cardiff,UK

GaliyaS.Irmukhametova, FacultyofChemistryandChemicalTechnology,al-Farabi KazakhNationalUniversity,Almaty,Kazakhstan

SandraKamga, WoundExpertiseCenter(WEC),Ikazia,Rotterdam,TheNetherlands

VitaliyV.Khutoryanskiy, SchoolofPharmacy,UniversityofReading,UK

AwoAfiKwapong, DepartmentofPharmaceuticsandMicrobiology,SchoolofPharmacy, UniversityofGhana,Accra,Ghana

KirstieLane, WestByfleetHealthCentre,WestByfleet,UK

KimberlyLeBlanc, WoundOstomyContinenceInstitute/AssociationofNursesSpecializedinWoundOstomyContinence,Ottawa,ON,Canada

BarunMajumder, AshfordandStPeter’sHospitalsNHSFoundationTrust,Chertsey,UK

DuniyaMajumder, Lanarkshire,Glasgow,UK

AlexandraP.Marques, TheDiscoveriesCentreforRegenerativeandPrecisionMedicine, HeadquartersatUniversityofMinho,Barco,Guimarães,Portugal

EmiMaruhashi, UniversityofLisbon,Lisbon,Portugal

GrigoriyA.Mun, FacultyofChemistryandChemicalTechnology,al-FarabiKazakh NationalUniversity,Almaty,Kazakhstan

Shiow-FernNg, CentreforDrugDeliveryResearch,FacultyofPharmacy,Universiti KebangsaanMalaysia,KualaLumpur,Malaysia

SilviaRossi, DepartmentofDrugSciences,UniversityofPavia,Italy

GiuseppinaSandri, DepartmentofDrugSciences,UniversityofPavia,Italy

SandeepSingh, AshfordandStPeter’sHospitalsNHSFoundationTrust,Chertsey,UK

SophiaTate, UniversityHospitalofWales,CardiffandValeUniversityHealthBoard, Cardiff,UK

AnneMarieE.vanWell, WoundExpertiseCenter(WEC),Ikazia,Rotterdam, TheNetherlands

SeriesPreface

Theseries AdvancesinPharmaceuticalTechnology coverstheprinciples,methodsand technologiesthatthepharmaceuticalindustryusestoturnacandidatemoleculeornew chemicalentityintoafinaldrugformandhenceanewmedicine.Theserieswillexplore meansofoptimizingthetherapeuticperformanceofadrugmoleculebydesigningand manufacturingthebestandmostinnovativeofnewformulations.Theprocessesassociated withthetestingofnewdrugs,thekeystepsinvolvedintheclinicaltrialsprocessandthe mostrecentapproachesutilizedinthemanufactureofnewmedicinalproductswillallbe reported.Thefocusoftheserieswillverymuchbeonnewandemergingtechnologiesand thelatestmethodsusedinthedrugdevelopmentprocess.

Thetopicscoveredbytheseriesincludethefollowing:

Formulation: Themanufactureoftabletsinallforms(caplets,dispersible,fast-melting) willbedescribed,aswillcapsules,suppositories,solutions,suspensionsandemulsions, aerosolsandsprays,injections,powders,ointmentsandcreams,sustainedreleaseand thelatesttransdermalproducts.Thedevelopmentsinengineeringassociatedwithfluid, powderandsolidshandling,solubilityenhancement,colloidalsystemsincludingthestabilityofemulsionsandsuspensionswillalsobereportedwithintheseries.Theinfluence offormulationdesignonthebioavailabilityofadrugwillbediscussedandtheimportanceofformulationwithrespecttothedevelopmentofanoptimalfinalnewmedicinal productwillbeclearlyillustrated.

DrugDelivery: Theuseofvariousexcipientsandtheirroleindrugdeliverywillbe reviewed.Amongthetopicstobereportedanddiscussedwillbeacriticalappraisal ofthecurrentrangeofmodified-releasedosageformscurrentlyinuseandalsothose underdevelopment.

Thedesignandmechanism(s)ofcontrolledreleasesystemsincludingmacromolecular drugdelivery,microparticulatecontrolleddrugdelivery,thedeliveryofbiopharmaceuticals,deliveryvehiclescreatedforgastrointestinaltracttargeteddelivery,transdermal

deliveryandsystemsdesignedspecificallyfordrugdeliverytothelungwillallbe reviewedandcriticallyappraised.Furthersite-specificsystemsusedforthedelivery ofdrugsacrosstheblood–brainbarrierincludingdendrimers,hydrogelsandnew innovativebiomaterialswillbereported.

Manufacturing: Thekeyelementsofthemanufacturingstepsinvolvedintheproduction ofnewmedicineswillbeexploredinthisseries.Theimportanceofcrystallization;batch andcontinuousprocessing,seeding;andmixingincludingadescriptionofthekeyengineeringprinciplesrelevanttothemanufactureofnewmedicineswillallbereviewed andreported.Thefundamentalprocessesofqualitycontrolincludinggoodlaboratory practice,goodmanufacturingpractice,QualitybyDesign,theDemingCycle,regulatory requirementsandthedesignofappropriaterobuststatisticalsamplingproceduresforthe controlofrawmaterialswillallbeanintegralpartofthisbookseries.

Anevaluationofthecurrentanalyticalmethodsusedtodeterminedrugstability,the quantitativeidentificationofimpurities,contaminantsandadulterantsinpharmaceutical materialswillbedescribed,aswilltheproductionoftherapeuticbio-macromolecules,bacteria,viruses,yeasts,molds,prionsandtoxinsthroughchemicalsynthesisandemerging synthetic/molecularbiologytechniques.Theimportanceofpackagingincludingthecompatibilityofmaterialsincontactwithdrugproductsandtheirbarrierpropertieswillalsobe explored.

AdvancesinPharmaceuticalTechnology isintendedasacomprehensiveone-stopshop forthoseinterestedinthedevelopmentandmanufactureofnewmedicines.Theserieswill appealtothoseworkinginthepharmaceuticalandrelatedindustries,bothlargeandsmall, andwillalsobevaluabletothosewhoarestudyingandlearningaboutthedrugdevelopment processandthetranslationofthosedrugsintonewlife-savingandlife-enrichingmedicines.

DennisDouroumis AlfredFahr JürgenSiepmann MartinSnowden VladimirTorchilin

Preface

Woundsandtheireffectivehealingconstituteacommonandcurrentglobalmedicalconcern withseveralchallenges,includingtheincreasingincidenceofobesityandtype2diabetes, anageingpopulationthathasincreasedtheincidenceofchronic(difficulttoheal)wounds, andtherequirementformoreeffectivebutalsocost-effectivedressings.Woundscanbe chronicoracuteandcanresultfromburns,amputation,surgicalprocedures,orunderlying medicalconditions.Innovativedressingsthattakeanactivepartinwoundhealinginamore rapidmannerandatreasonablecostarecurrentlyanunmetpublichealthneed.Although thereareseveraldressingsonthemarket,notallofthemtakeanactivepartinwound healing;instead,theydependonthebody’snaturalphysiologicaltissueprocesses,which arenormallycompromisedinpatientswithunderlyingmedicalconditionsandinthosewho arehighlytraumatized,suchascombatpersonnelandmasscasualties.

Therefore,interesthasshiftedinacademicresearchlaboratories,industry,andgeneral clinicalpracticetowardsmoreadvancedtherapeuticdressingsthatarebiologically activeandusuallyinvolvemulti-disciplinaryapproachesspanningmolecularbiology, biomaterial/polymerscience,biochemistry,formulationscience,andbiopharmaceutics. Theseincludemedicateddressings,biomaterial-basedbiologicaldressings(biologicaland naturallyderived),tissue-engineeredscaffolds,aswellasnanotechnology.

Thisbooksystematicallycoversvariousaspectsoftheaboveadvancedwoundhealing therapiesandisdividedintothreemainthemes.Thebookcomprises17chapterswrittenby variousauthorswhoarewidelyrecognizedintheirfieldsofexpertise.Thefirstsixchapters focusonthephysiologicalandmolecularbasisofwoundsandtheirhealing,includingthe varioustypesofchronicwoundsaswellassomeofthecomplicatingandriskfactors,suchas infectionsanddeadtissues,andhowtomanagethesefromaclinicalperspective.Chapters 7–9focusonadvancedmoistmoderndressingssuchaswafersandhydrogelsaswellas onnanotechnology-basedsilverdressings.Finally,Chapters10–17addressmoreadvanced andnovelapproachestowoundhealing,includinggenetherapy-baseddressings,tissue engineering,deliveryofgrowthfactors,electrospundressings,biomaterial-baseddressings,

andtheuseofthree-dimensional(3D)printedscaffoldsembeddedwithcellsandother activeentitiesthattakepartintissueregeneration.

Mostimportantly,Iwouldliketopersonallythankalloftheauthorsfortheirwillingnesstocontributetothisbookinthefirstplace,andforpreparingtheirchapterswithdue diligenceandasenseofpurposetomeettheagreeddeadlines.

JoshuaBoateng

1 ChronicWoundHealing:Molecular andBiochemicalBasis

SophiaTate1 andKeithHarding2

1 UniversityHospitalofWales,CardiffandValeUniversityHealthBoard,Cardiff,UK

2 DivisionofPopulationMedicine,CardiffUniversitySchoolofMedicine,Cardiff,UK

1.1Introduction

Awoundcanbedefinedasabreakintheepithelialintegrityofthetissue,oradisruption ofnormalanatomicalstructureandfunction[1].Usuallyawoundprogressesthrough severalsequential,thoughoverlapping,stagesofcellularandbiochemicalactivityto achievehealing.Achronicwoundmaybedefinedasonethatisfailingtoprogress throughthewoundhealingprocessinananticipatedtimeframe[2].Awoundthatdoes notshowsignificantimprovementwithin4weeks,orhealcompletelyin8weeks,maybe consideredachronicwound[3].Therearefourstagesdescribedinnormalwoundhealing: haemostasis,inflammation,proliferation,andremodelling.Thehealingofachronic woundmaybearrestedinanyofthesestages,butmostcommonlyduringinflammationor proliferation[4].Thischapterwillbrieflydescribenormalwoundhealing,considersome subtypesofchronicwound,andthenexaminethedifferentmolecularandbiochemical processesthatoccur.

1.2AcuteWoundHealing

Theprocessofacutewoundhealingiswelldescribedandwidelyreportedintheliterature, andissummarisedinFigure1.1.

TIME

INJURY

Haemostasis

Vasoconstriction

Platelet activation

Coagulation cascade

PDGF, TGF-β, IL-1, TNF-α

Inflammation

Vasodilation, increased vascular permeability

Neutrophils

Macrophages

PDGF, TGF-β, IL-1,TNF-α

Proliferation

Fibroblasts

Collagen deposition

Keratinocytes

Re-epithelialisation

Endothelial cells

Angiogenesis

VEGF, FGF, PDGF, EGF, TGF-α, IGF

Remodelling

Collagen replacement and cross-linking

Myofibroblasts

Wound contraction

TGF-α, MMPs HEALED WOUND

Figure1.1 Asummaryofacutewoundhealing.EGF,epidermalgrowthfactor;FGF,ﬁbroblast growthfactor;IGF,insulin-likegrowthfactor;IL-1,interleukin1;MMP,matrixmetalloproteinase;PDGF,platelet-derivedgrowthfactor;TGF,transforminggrowthfactor;TNF-�� ,tumour necrosisfactor �� ;VEGF,vascularendothelialgrowthfactor.

Thefirststep,haemostasis,ischaracterisedbyvasoconstrictionandcoagulation;ittakes placesoonafterinjuryandiscompletewithinhours.Thetissueinthewoundisexposedto bloodbecauseofdisruptionofthebloodvesselsandlymphaticsduringinjury.Plateletsare activatedwhentheycomeintocontactwithcollagenandinitiatethecoagulationcascade, resultinginthedepositionofahaemostatic‘plug’[5].Anumberofcytokinesarereleased

ChronicWoundHealing:MolecularandBiochemicalBasis3 bythedegranulationofactivatedplatelets.Ofparticularimportanceareplatelet-derived growthfactor(PDGF)andtransforminggrowthfactor-beta(TGF-β).PDGFisachemoattractantofneutrophils,macrophages,smoothmusclecells,andfibroblasts[1].TGF-β is alsoinvolvedinthechemotaxisofmacrophages,fibroblasts,andsmoothmusclecells,and hasaroleinactivatingthesecellstoexpressothercytokinesandenzymeswhicharecrucial toenablethewoundhealingtoprogress[1].

Aftertheinitialvasoconstrictionduringhaemostasis,thereisvasodilationandincreased vascularpermeabilityasthestageofinflammationbegins.Thisisregulatedbymastcell degranulation,whichreleaseshistamineandothervasoactivemediators[1].Debris,dead cells,andbacteriaareclearedfromthetissuebyneutrophils,andlaterbymacrophages. Inflammationisusuallycompleteafter48–72h,butmaylastaslongas5–7days[6].

Thenextstageisproliferation,whichcontinuesforweeks.Thehallmarkoftheproliferativephaseisthemigrationoffibroblastsintothewound,wheretheyareactivatedto producecollagenIII,fibrin,fibronectin,andhyaluronicacidinthenewextracellularmatrix [7].Granulationtissueisdepositedtofillthedefect.Keratinocytes,stimulatedbyepidermalgrowthfactor(EGF)andtransforminggrowthfactor-alpha(TGF-α),migratetothe woundedges,andeventuallyclosethedefect[1].Angiogenesisisimportanttosupport theincreasedmetabolicactivityinthewound.Anumberofgrowthfactorsstimulatethe neovascularisation,includingvascularendothelialgrowthfactor(VEGF).Epidermalcells, fibroblasts,macrophages,andvascularendothelialcellsproducethesefactorsinresponse toconditionsinthewoundenvironment,suchaslowpHandreducedoxygentension[1].

Thefinalstage,remodelling,beginsafteraboutaweekandmaylastforyears.This phaseischaracterisedbytheremovaloftypeIIIcollagenfromtheextracellularmatrix andthedepositionofmaturetypeIcollagen[8].Collagenaseenzymesfromfibroblasts, neutrophils,andmacrophagesareimportantinthisstage[1].Woundcontractionismediated bydifferentiatedfibroblasts(myofibroblasts)inresponsetoTGF-α,andthepresenceof matrixproteinssuchasextra-domain-AfibronectinandtenascinC[9].Onceremodelling hasoccurred,thereisapoptosisoffibroblasts,leavingrelativelyacellularscartissue[9].

1.3CategoriesofChronicWound

Althoughchronicwoundsmayseemvariedintheirpresentationandcharacteristics,often theunderlyingaetiologicalprocessesaresimilar.Somecommonchronicwoundcategories areconsideredhere.Ultimately,thefinalcommonpathwayisanopenwoundthathas beencolonisedwithbacteria,initiatingadamaginginflammatoryresponsethatimpedes healing[10].

1.3.1PressureUlcers

Pressureulcersareanexampleofchronicischaemia–reperfusioninjury.Repeatedtissue traumaoccursininsensateareaswhenthepressureinthetissueexceedscapillaryperfusion pressure[10].Thisresultsinskinbreakdown,whichisfollowedbybacterialcolonisation, oftencompoundedbythelocationofsuchulcersneartotheperineum.Thereisfailure oftheprocessesofangiogenesis,extracellularmatrixdeposition,andwoundcontraction, resultinginthedevelopmentandpersistenceofachroniculcer[11].Thesestepsinwound

healingareusuallydrivenbygrowthfactors,andthedestructionorreducedsynthesisof theseproteinsinpressureulcershasbeeninvestigated.Inastudyusinganenzyme-linked immunosorbentassaytechniquetoquantifythelevelsofgrowthfactorsinwoundfluidfrom pressureulcers,Cooperetal.[12]foundthatPDGF,fibroblastgrowthfactor(FGF),EGF, andTGF-β levelswerevariable,anddecreasedcomparedwiththelevelsofgrowthfactors inacutewounds.

1.3.2VenousStasisUlcers

Venousstasisulcersoccurwhendamagedordefectivelegveinvalvesresultinvenous hypertensionandoedema.Eventuallythevenouspressureexceedsthecapillaryperfusion pressureoftheskin,andthetissuebecomesischaemic.Theincreaseinintraluminalpressure affectsthepermeabilityofthevesselwalls,andtheveinsleakfibrinandotherplasmacomponentsintotheperivascularspace[9].Accumulationoffibrinimpairshealingbyimpairing collagensynthesis,andbyformingperi-capillaryfibrincuffsthatimpedenormalvessel function[9].Oftenavenousulcerisprecipitatedbyminortrauma,forexampleascratch orinsectbite.Theskinbreakdownisacceleratedbythehypoxicconditions,andsecondary bacterialcolonisation.Thisincreasesthetissueinjuryandinflammationatthewoundsite, andimpairsepithelialisation[11].

1.3.3IschaemicUlcers

Atherosclerosisand/orembolisminlegarteriesleadstonarrowingofthelumensofthe vesselsandischaemiaofdistaltissue.Minortraumamaythenresultinanulcer.Healingis slowbecauseofthelowoxygenconcentrationinthetissue,andtheresultantopenwound iscolonisedbybacteria.Thisincreasesinflammationinthewound,andthetissuedefect persists.TheeffectsofhypoxiaaredescribedinmoredetailinSection1.5.4.

1.3.4DiabeticFootUlcers

Diabeticfootulcersareanothercategoryofwoundswhicharecommonlychronicintheir course.Thediabeticfootmaybesubjecttorepeatedtraumaasaresultofsensoryloss. Theremayalsobeadegreeofischaemiabecauseofmicrovasculararteriopathy.Oncethe skinbarrierisbreached,low-gradebacterialcolonisationiscommon.Tissuefragmentsand bacterialproductsperpetuatetheinflammatoryresponse.Theeffectsofhyperglycaemiaare describedinmoredetailinSection1.5.2.

1.4HowaChronicWoundDevelops:IntrinsicComponents

Thereareseveralhallmarksofchronicwoundswhencomparedwithnormalacutewounds [9].Inanormalwoundbed,therewillbeahighconcentrationofgrowthfactors,with healthycellpopulationsinanorganisedextracellularmatrix.Bycomparison,chronic woundbedstendtohavelowconcentrationsofgrowthfactorsandadisorganisedextracellularmatrix.Thisisbecauseofexcessiveproteolysisdrivenbyapersistentinflammatory state,oftenaresponsetoabacterialbiofilmorlow-gradeinfection.Impairedangiogenesis

Hyperproliferative but non-advancing epithelium

High bacterial load in the wound

Pro-inflammatory macrophages Impaired neovascularisation

Increased vascular permeability – extravasation of monocytes, neutrophils, and plasma

Figure1.2 Thelocalenvironmentinthechronicwound.MMP,matrixmetalloproteinase; ROS,reactiveoxygenspecies.

andneovascularisationmeanthatcellsinthewoundenvironmentarestarvedofoxygenand nutrients.Theresultisimpairedfibroblastandepithelialcellproliferationandmigration, anddelayedhealing.Figure1.2summarisesthesecomponents.

1.4.1CellPhenotype

Thecellsinchronicwoundshaveanalteredphenotype,withfewergrowthfactorreceptors andlessmitogenicpotential[9].Theydonotthereforerespondtothewoundenvironment inthesamewayascellsobservedinahealthyacutewound.Somespecificobservationsin differentcelltypesarebrieflydescribedbelow.

1.4.1.1Fibroblasts

Inahealthywound,fibroblastsrespondtochemicalsignalsintheformofgrowthfactors, suchasPDGF,insulin-likegrowthfactor(IGF),andFGF,tomigratetowardsthesiteof injury,divide,andsynthesisekeyextracellularmatrixproteinssuchascollagenIII,fibrin,fibronectin,andhyaluronicacid[7].Fibroblastsfrommanychronicwoundtypeshave areducedresponsetogrowthfactors.Studiesoffibroblastsfromchronicdiabetic[13], chronicnon-diabetic[13],andchronicvenousulcers[14]havedemonstratedlowerrates ofcelldivisioninresponsetoPDGF,IGF,andFGF,whichusuallypromoteproliferation, whilecellmotilityisalsoreducedatthesametime.Thesefindingsarethoughttobedue toreducedgrowthfactorreceptordensity[9].Aswellashavingreducedactivity,fibroblastsfromchronicwoundsshowsignsofprematuresenescence.Inastudywherefibroblast

Senescent fibroblasts

culturesweregeneratedfrompunchbiopsiestakenfromvenouslegulcers,andcompared withthosefromuninjuredskininthecontralaterallimb,thecellsfromthewoundshowed areductioningrowthpotentialandalteredgeneexpression.Thisobservationwasindependentoftheageofthepatient[15].

1.4.1.2Keratinocytes

Inhealthyskin,basalkeratinocytesatthedermal–epidermaljunctionundergocelldivision periodically,andthenthesedaughtercellsdifferentiatetoformthesupra-basalepidermallayers.Keratinocytesexpresskeratinproteins,withthepatternofkeratinfilament subtypesindicatingthedegreeofdifferentiationofthekeratinocyte[16].Inresponseto injury,keratinocytesintheadjacentskin,aswellasthoseinthesupra-basallayer,startto expresskeratins6and16,demonstratingamoreactivatedphenotype,whichthenreverts tonormalwhenthewoundhasbeenclosed[16].Inaddition,cellsattheleadingedgeof thewounddepositanextracellularmatrixprotein(laminin-332),whichmediatesthekeratinocytemigrationandanchorscellstothebasementmembrane[9,16].Keratinocytesin chronicwounds,suchasdiabeticandvenousulcers,arenotabletocompletethesekey stepsinre-epithelialisation.Whilsttheyhaveanactivatedphenotypeandarehighlyproliferative,theyarenotwelldifferentiated[16,17].Theyhaveimpairedabilitytomigrate, whichisthoughttobebecauseofdecreasedproductionoflaminin-332[9].Theyalsohave decreasedexpressionofthegrowthfactorsVEGFandTGF-α,althoughtheyshowincreased expressionofPDGFwhencomparedwithkeratinocytesinhealthywounds[9].Overall, thisimbalanceingeneexpressionresultsinthedisorganisedhyperkeratosisobservedatthe non-advancingedgesofchronicwounds.

1.4.2ImmuneCellsandInflammatoryMediators

1.4.2.1Neutrophils

Thechemoattractantsreleasedbyplateletdegranulationduringhaemostasismeanthat neutrophilsarerecruitedtothewoundearlyandarerequiredforthecontrolofpathogens atthesiteofinjury.Onceactivated,theneutrophilsadheretotheendotheliumofthe bloodvesselsatthewoundsite,andmoveintothewoundbytransmigrationthrough anintracellularjunctionandthenthroughtheextracellularmatrix.Inordertodothis, andalsotophagocytosebacteriaanddamagedextracellularmatrix,neutrophilshave numerousenzymescontainedincytoplasmicgranulesandsecretoryvesicles[18].These includeproteases,suchaselastaseandcathepsinB,D,andG,andantimicrobials,suchas myeloperoxidaseandlysozyme[18].

Neutrophilsalsoreleaseanumberofcytokines,includinginterleukin(IL)-1,IL-6,and TNF-α,antimicrobialsubstances,forexamplereactiveoxygenspecies(ROS),andgrowth factors[19].Theyareimportantintherecruitmentofotherimmunecells,suchasmonocytes,andalsoinpromotingproliferationofkeratinocytes,fibroblasts,andendothelialcells.

However,whilstneutrophilactivityisessential,itmustbecarefullyregulatedand uncontrolledactivityisdetrimental.Excessivenumbersofneutrophilshavebeenobserved innon-healingwoundsandthisresultsinapro-inflammatoryenvironment.Overproduction ofROScausesdamagetotheextracellularmatrix,increasesmatrixmetalloproteinase

ChronicWoundHealing:MolecularandBiochemicalBasis7 (MMP)activation,andleadstoearlycellsenescence[19].LevelsofMMPsandother neutrophil-producedproteasessuchasneutrophilelastaseareincreasedinchronicwound fluidcomparedwithacutewoundfluid[20].Increasedproteaseactivitybreaksdown growthfactorsinthewoundenvironment,reducingtheireffects.Adhesionmoleculessuch asfibronectinarealsobrokendown,impairingthecelladhesionthatisneededforwound closure[21].

1.4.2.2Macrophages

Monocytesarriveinthewound5–6hafterinjury,anddifferentiateintomacrophages.In additiontomonocytesrecruitedfromthecirculationbychemokines,thereisapopulationofresidenttissuemacrophagesthatproliferateinresponsetoinjury.Macrophages areimportantinallstagesofwoundhealing,andtheiractionsandphenotypechangeas woundhealingprogresses[22].Intheinflammatorystage,activatedmacrophagesclear damagedtissueandcontrolpathogensthroughphagocytosisandantigenpresentationtoT cells.Theysecreteanumberofpro-inflammatorycytokinesandgrowthfactorsIL-1,FGF, VEGF,andPDGF[19].Thesepro-inflammatory‘M1’macrophagesundergoapoptosisa fewdaysafterinjury.However,asecondpopulationof‘M2’macrophagessurvivetothe proliferativephase[19].Theirphenotypechanges,andtheyhavearoleinthestimulation ofkeratinocytes,fibroblasts,andendothelialcellstore-epithelialisethedefect,depositnew extracellularmatrix,andcarryoutneovascularisation[22].Duringthisphase,macrophages areimportantintheproductionofTGF-β andVEGF,theeffectsofwhicharediscussedin moredetailinSection1.4.4.

Inchronicwounds,macrophagenumbersareincreased[23];however,thecellspresent arethoughttobedysfunctional.Theswitchfromthepro-inflammatory‘M1’phenotypeto theanti-inflammatory‘M2’phenotypeisimpaired[19].Studiesindiabeticmousemodels haveshownthatifmacrophagesdonotundergophenotypicconversionthereisareduction inkeygrowthfactors(TGF-β,VEGF,IGF-1),andthereforefailuretomoveintotheproliferativephaseofwoundhealing[24].Additionally,themacrophagesinthesechronicwounds haveareducedphagocyticcapacity,andtheresultingbuild-upofdebrisandpathogenic materialperpetuatesthepro-inflammatorystateinthewound[25].

1.4.2.3TumourNecrosisFactorAlpha

TNF-α issecretedbymanycelltypesinthewoundenvironment,includingkeratinocytes, fibroblasts,vascularendothelialcells,andinflammatorycells(neutrophilsandmacrophages).TNF-α stimulatesitsownrelease,aswellastheproductionofIL-1,andupregulatestheproductionofMMPswhilstdownregulatingtheproductionoftissueinhibitorsof MMPs(TIMPs)bymacrophages,keratinocytes,andfibroblasts.Inlowconcentrationsfor ashortperiod,thisisbeneficial,aswoundhealingisenhancedbytheremovalofdamaged tissueandthestimulationofinflammatorycellsandresultinggrowthfactorproduction [10].However,prolongedandincreasedTNF-α secretiondelayswoundhealingasthe overallresultofsustainedTNF-α signallingisthedegradationoftheextracellularmatrix, aswellasanumberofgrowthfactorsandtheirreceptors[10].

WhilstthereleaseofTNF-α ispartofthenormalcytokineresponsetoinjury,theusual patterninanacutewoundisthattheincreaseinTNF-α islimitedandtransient.Inchronic

wounds,thepro-inflammatorycytokinecascadeisprolongedandamplifiedbecauseofthe persistenceofnoxiousstimuli[10].Studiesofwoundfluidfromchronicwoundshavefound markedlyelevatedlevelsofTNF-α comparedwithhealthysurgicalwounds[26].

1.4.2.4Interleukin1

IntheskinIL-1ismanufacturedandstoredinkeratinocytes,readyforreleasewheninjury occurs.Itisthereforepresentfromtheverybeginningofthehaemostasisandinflammatory stagesofwoundhealing.LevelsarefurtherincreasedbythereleaseofIL-1fromother inflammatorycells,suchasmacrophages,oncetheyareactivatedatthesiteofinjury.IL-1 isachemokineforneutrophils,whicharerequiredininjurytoremovepathogens.Chronic woundshaveincreasedlevelsofIL-1[26]and,inmanycases,thisisatleastpartlyin responsetothepresenceofbacteria.Thesewoundsalsohaveelevatedlevelsofproteolytic enzymes,suchascollagenases,gelatinases,andstromelysins,whoseproductionisinduced byIL-1andTNF-α [26].

1.4.3ReactiveOxygenSpecies

ROSarereleasedfromendothelialcellsinresponsetoTNF,PDGF,andthrombin,from fibroblastsinresponsetoIL-1,andalsofromneutrophilsandmacrophages[27].Theyare essentialinoxidativebacterialkilling,andenhancethechemotaxisofneutrophils;theyare, therefore,importantinthepreventionofwoundinfection.ROSproductionbynicotinamide adeninedinucleotidephosphatehydrogen(NADPH)-linkedoxygenaseishighlyoxygen dependent[27].However,likemostprocessesintheinflammatorystageofwoundhealing, regulationiscritical,asveryhighconcentrationsofROSaredamaging.Antioxidantssuch asnitricoxideareproduced,andreductasesareactivatedtopreventoxidativedamage. Again,theseprocessesaredependentonoxygen[27].Thechronicwoundenvironmentis oftenhypoxic,eitherasaresultofsystemicorregionaldisease,forexampleatherosclerosis orvenoushypertension,orasaresultoflocalfactorssuchasinfection,oracombinationof both.Thereisoftenarepetitivecycleofischaemiaandreperfusion,suchaswhenalegwith apoorarterialbloodsupplyiselevatedandthendependent.Insuchcircumstances,thereis anetbuild-upofROSandanincreaseininflammationandtissuedamage[27].

1.4.4GrowthFactors

1.4.4.1ThePlatelet-DerivedGrowthFactorFamily

PDGFismadeupofafamilyofhomo-orheterodimericgrowthfactors,whichbind tothreedifferenttransmembranetyrosinekinasereceptors[28].PDGFisreleasedby plateletdegranulationduringthehaemostasisstageofwoundhealing,andisfoundin highconcentrationsinwoundfluidearlyafterinjury[29].Ithasachemotacticeffecton neutrophils,monocytes,fibroblasts,andsmoothmusclecells[30].Inaddition,PDGF stimulatesfibroblaststoproliferateandsynthesiseextracellularmatrixcomponents[28].It promotesangiogenesisinhypoxicconditions,andinvivoexperimentshavedemonstrated thatPDGFincreasespericyteandsmoothmusclecellrecruitmenttothenewcapillaries, increasingstructuralintegrity[30].