J Cosmet Dermatol. 2019;00:1–11.  | 1wileyonlinelibrary.com/journal/jocd

Received:26April2019  |  Revised:22May2019  |  Accepted:24May2019DOI: 10.1111/jocd.13051

O R I G I N A L C O N T R I B U T I O N

In vivo evaluation of the effectiveness of biocellulose facial masks as active delivery systems to skin

Paola Perugini PhD1,2  | Mariella Bleve PhD2 | Renato Redondi PhD3 | Fabiola Cortinovis4 | Antonio Colpani PhD5

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution‐NonCommercial‐NoDerivsLicense,whichpermitsuseanddistributioninanymedium,providedtheoriginalworkisproperlycited,theuseisnon‐commercialandnomodificationsoradaptationsaremade.©2019TheAuthors.Journal of Cosmetic DermatologyPublishedbyWileyPeriodicals,Inc.

1DepartmentofDrugSciences,UniversityofPavia,Pavia,Italy2Etichubs.r.l,Academicspin‐offofPaviaUniversity,Pavia,Italy3DepartmentofManagement,InformationandProductionEngineering,UniversityofBergamo,Bergamo,Italy4ASST(AziendaSocioSanitariaTerritoriale)PapaGiovanniXXIII,Bergamo,Italy5iBeauty,Brembatedisopra,Italy

CorrespondencePaolaPerugini,DepartmentofDrugSciences,UniversityofPavia,Pavia,Italy.Email:paola.perugini@unipv.it

AbstractBackground: Inrecentyears,bacterialcellulose(BC),orbiocellulose,anaturalpoly‐mersynthesizedbycertainbacteria,hasattractedgreatinterestindermatologyandcosmeticapplications.Severalbioactive ingredientsarecurrently loaded intoBCmasks.However,onlyafewstudieshavereportedtheeffectivenessofsuchdeliverysystems.Aim: Theaimofthisstudywastoevaluatetheeffectonskinparametersofthreebiocellulosemasksformulatedtohavedifferentcosmeticeffects(anti‐aging,lifting,andcellrenewal).Inparticular,skinmoisturizing,skincolor,skinviscoelasticproper‐ties,skinsurfacesmoothness,wrinklereduction,dermalhomogeneity,andstratumcorneumrenewalwereevaluated.Materials and methods: Thestudyinvolved69healthyCaucasianfemalevolunteersbetween25and64years,whoweredividedintothreedifferentstudies.Biocellulosefacial masks were applied using the split‐face method three times a week for4‐8weeksdependingonthestudy.Results: The resultsobtained from thisworkhighlight thatbiocellulosemasksareverywelltolerated.Asignificantdecreaseinskinroughnessandwrinklebreadth,andanimprovementindermalhomogeneityandfirmness,wasobservedafter2monthsoftreatmentwith“anti‐aging”masks.Asignificantimprovementinskinfirmnessandelasticitywasobservedafter1monthoftreatmentwith“lifting”masks.Furthermore,a1‐monthtreatmentwith“cellrenewal”maskspromotedtheproductionofnewskincellsthroughamildexfoliatingaction.Conclusions: Thisstudyhighlightsthatbiocellulosemasksareeffectivedeliverysys‐temstosuccessfullyreleaseintotheskinseveraltypesofactivecompoundsexertingmanybeneficialeffects.

K E Y W O R D S

bacterialcellulose,cosmeticfacialmask,efficacyevaluation,invivostudy,tolerabilityevaluation

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1  | INTRODUC TION

Facialmasksaretraditionalcosmetictreatmentsandhaverecentlyundergonemodifications,withnewaspects,ingredients,andvehi‐clesofferingnewperspectivesforskincare.Facialmasksareverypromising cosmetic formulations as they are effective and easyand quick to apply. Furthermore, they are accessible, being for‐mulatedasagel,cream,sheet,orpaste,andshowinstanteffectsontheskin.1

Among several types of face‐mask formulations, sheetmasks have some distinctive traits. They are face‐shaped fabricsheetssoakedinactivesolution,aregenerallyusedonce,andareindividuallypackaged,makingthemfast,convenient,andeasytouse.Furthermore,theycanpreventquickevaporationofthewaterphase, thus allowing the ingredients to penetrate deep into theskin.

Althoughalargenumberofsyntheticmaterialshavebeendevel‐opedforuseinfacemasks,therehasbeengrowinginterestinthedevelopmentofmaterialsbasedonnaturalpolymers,mainlyduetotheirnaturalbiocompatibilityandbiodegradability,butalsototheirrenewable character and specific properties.Amongnatural poly‐mers,cellulosefibershavefoundextensiveapplicationsinthefieldsofbiomedicineandcosmetics.2‐8

Bacterial cellulose (BC) is produced by several species of bac‐teria, such as those belonging to the genera Gluconacetobacter (formerly Acetobacter), Aerobacter, Agrobacterium, Azotobacter,Achromobacter,Alcaligenes,Rhizobium,Pseudomonas,Salmonella,andSarcina.9,10Amongthesespecies,Gluconacetobacter xylinus can pro‐ducearelativelyhighlevelofBCandhasbeenwidelystudiedasamodelBC‐producingmicroorganism.

ThematerialpropertiesofBCcanbetailoredbyusingvariousinsitu techniques, suchas theadditionofvarioussubstances, thechangeofculturingconditions,andtheuseofgeneticallymodifiedstrains,aswellasexsitustrategies,suchasphysicalandchemicalmodification,specializeddryingconditions,andexposuretoelectro‐magneticirradiation.11,12

Bacterialcelluloseisanalternativecellulosicsubstrateforfacialmasks,mainlydueto itsultrafinenetworkstructure.Bacterialcel‐lulosedisplaysuniquephysicochemicalproperties, includingahighYoung's modulus, water‐holding capability, crystallinity, porosity,transparency,water‐retentioncapability, tensile strength, andbio‐compatibility,aswellasanultrafinefibernetwork.13

Like all celluloses, BC is a polymer of glucose, in which thepyranose rings are joined together by b‐1,4 bonds, giving rise tomacromolecules.Bacterialcelluloseisanaturalandtotallybiocom‐patiblepolymer.14,15AlthoughBCandplantcellulosepossessasim‐ilarchemicalstructure,theself‐assemblingnanofibrousstructureofBCisdifferenttothatofplantcellulose.Despitethemanypositiveeffectsofbiocellulose, it isessential toextensivelycheckallstepsofface‐maskproductioninordertomeetthelegalrequirementsofqualityandsafetyspecifiedforcosmeticproducts.16

Bioactiveingredients,includingthosefoundinmoisturizers,ex‐foliants,andskin‐lighteningcosmetics,aswellasherbalingredients,

vitamins, proteins, minerals, and more unusual ingredients suchas pearl, snail extract, and seaweed, are currently loaded intoBCmasks.However,thereareveryfewstudiestosupporttheeffective‐nessoftheseingredients.17‐20

Themainaimof thiswork is topresentascientificprotocol inordertoevaluatetheefficacyoftopicaltreatmentsbasedonaninvivouseofbiocellulosemasks(BM)withdifferentclaimedeffects,namelyanti‐agingBM(ABM),liftingBM(LBM),andpurifyingandre‐generatingBM(PRBM).Theobjectiveoftheseexperimentalstudieswastotesttheeffectsofcellulosesheetmasksloadedwithdifferentactiveingredientsonskinmoisturization,skincolor,skinviscoelasticproperties,skinsurfacesmoothness,thepresenceofwrinkles,der‐malhomogeneity,andcellrenewal.

2  | MATERIAL S AND METHODS

2.1 | Materials

BacterialcellulosewasobtainedbytheincubationofG xylinus in a sterilebrothat30°Cforaperiodofupto120hoursatapHof6.5‐7.0instaticconditions.Afterpartiallyde‐wateringthegel,theBCsheetsweredie‐cuttoobtainamaskshapewithholesfortheeyes,nose,andmouth.Then,BCsheets (BCS)weresupplied instackssoakedwithapreservedwatersolution(phenoxyethanol—0.075%,iodopro‐pynylbutylcarbamate—0.0015%).

2.2 | Preparation of face masks

Activebiocellulosemasks (ABM,LBM,PRBM)andplacebobiocel‐lulosemasks (PLBM)were prepared, starting fromBCS thatwerefolded and embeddedwith 20mL of active solution and placebosolution,respectively.Thefoldedmaskswereinsertedintopolyeth‐yleneterephthalate(PET)/aluminum/polyethylene(PE)sachetsandthenthermosealed.

Theplacebosolutioncontained95.7%water,3%butyleneglycol,0.9% phenoxyethanol, 0.1% ethylhexylglycerin, and 0.3% xanthangum.

Thesolutionsfortheactivemaskscontainedthefollowingactiveingredients:

• Anti‐agingmask:Adansonia digitatafruitextract,Hibiscus sabdar-iffa flower extract, acetyl decapeptide‐3, Coffea arabica seed‐cakeextract,hydrolyzedhyaluronicacid,palmitoyltripeptide‐38,Kigelia africana fruit extract, Acacia senegal gum, dimethylami‐noethanoltartrate,andCrocus chrysanthusbulbextract;

• Lifting mask: hydrolyzedChenopodium quinoa seed, hydrolyzedhyaluronicacid,andacetyldecapeptide‐3;

• Purifyingandregenerativemask:Portulaca oleracea flower/leaf/stemextract,sodiumhyaluronatecross‐polymer,Chlorella vulgaris extract,kaolin,andmagnesiumsulfate.

Inparticular,fruitextractofA digitata and H sabdariffaflowerextractwerechosenbecauseof thehighconcentrationofcompoundswith

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antioxidantefficacythatarepresentinbothofthem,hydrolyzedhyal‐uronicacidactsimprovinghydrationandskinelasticity.Furthermore,peptideswerechosenbecauseoftheirbiomimeticaction,contributingto theactivationof regenerationprocessesand initiationofmecha‐nismsthatpreventaging.21‐25

AllmaskswereproducedfollowingGoodManufacturingPractices(GMP)guidelinesandwerecheckedforquality,microbialcontent,andtheeffectivenessofthepreservativesystemthroughtheChallengetest,andforallmasks,aconfirmationoftolerabilitywasperformedthroughaninvivotest(PatchTest)inordertoensureasafeandhigh‐qualityproductsuitabletobeappliedtovolunteers.16,26

2.3 | In vivo study design

The study was carried out according to the Helsinki declaration(EthicalPrinciplesforMedicalResearchInvolvingHumanSubjects).27 Atotalof69healthyfemalevolunteersbetween25and64yearsoldwererecruitedaccordingtothefollowinggeneralinclusionandex‐clusioncriteria.

Generalinclusioncriteria:

• Goodgeneralhealth.• Absenceofcutaneousdiseases.• Absenceofcutaneouslesionsorotherlesionsintheareaofinter‐estthatcouldinterferewiththestudyevaluation.

• Nohistoryofhypersensitivitytothecommoncomponentsofthecosmeticformulations.

• Notpregnantorbreastfeeding.• Subjectsagreednottoundergoothertreatments inthetreatedareafortheentiredurationofthetest.

• Subjectssignedtheinformedconsentform.

Generalexclusioncriteria:

• Subjectsthatdidnotmeettheinclusioncriteriaabove.• Subjectsundergoingpharmacologicaltherapy.• Subjectsthathadparticipatedinasimilarstudylessthan60dayspriortothestartofthepresentstudy.

• Subjectswithknownallergies.

Volunteers selectedaccording to the specific inclusioncriteriaweredividedintothreedifferentstudies:

• ABM:a totalof24 subjectsbetween42and64yearsoldwithfacialskinaging (lines,wrinkles, thinskin,and lossofskintone)wereenrolledina2‐monthstudyofthecontinuoususeofABMandPLBM;

• LBM:A totalof20subjectsbetween38and57yearsoldwithfacialskinsaggingwereenrolledina1‐monthstudyofthecontin‐uoususeofLBMandPLBM;

• PRBM:Atotalof25subjectsbetween25and40yearsoldwithdullfacialskinwereenrolledina1‐monthstudyofthecontinuoususeofPRBMandPLBM.

Ineachstudy,subjectsweretrainedtoapply,bythesplit‐facemethodinaself‐controlledstudy, theplacebomaskononesideoftheface,andtheactivemask(ABM,LBM,orPRBM)ontheotherside.Inthisway,acomparisonwasmadebetweentheactiveandplacebomasksonthesamesubjectsoastoremoveorreduceasmuchaspossibletheeffectofunintentionalfactorsotherthantreatmentonthesubjectresponse.Eachsubjectthereforeservedasherowncontrol,therebygivinggreaterstatisticalpowertothestudy.

Maskswereappliedatspecifiedintervalsthreetimesaweekfor4or8weeksdependingon thestudydesign.The twohalfmaskswereplacedon the faceaftermildcleansingand lefton forup to20minutes.Thespecificfacesidestowhichtheactiveandplacebomaskswereappliedwererandomizedamongvolunteers.

2.4 | Instrumental efficacy evaluation

Assessmentswereperformedat thebeginningof each study (T0)andatpredeterminedsubsequenttimes(T1orT2)dependingontheaimofthestudy.Theinvestigatedareasweretheforeheadonbothsidesoftheface,theforeheadfoldforwrinkleinvestigation,andthecheekbonefortheinvestigationoftheskinrenewaleffect.

Allmeasurementsweremade in an air‐conditioned roomwithcontrolled temperature and humidity (T = 22°C, relative humidity[RH]=70±5%).Subjectswerepreconditioned in the roomforatleast15minutesbeforethemeasurementsweremade.

The instruments used in the evaluation of skin parameters in‐volvecontactbetweentheskinandaseriesofprobesthatdonotcausediscomfort,pain,ordamagetheskin.

2.5 | Evaluation of tolerability and moisturizing effect

AssessmentsofthetolerabilityandmoisturizingeffectsofthefacialmaskswereperformedatT0andatT1(after12applications).Theinvestigatedareawastheforeheadonbothsidesoftheface.

The tolerability of themaskswas assessed bymeasurementof the erythema index (EI) using a reflectance spectrophotome‐ter(MexameterMX18,Courage&Khazaka),whichmeasureslev‐els ofmelanin andhemoglobin (erythema), the two componentsthataremainlyresponsibleforthecoloroftheskin.Tomakethemeasurement,aprobewithadiameterof5mmemitsthreewave‐lengthsof light (568,660, and870nm) inorder toquantify theamountof theselectedbiochromophores (melanin,hemoglobin),thusprovidinganarbitrarymelaninindex(MI;range=0‐999)andEI(range=0‐999).

Thestratumcorneumwatercontent(SCWC),whichreflectsthehydratingeffectofthefacemask,wasevaluatedbyaCM825cor‐neometer(cutometerMPA580,Courage&Khazaka).Corneometryisatechniqueusedtoassessthehydrationoftheouterlayeroftheepidermis,whichisknownasthestratumcorneum.28Sinceskinisadielectricmedium,allvariationsinhydrationresultinacorrespond‐ingchangeintheskin'selectricalcapacity.29Thedeviceusedinourtrialwasequippedwitha49mm2surfaceprobethatallowsprecise

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measurementin1secondwithinadepthrangeof10‐20μminthestratumcorneum.Theparameterswereexpressedusinganarbitraryscorescale(0‐100AU).

The assessment of skin barrier integrity and the possible oc‐clusive or irritant effect of the facial masks used in this studywereperformedbythemeasurementoftransepidermalwaterloss(TEWL)usingaTM300Tewameter(cutometerMPA580,Courage&Khazaka).30TEWLwasquantifieding/m2hbyaskinevaporimetermadeofa small cylindricalopenchamber (1cm indiameter,2cminheight)withtwohygrometricsensorsconnectedtoamicropro‐cessorpluggedintoacomputerworkstation.ThedeviceallowstherecordingofTEWLvalues(rangingfrom0to90g/m2h)aswellastherelativehumidity(rangingfrom0%to100%)andprobetemperature.

2.6 | Biocellulose mask specific activity

2.6.1 | Anti‐aging effect (ABM)

AssessmentswereperformedatT0,after12applications(T1),andattheendoftreatmentafter24applications(T2).Theinvestigatedareasweretheforeheadonbothsidesofthefaceandtheforeheadfoldforwrinkleinvestigation.

Skin surface roughness

Skin surface roughnesswas evaluated using a PRIMOSpico instru‐ment(GFM),whichallowstheacquisitionofa3Dimageoftheskinsurface by an optical analysis system and its transformation usingdedicatedsoftware.The3Dimageacquiredwiththeinstrumentcanbe transformedusingspecific software intoacolor image inwhicheachshadeofcolor(frombluetoyellow)isrelatedtoaheight;thus,thepresenceofagroovewillbeindicatedbyacolorgradientthatcantransitionfrombluetogreenaccordingtoitsdepth.Parameterschar‐acterizingskinmicro‐reliefswhichcanbemeasuredataglancethankstothis3Dtopographicrepresentationoftheskinarethefollowing:

Rarepresentstheaverageskinroughness.Itistheintegralofthefunctiondescribingtheskinroughnessprofilecurve;thehigherthevalueofRa,thehighertheskinroughness.

Rmaxrepresentsthemaximalskinroughness.Singleroughnessdepths are first determined by measuring the peak‐to‐valley dis‐tancewithineachevaluatedsampling length.Rmax isthegreatestpeak‐to‐valleydistancewithinanysinglesampling length,which isdefinedastheverticaldistancebetweenthetopofthehighestpeakandthebottomofthelowestvalley.

Dermis homogeneity

Ultrasound images were produced and further processed by a22MHzDUBsystem(tpmTabernapromedicumGmbH),whichal‐lowstheacquisitionofanultrasoundimageoftheskinandthemeas‐urement of skin tissue thickness inμm (A‐scan andB‐scanmode,respectively), thuspermitting the visualizationof structures up to8mm in depth. The dermis is echogenic, with echoes originatingfromthefibernetworkcomprisedofcollagenandelasticfibers;fatandedemaareecho‐poorareas.

2.6.2 | Lifting effect (LBM)

Assessments were performed at T0 and at the end of treatmentafter12applications (T1).The investigatedareawas the foreheadonbothsidesoftheface.

Viscoelastic properties of the skin

Theeffectoftheproductsonskinmechanicalpropertieswaseval‐uated using anMPA580 cutometer (Courage& Khazaka). In thistechnique, theskin isevaluatedthroughthemeasurementof theverticaldeformationoftheskininducedbyvacuumaspirationwithanegativepressureof450mbar,which isapplied to theskin forbetween1and3secondsthroughaprobewithadiameterof2mm.Eachaspirationisfollowedbyareleasetime,allowingtheskintoreturntoitsrestingconditions.Theconsideredparameterswere:

• R0:Skinfirmness.Thisparametermeasuresthemaximumampli‐tudethattheskinreachesfollowingtheapplicationofsuctionbythecutometerandrepresentsthepassiveresponseoftheskintoappliedforce.R0representstheratiobetweenskinloosenessandskintightness;thelowerR0is,thehigherthefirmnessoftheskin.

• R2:Anindexrepresentingthecapabilityoftheskintoreturntoitsrestconditionafterbeingexposedtomechanicalstress,thatis,itisanindexofthetotalelasticityoftheskin.ThecloserR2isto1,themoreelastictheskinis.

• R5: The ratio between the skin's immediate recovery and itsimmediate deformation. Therefore, it is a measure of the netelasticity.

• R7:Theportionoftheskinelasticitycomparedtothecompleteskindeformationcurve.ThecloserR7isto1,themoreelastictheskinis.

2.6.3 | Purifying and renewal effect (PRBM)

Assessments were performed at T0 and at the end of treatmentafter 12 applications (T1). To study the stratum corneum renewaleffect,theinvestigatedareawasthecheekbone.

Stratum corneum renewal effect

Thestratumcorneumrenewaleffectwasinvestigatedbytheuseoftapestripping,whichisoneofthemostfrequentlyusedapproachestosamplethestratumcorneum.31Thismethodinvolvestheapplica‐tionofadhesive tapewithadiameterof14mm (D101D‐SquameStrippingDiscs,Cuderm)totheskin,anditssubsequentremovaltostripoffalayerofstratumcorneum.Aconstantpressureof225g/cm2 is impressed on the disk surface. The infrared densitometry(IRD)techniquewasusedtoquantifytheabsoluteamountofstra‐tumcorneumremovedbyeachtapestrip.

Inparticular,aSquameScan™device(CuDerm)wasusedtomea‐sure theproteincontent (PC)of thestratumcorneumsamples re‐moved by tape stripping. This apparatus has a diodewhich emitslightatapeakwavelengthof850nmthatshinesthroughthetape

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stripontoadevicewhichmeasurestheamountoftransmittedlight.TheresultsareexpressedasPCinµg/cm2.

2.7 | Statistical analysis

Data were processed both for descriptive statistical analysis andstatisticalanalysiswithcomparisontestsforparametricdata.Inpar‐ticular,intheself‐controlledstudymethodologyusedinthiswork,apaired ttestwasapplied.Foreachparameteranalyzedintheclinicalstudies, a comparisonwasmadebetween thevaluesmeasuredattimeTi (4or8weeks)with respect to the initial value at timeT0andanevaluationofthenetactive‐placeboeffect,forbothactiveandplacebomasks.Foreachdataseriescomparedusingthettest,normal distributionwas verified by theKolmogorov‐Smirnov test.Asignificancelevelof5%waschosen,sochangeswereconsideredstatisticallysignificantforP < 0.05.

3  | RESULTS

Allvolunteersusedanactivemaskononehalfofthefaceandapla‐cebomaskontheotherhalfofthefacefortheentiredurationofthetest.Arandomizedapplicationprocedurewasapplied.

3.1 | Tolerability and moisturizing effect of biocellulose masks

The instrumental evaluation of the EI can give information aboutmasktolerability.

Figure1reportsthemainresultsfromtheerythemaanalysisatT0 and after 12 applications in the facial area for treatmentwithactivemasks(ABM,LBM,PRBM)andPLBM.

ThevariationoftheEI(erythemaindex)after4weeksofappli‐cationwasfoundtobestatisticallysignificantonlyintheABMtest,

inwhichanincreaseof4.45%wasobserved.IntheABMstudy,thefinalobservationwasmadeatT2(after8weeks).Atthistime,thevariation in erythemawith respect to T0was 3.71% andwas notstatisticallysignificant(P=0.1677).Theseresultshighlightthatthetestedproductswereverywelltolerated.

SCWC and TEWL were investigated since they are relevantparameters for the evaluation of skin‐moisturizing effectiveness.Furthermore,asignificantreductionintheTEWLmaybesymptom‐aticofanocclusiveeffectoftheformulationinuse,whileanincreasecanhighlightaspecificalterationreactionoftheskinbarriercausedbytheuseoftheproduct.Therefore,TEWLisconsideredtobeanimportantmeasureofepidermalbarrierfunction.

Figure 2 reports the main results for SCWC and TEWL after12applicationsintheareastreatedwithactivemasks(ABM,LBM,PRBM)andintheareatreatedwithaPLBM.

F I G U R E 1  Comparisonofthevariationoftheerythemaindexintheareastreatedwithanti‐agingbiocellulosemasks(ABM),liftingbiocellulosemasks(LBM),purifyingandregeneratingbiocellulosemasks(PRBM),andplacebobiocellulosemasks(PLBM).*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

F I G U R E 2  Comparisonofthevariationinstratumcorneumwatercontent(SCWC)andtransepidermalwaterloss(TEWL)intheareastreatedwithABM,LBM,PRBM,andPLBM.*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

F I G U R E 3  Confidenceintervalsofthenetactive‐placeboeffect.Theareabetweenthelowerandupperboundscorrespondstotheregionoftheacceptanceofthenullhypothesis,thatis,thatthereisnodifferencebetweentheactiveandplacebomasks

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The results show that, after 12 applications, all active masks,namely ABM, LBM, and PRBM, led to statistically significant in‐creasesoftheSCWC,of9.1%(P=0.006),11.54%(P=0.0045),and10%(P=0.0001),respectively.TheincrementoftheSCWCparam‐eterintheplaceboareawasstatisticallysignificant(P=0.0324)only

for PRBM,with an increase of 5.24%. Variations in TEWL valueswerestatisticallysignificantbothintheLBMandinthePRBMtestsforbothactiveandplacebomasks(Figure2).Inanycase,TEWLval‐uesremainedwithintherangeofhealthyskin,confirmingthehightolerabilityoftheproducts.

Thenetactive‐placeboeffectforallstudiesisshowninFigure3.Thenetactive‐placeboeffectfortheSCWCparameterwasalwaysstatistically significant andwasmore than 10% for the ABM andLBMandlessthan5%forthePRBM.Nostatisticallysignificantdif‐ferencesinthenetactive‐placeboeffectwerefoundforTEWLorEI.

3.2 | Specific activity of biocellulose masks

3.2.1 | Anti‐aging effect

Inordertoevaluatetheanti‐agingeffectofABM,24subjectsbe‐tween42and64yearsoldwithsignsoffacialskinagingwereen‐rolledtousetheactivemaskandaplacebomaskthreetimesaweekfor2monthsusingthesplit‐facemethod.

Skin texture improvement and wrinkle‐reduction effect

The invivoevaluationof thesurfacetopographyofhumanskin isof great interest for dermatological research, as such topographyrepresents the3Dorganizationof the dermis and the subcutane‐oustissue.Thesurfacetopographycanbeconsideredtoreflectthe

TA B L E 1  Meanvalues,variation,neteffect,andstatisticalsignificanceofaverageskinroughness(Ra;µm)andmaximalskinroughness(Rmax;µm)obtainedforallsubjectsintheareastreatedwithanti‐agingbiocellulosemasks(ABM)andplacebobiocellulosemasks(PLBM)after4wk(T1)and8wk(T2)oftreatment

Parameter Study

Mean T1‐T0 T2‐T0

T0 T1 T2 Variation Variation (%) P‐value Variation Variation (%) P‐value

Ra ABM 31.13 29.46 27.63 −1.667 −5.13 0.0101 −3.500 −10.99 0.0000

PLBM 29.79 29.25 29.08 −0.542 −1.29 0.2965 −0.708 −1.92 0.5461

NetEffect 1.33 0.21 −1.46 −1.125 −3.84 0.1180 −2.792 −9.07 0.0000

Rmax ABM 217.88 209.04 194.46 −8.833 −3.62 0.1253 −23.417 −10.33 0.0000

PLBM 201.33 200.46 201.25 −0.875 0.75 0.8678 −0.083 0.72 0.9891

NetEffect 16.54 8.58 −6.79 −7.958 −4.37 0.1846 −23.333 −11.04 0.0007

F I G U R E 4  Variationandstatisticalsignificanceofaverageskinroughness(Ra)andmaximalskinroughness(Rmax)intheareastreatedwithABMandPLBM.*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

F I G U R E 5  3Dskinimagesofaforeheadbeforetreatment(left)andafter2mooftreatmentwithABM(right)

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functionalstatusoftheskin;itisanexpressionoftheabilityoftheskin to respond to mechanical stimuli and threats. Various treat‐mentsexisttoreverseoratleastdecreasechangesinskintexture.Inordertoassessthesuccessofananti‐agingtreatment,wrinklesandthevariationinmicro‐reliefdepthneedtobemeasuredbeforeandaftertheapplicationofthecosmeticproduct.

The 3D system permitted the evaluation, bymatching, of thesameskinareainthethreesubsequentanalyses.TheparametersRa(theaverageskinroughnesscorrelatedtosensoryperception)andRmax(thegreatestpeak‐to‐valleydistanceinasinglewrinkle)weremeasured.ThegreaterthevaluesofRaandRmax,thegreatertheskinroughnesscausedbyskintexture,lines,andwrinkles.

TheresultsobtainedarereportedinTable1.Theresultsshowthat theapplicationof theABMledtoasig‐

nificantdecreaseintheskinroughnessof5.13%(P=0.0101)after12applications,andasignificantdecreaseof10.99%(P=0.0000)after2monthsoftreatment.ThevariationofthisparameterintheplaceboareawasnotsignificantatT1oratT2 (Figure4).ThenetABM‐PLBMeffectshowedatrendofdecreasingskinroughnessthatwasnotstatisticallysignificantatT1(P=0.1180)butwasstatisticallysignificant at T2,when a decrease of about 9% (P = 0.0000)wasobserved,asshowninFigure5.

Atthesametime,asignificantdecreaseinthebreadthofwrin‐klesof10.33%(P=0.0000)wasdetectedafter2monthsoftreat‐ment. The variation of this parameter in the PLBM area was notstatisticallysignificantatT1orT2(Figure4).

Theseeffectscouldbeduetothepresenceofvariousactive in‐gredients in theABM.Fruit extractofA digitata, alsoknownas thebaobab tree, contains a high concentration of vitamin C—seventimesmore thanorange fruit—and flavonoid compounds showing ahigh antioxidant efficacy.21 Hibiscus sabdariffa flower extract is richinphenolicacids, flavonoids, andanthocyanins,whichexertprotec‐tiveeffects againstoxidative stress.22 Furthermore, acetyldecapep‐tide‐3andpalmitoyl tripeptide‐38arebiomimeticpeptides identicaltoaminoacidsequencessynthesizedbyhumansandsocouldinteractwithgrowthfactorreceptorsandprovideclinicalanti‐agingeffects.23

The3DimagesshowninFigure5,whichwerecreatedusingthesoftware, clearly show the reduction inwrinkle breadth following2monthsoftreatmentwithABM.

Improvement in dermis homogeneity and thickness

Theevaluationscarriedoutbyultrasoundpermittedthetotalthick‐ness of the epidermis and dermis to be visualized andmeasured.

F I G U R E 6  B‐modeultrasoundimagesofskinatT0(A),after12applicationsofABM(B),andattheendofthestudyafter24applicationsofABM(C).Theepidermisanddermisarevisibleasecho‐rich(green)areas

(A) (B) (C)

F I G U R E 7  VariationandstatisticalsignificanceofdermisthicknessobtainedforallsubjectsintheareastreatedwithABMandPLBMafter4wk(T1)and8wkoftreatment(T2).*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

F I G U R E 8  Confidenceintervalsoftheactive‐placeboneteffect.Theareabetweenthelowerandupperboundscorrespondstotheregionofacceptanceofthenullhypothesis,thatis,thatthereisnodifferencebetweentheactiveandplacebomasks

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MeasurementsofskinthicknesswereperformedusingtheA‐scanmodeonallsubjectsatbaselineandafter12and24applications.

Figure6showsexamplesofultrasoundimagesofskinatbaselineandafter12and24applicationsofABMtreatment.

Thecomparisonofultrasoundimagesbeforeandaftertreatmentclearlyshowsadifferenceintheorganizationofthetissuebelowtheepidermisresultinginanimprovementofdermishomogeneityandfirmness.Theseresultswereconfirmedbymeasurementsofdermisthickness.

The ABM led to an average reduction in dermis thickness ofmorethan3%(P=0.0001)after12applicationsandofmorethan4%(P=0.0000)after24applications.Intheareastreatedwiththeplacebo,nosignificantdifferenceswereobservedatT1,whileasig‐nificantreductionofdermisthicknessofmorethan1%(P=0.0117)wasobservedatT2(Figure7).

ThenetABM‐PLBMeffectsforRa,Rmax,anddermisthicknessareshowninFigure8.ThetrendofdecreaseinwrinklebreadthwasnotstatisticallysignificantatT1;however,itwasstatisticallysignif‐icant at T2,when a decrease of about 11% (P = 0.0007)was ob‐served.TheneteffectABM‐PLBMshowedastatisticallysignificantreductioninthedermisthicknessatT1ofabout3.25%(P=0.0000).

3.2.2 | Lifting effect

InordertoevaluatetheliftingeffectofLBM,20subjectsbetween38and57yearsoldwithfacialskinsaggingwereenrolledtousethismaskandaplacebomaskthreetimesaweekfor1monthusingasplit‐facemethod.

Parameter Study

Mean T1‐T0

T0 T1 Variation Variation (%) P‐value

R0 LBM 0.262 0.198 −0.0634 −24.22 0.0000

PLBM 0.247 0.222 −0.0252 −10.63 0.0064

Neteffect 0.014 −0.024 −0.0381 −13.59 0.0002

R2 LBM 0.561 0.621 0.0594 10.78 0.0004

PLBM 0.593 0.633 0.0399 7.29 0.0553

Neteffect −0.031 −0.012 0.0195 3.49 0.4905

R5 LBM 0.426 0.498 0.0724 18.21 0.0103

PLBM 0.473 0.476 0.0034 4.68 0.9072

Neteffect −0.047 0.022 0.0691 13.53 0.0963

R7 LBM 0.252 0.280 0.0279 11.42 0.0050

PLBM 0.275 0.278 0.0029 2.67 0.8302

Neteffect −0.023 0.002 0.0250 8.75 0.1509

Note: R0=skinfirmness;R2=grossskinelasticityindex;R5=netelasticity;R7=viscoelasticityindex.

TA B L E 2  Values,variation,neteffect,andstatisticalsignificanceofskinelasticityobtainedforallsubjectstreatedwithLBMandPLBM

F I G U R E 9  VariationandstatisticalsignificanceofskinelasticityintheareastreatedwithLBMandPLBM.*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

F I G U R E 1 0  Confidenceintervalsofthenetactive‐placeboeffect.Theareabetweenthelowerandupperboundscorrespondstotheregionofacceptanceofthenullhypothesis,thatis,thatthereisnodifferencebetweentheactiveandplacebomasks

     |  9PERUGINI Et al.

Severalelasticityparameterswereevaluated.Table2showsthemeanvalues,variation,neteffect,andstatisticalsignificanceofskinelasticityobtainedforallsubjectsintheareastreatedwithLBMandPLBMafter4weeksoftreatment(T1).

TheresultsshowthattheapplicationofLBMledtoanimprove‐mentinskinfirmness,withasignificantdecreaseintheR0param‐eterof24.22%(P=0.0000)observedafter1monthof treatment(Figure9).Thevariationofthisparameter intheplaceboareawassignificant (P=0.0064),andthenetactive‐placeboeffectshoweda statistically significant decrease of about 13.59% (P = 0.0002;Figure10).

The application of LBM led to a significant increase in theR2parameter of about 10.78% (P = 0.0004) after 1 month of treat‐ment.Thevariationof thisparameter in theplaceboareawasnotsignificant.

TheresultsshowthattheapplicationofLBMledtoasignificantincrease in the R5 parameter of about 18.21% (P = 0.0103) after1monthoftreatment.Thevariationofthisparameterintheplaceboareawasnotsignificant.

TheresultsshowthattheapplicationofLBMledtoasignificantincrease in the R7 parameter of about 11.42% (P = 0.0050) after1monthoftreatment.Thevariationofthisparameterintheplaceboareawasnotsignificant.

The net active‐placebo effect was not statistically significantafter12applicationsofLBMfortheparametersR2,R5,andR7,asshowninFigure10.

Among several ingredients contained in the LBM, hydrolyzedhyaluronic acid could be responsible for the improvement of skinelasticity. Furthermore, the presence of this ingredient togetherwithacetyldecapeptide‐3couldleadtoclinicaleffectssuchastheslowingofaging,duetotheactionofacetyldecapeptide‐3onthe

Sample Study

Mean (µg/cm2) T1‐T0

T0 T1 Variation Variation (%) P‐value

PCtot PRBM 80.59 51.99 −28.60 −33.02 0.0000

PLBM 79.62 76.08 −3.54 −0.92 0.4294

Neteffect 0.97 −24.09 −25.06 −32.09 0.0000

PCS1 PRBM 23.59 17.50 −6.09 −20.00 0.0008

PLBM 23.57 23.98 0.42 9.41 0.7950

Neteffect 0.03 −6.48 −6.51 −29.41 0.0003

PCS2 PRBM 21.74 13.44 −8.30 −35.64 0.0000

PLBM 21.00 20.00 −1.00 0.74 0.4974

Neteffect 0.74 −6.56 −7.30 −36.38 0.0002

PCS3 PRBM 18.76 11.32 −7.44 −36.53 0.0000

PLBM 18.52 16.65 −1.87 −7.63 0.0954

Neteffect 0.24 −5.33 −5.57 −28.89 0.0015

PCS4 PRBM 16.50 9.73 −6.77 −38.41 0.0000

PLBM 16.53 15.45 −1.08 0.46 0.3714

Neteffect −0.03 −5.72 −5.69 −38.87 0.0004

TA B L E 3  Totalandsingletapestrippingproteincontent(PC)datashowingvariation,neteffect,andstatisticalsignificance

F I G U R E 11  Variationandstatisticalsignificanceoftheproteincontent(PC)oftotalandsingletape‐strips(Si)intheareastreatedwithPRBMandPLBM.*,P<0.05issignificant;**,P<0.01isstronglysignificant;***,P<0.001isverystronglysignificant

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F I G U R E 1 2  Confidenceintervalsofthenetactive‐placeboeffectforproteincontent(PC).Theareabetweenthelowerandupperboundscorrespondstotheregionofacceptanceofthenullhypothesis,thatis,thatthereisnodifferencebetweentheactiveandplacebomasks

10  |     PERUGINI Et al.

formation of wrinkles.24,25 Skin roughness decreased significantlyafter1monthoftreatmentwithLBM(by6.7%).Thevariationofthisparameterintheplaceboareawasnotsignificant.UnlikeforABM,nosignificantdifferencewasdetectedinwrinklebreadthfollowingtheuseofLBM.

3.2.3 | Skin purifying and renewal effect

InordertoevaluatetheskinpurifyingandrenewaleffectofPRBM,subjectsbetween25and40yearsoldwithdullfacialskinwereen‐rolledina1‐monthstudyinvolvingthecontinuoususeofPRBMandPLBM.

Atthebeginning(T0)andtheendofthestudy(T1),fourdifferenttapestrippings(S1,S2,S3,S4)wereperformedsequentiallyonthesameareaofthecheekbonetreatedwithPRBMandPLBM.Thetapestrippingprocedureisabletoremovethefirstlayersofthestratumcorneum.

Table3reportstotalandsingletapestrippingPCdatainµg/cm2.TheresultsshowthatPRBMledtoasignificantdecreaseofthe

averagePCandthenof thestratumcorneumcohesionof33.02%(P=0.0000)after1monthoftreatment.ThevariationoftheaveragePCintheplaceboareawasnotstatisticallysignificant(P=0.4294)andwasequalto−0.92%(Figure11).

Thenet active–placeboeffecton the averagePCwas statisti‐callysignificantandcorrespondedtoavariationofabout−32.09%(P=0.0000;Figure12).

The stratum corneumappears to bemore cohesivewith a re‐ductionoftheremovedPC,whichresultsinanimprovementinthesmoothnessoftheskinsurface.ThePRBMseemstoexertarenewaleffectontheepidermisthroughamildexfoliatingaction.

ByanalyzingthePCofeachsingletape,itisclearthat,atthebe‐ginningofthestudy,theamountofproteinswasthesameforbothsidesoftheface.After1monthoftreatmentwithPRBM,a loweramount of proteinwas removed by each tape stripwith a strongstatistical significance,ashighlighted inFigure11,andthenetac‐tive‐placeboeffectwasalwaysstatisticallysignificantforeachstrip(Figure12).

Thesmoothingeffectcouldbeduetoanexfoliatingmechanism.Additionally, if thiseffect ispresent,noerythemaishighlighted inthePRBM‐treatedarea,probablyduetothepresenceofP oleracea extract,which alleviates skin irritation and allergic responses andhaspharmacologicaleffectssuchasantibacterialandanti‐inflamma‐toryeffects.Thus,thisextractisusedmainlyinacnecareproducts,cosmeticsforsensitiveskin,etc.31

4  | CONCLUSIONS

Bacterialcelluloseisaninnovativesubstrateforfacialmasks,mainlyduetoitsfavorablepropertiessuchasveryhighwater‐holdingcapa‐bility,tolerability,andbiocompatibility.

Eventhoughthereisagrowingskincaremarketforcosmeticsheetmasks,thereareveryfewstudiesaddressingtheireffectivenessin

theliterature.Thisworkmainlyfocusedontheinvivoevaluationoftheeffectivenessofthreedifferentfacemasksbasedonbiocellulosesheetsthatwereformulatedtohavespecificcosmeticeffects(anti‐aging,lifting,andcellrenewal).

Scientificprotocolsusingnoninvasiveskinbioengineeringtech‐niquesandadequatestatisticalmethodswereemployedinordertodiscriminatetheefficacyofthedifferentmaskformulationstestedwithrespecttoplacebocomparison.

Inparticular,theself‐controlledstudymethodologyusedinthisworkrevealedtobethebestapproachto investigateboththeef‐fectivenessofactivemasksandtheevaluationofthenetactive‐pla‐ceboeffect.

Theresultsobtainedinthisstudyhighlightthepossibilitytode‐velopcosmeticmaskswithveryhighquality, tolerability, andeffi‐cacybasedonbiocellulosesheetembeddedwithseveralbioactiveingredients, such as peptides, natural extracts, and biopolymers.A statistically significant decrease in skin roughness and wrinklebreadth, and a statistically significant improvement in dermis ho‐mogeneityandfirmness,wasobservedafter2monthsoftreatmentwith “anti‐aging” biocellulose masks. Furthermore, a statisticallysignificant improvement of skin firmness and elasticity was ob‐servedafter1monthoftreatmentwith“lifting”biocellulosemasks.Moreover,1‐monthtreatmentswith“cellrenewal”“purifyingandre‐generatingbiocellulosemask,whichpromotetheproductionofnewskin cells through amild exfoliating action, significantly improvedstratumcorneumcohesion.

Inconclusion,thisstudyhighlightsthatbiocellulosemasksrep‐resentaneffectivetailoredsystemtoexertstatisticallysignificantbeneficialeffectstotheskin.

ORCID

Paola Perugini https://orcid.org/0000‐0002‐8774‐8534

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How to cite this article:PeruginiP,BleveM,RedondiR,CortinovisF,ColpaniA.Invivoevaluationoftheeffectivenessofbiocellulosefacialmasksasactivedeliverysystemstoskin.J Cosmet Dermatol. 2019;00:1–11. https://doi.org/10.1111/jocd.13051