s41368-023-00274-9 by Biogenomics

Emergingrolesofexosomesinoraldiseasesprogression

JiayiWang1,JunjunJing2,ChenchenZhou3 andYiFan 1 ✉

Oraldiseases,suchasperiodontitis,salivaryglanddiseases,andoralcancers,significantlychallengehealthconditionsduetotheir detrimentaleffectsonpatient’sdigestivefunctions,pronunciation,andestheticdemands.Delayeddiagnosisandnon-targeted treatmentprofoundlyinfluencepatients’ prognosisandqualityoflife.Theexplorationofinnovativeapproachesforearlydetection andprecisetreatmentrepresentsapromisingfrontierinoralmedicine.Exosomes,whicharecharacterizedasnanometer-sized extracellularvesicles,aresecretedbyvirtuallyalltypesofcells.Astheresearchcontinues,thecomplexrolesoftheseintracellularderivedextracellularvesiclesinbiologicalprocesseshavegraduallyunfolded.Exosomeshaveattractedattentionasvaluable diagnosticandtherapeutictoolsfortheirabilitytotransferabundantbiologicalcargosandtheirintricateinvolvementinmultiple cellularfunctions.Inthisreview,weprovideanoverviewoftherecentapplicationsofexosomeswithinthe fieldoforaldiseases, focusingoninflammation-relatedbonediseasesandoralsquamouscellcarcinomas.Wecharacterizetheexosomealterationsand demonstratetheirpotentialapplicationsasbiomarkersforearlydiagnosis,highlightingtheirrolesasindicatorsinmultipleoral diseases.Wealsosummarizethepromisingapplicationsofexosomesintargetedtherapyandproposedfuturedirectionsforthe useofexosomesinclinicaltreatment.

InternationalJournalofOralScience (2024)16:4 ;https://doi.org/10.1038/s41368-023-00274-9

INTRODUCTION

Theoralcavityistheinitialsegmentofthedigestivesystem, secondsourceofrespiration,andacrucialorganforpronunciation,mastication,andfacialesthetics.Poororalhealthmayhave animpactonaperson’soverallhealth,causingpain,discomfort, anddisﬁgurement.1 Oraldiseases,suchasdentalcaries,periodontaldiseases,andoralcancers,affectnearly3.5billionpeople, whichareglobalburdensthatcausepatientssuffering,especially thosewithalowsocioeconomicstatus.2 Toalleviatethese burdens,researchershavecomeupwithinnovativemethodsfor earlydiagnosisandeffectivetreatment.

Extracellularvesicles(EVs)arederivedfromcellularmembranes andreleasedintotheextracellularspace;theyplaycriticalrolesin intercellularcommunication.3 Therearetwomaincategoriesof EVs,includingexosomesandectosomes.4 Exosomes,whichare principalconstituentsofEVs,arederivedfromtheendosomal systemandpossessbilayerlipidencapsulation,withdiameters rangingfrom30to150nm.5 Comparedwithectosomes,which assemblecargosonthecytosolicsurfaceandtransientreleasein “outwardbudding” , 6 exosomesexhibitmoreintricateinteractions withcyto-inclusionsandhavegarneredsigniﬁcantattentionfrom biologicalresearchersworldwidefortheirabundantcargosand variousfunctionsassociatedwithphysiologicalorpathological processes.5

Thebiogenesisofexosomesistightlyregulatedthrougha complexnetworkofprocesses.Uponendocytosis,thepotential cargosareinternalizedbythecellsandgiverisetoearly-sorting endosomes(ESEs).Thesubsequentinteractionswithorganelles, suchastheendoplasmicreticulum(ER)andGolgiapparatus,lead

tothematurationofESEsintolate-sortingendosomes(LSEs). Followingthis,thecargosaccumulatenearthelimitingmembrane ofmultivesicularbodies(MVBs)andgenerateintraluminalvesicles (ILVs),whichwilleventuallybereleasedasexosomesvia exocytosis.5,7,8 ItshouldbenotedthatsomeILVsmayalso undergointeractionswithlysosomesorautophagosomes.5,7,8 Throughouttheentireprocess,variousregulators,suchasthe endosomalsortingcomplexrequiredfortransport(ESCRT), tetraspaninCD9/CD63/CD81,andAlix/programmedcelldeath 6-interactingprotein(PDCD6IP),areinvolvedintheintricate mechanismsofsortingandsecretion.5,7–9 Accordingly,an extensivebodyofresearchhasconfirmedthatthe finalversion oftheexosomescontainsdiversifiedcontents,includingamino acids,proteins,nucleicacids,andcellularmetabolites.These moleculesplaydistinctrolesinintercellularsignalingtransmission, immune-modulation,stromaladaptation,andmultiplebiological events.9

Therefore,exosomesexhibitsignificantpotentialinmanaging diseaseprocesses.Previousresearchhaselucidatedtheclose correlationbetweenexosomesandoraldiseases,including periodontalinflammation,oralsquamouscellcarcinomas(OSCCs), oralmucosadiseases,etc.Theapplicationofexosomesinvolves monitoringtheprogressionofthediseases,earlydiagnosis, detectionthroughspecificmanifestationsin fluid,andadvanced targetedtherapyviaprecisemoleculedelivery.Thisreview concludestherecentstudiesontheapplicationsofexosomesin oraldiseasesandaimstoprovideacomprehensiveinsightinto thelatestdevelopmentsinexosomealterations,functions,and applicationsinoral-relatedphysiologicalandpathological

1StateKeyLaboratoryofOralDiseases&NationalCenterforStomatology&NationalClinicalResearchCenterforOralDiseases&DepartmentofCariologyandEndodontics,West ChinaHospitalofStomatology,SichuanUniversity,Chengdu,China; 2StateKeyLaboratoryofOralDiseases&NationalCenterforStomatology&NationalClinicalResearchCenter forOralDiseases&WestChinaHospitalofStomatology,SichuanUniversity,Chengdu,Chinaand 3StateKeyLaboratoryofOralDiseases&NationalCenterforStomatology& NationalClinicalResearchCenterforOralDiseases&DepartmentofPediatricDentistry,WestChinaHospitalofStomatology,SichuanUniversity,Chengdu,China Correspondence:YiFan(yifan@scu.edu.cn)

Received:26October2023Revised:11December2023Accepted:26December2023

conditions.Theultimategoalistoidentifynewopportunitiesfor theeffectiveutilizationofexosomesinthepreventionand treatmentoforaldiseases.

EXOSOMESINTHEPROGRESSIONOFORALDISEASES

Asresearchershavedemonstratedtheintricaterolesofexosomes inbiologicalevents,theirdistinctalterationsaresigniﬁcant componentsinthepathologicalprocessesoforaldiseases.At theinitialstage,analyzingtheexosomalinformationhelpsto improveourearlyawarenessoforaldiseases.Duringthe developmentandprognosisstages,summarizingtheexosomerelatedmanifestationscanenhanceourcomprehensionofthe selectionandresponseoftreatments.Therefore,exosomesserve asacrucialindicatorinthediagnosis,monitoring,andtreatment ofseveraloraldiseases.Inthispart,wesummarizetherolesof exosomesindeterminingtheprogressionoforaldiseases,witha focusonperiodontaldiseasesandoralcancers.

Periodontalandbone-relatedpathologicalstatus

Periodontalinflammationandboneresorption.Followingexposuretoriskfactors(intrinsicand/oracquired),thepathological changesinperiodontitisareinitiatedbyimmune-inflammatory responses,leadingtothereleaseofinflammatorymolecules,such ascytokinesandmatrixmetalloproteinases(MMPs).These moleculesexerttheireffectsonperiodontaltissues,thereby inducingclinicalmanifestations.10 Osteoclastsareactivatedin inflammatorymicroenvironmentsandleadtothedestructionof thesurroundingalveolarbone.Subsequently,theseirreversible destructionsofperiodontalstructuresacceleratetheprogression ofperiodontitisandleadtotoothmobilityoreventoothloss, severelyimpactingthepatients’ qualityoflife.11 Theprevious studieshavedemonstratedthesignificanceofinflammatory factorsinperiodontitis.10 Itisnoteworthythatexosomesplay importantrolesinthemodulationandalterationofperiodontal inflammationandboneresorption.12

Periodontalligamentstemcell(PDLSC)-derivedexosomes signiﬁcantlyimprovedangiogenesisininﬂammatoryregionsby upregulatingthevascularendothelialgrowthfactor(VEGF)in humanumbilicalveinendothelialcells(HUVECs)viamiR-17-5p.13 SalivaryexosomalmiR-223-3pincreasedtheinterleukin(IL)-1β and IL-6levelsbymediatingNLRP3geneexpressionandpyroptosis.14 Oxidativestressisinvolvedinperiodontitisprogressionwith abnormalreactiveoxygenspecies(ROS).Proteinargininemethyltransferase1(PRMT1)inducedbyoxidativestressinhibited exosomesecretionfromperiodontalligamentcells(PDLCs), resultinginreducedosteogenicdifferentiation.15

Theinteractionsbetweenthehostin fl ammatoryresponse andmicrobearealsocloselyinvolvedintheprogressionof periodontitis. 10 SimilartoexosomalmicroRNAs(miRNAs) derivedfromhostcells,smallRNAsofmiRNAsize(msRNAs) frompathogens( A.actinomycetemcomitans,P.gingivalis,andT. denticola )weredetectedinthebacterialoutermembrane vesicles. 16 P.gingivalis canalsoassignasenescence-associated secretoryphenotype(SASP)todendriticcells(DCs)andTcells byexosomes,andultimately,thi sresultsinalveolarboneloss. 17 Moreover,mechanicalstimuliareapromoterofperiodontal in fl ammatoryandtissue/boneimpairment,mainlyactingon PDLCs. 18 PDLCssecreteexosomalmiR-9-5pwhenfacingcyclic stretchingandpromotetheM1(pro-in fl ammatory)polarization ofmacrophagesthroughthemiR-9-5p/SIRT1/NF-kBpathwayin murinemodels. 19 TheM1macrophagewasalsoinducedby periodontalligament fi broblast-derivedexosomeswhena compressiveforcewasapplied.Theunderlyingmechanism wasassociatedwiththeYes-associatedprotein(YAP), 20 whichis acrucialcomponentintheHi pposignalingpathwayand performssigni fi cantrolesincellularmechanotransduction. 21 In theimmune-modulationofthein fl ammatoryresponse,the

YAP/Hippopathwayactsasakeyupstreamregulator. 22 These studieshighlightthecomplexinteractionsbetweenexosomes andtheclinicalmanifestationsofperiodontaldiseases,accordingtowhichweshouldtakemorestepstoattenuatethe progressionofperiodontitis.

Orthodonticmovement.Orthodontictreatmentinvolvesthe complexprocessesofalveolarboneremodelinguponthe applicationofmechanicalforces.Duringmovement,theexpressionofgingivalcrevicular fluid(GCF)exosomalmiR-29is significantlyincreased.23 Meanwhile,thePDLSC-derivedexosomal miRNAsarelargelyaltered,24 25 andthequantityofexosomal proteinsannexinA3(ANXA3)increases,whichinducesosteoclast differentiationbytheactivationofextracellularregulatedprotein kinase(ERK).26 Whenorthodonticmovementstopsandteeth positionsatisfiesourneeds,PDLSC-derivedexosomesalso contributetoteethstabilization.Forinstance,Simvastatin,a bone-formation-enhancingdrug,hasbetterbioavailabilityin conjunctionwithPDLSC-derivedexosomes.27

Oralsquamouscellcarcinomas

Withincreasingincidenceandmortalityrates,oralcancersrankas the13thmostcommoncancerworldwide,withanestimated 377,713newcasesand177,757deathsin2020.28,29 The consumptionoftobaccoandarecanutsignificantlyincreases theriskoforalcancers.Specifically,oralcancerscauselarger burdensindevelopingcountriesduetodelayeddiagnosisand limitedtreatmentopportunities.29 Amongallthetypesoforal cancers,OSCCsarethemostcommon,withanestimated40% higherincidenceratein2040.30 Summarizingthealterationsinthe exosomesinOSCCscouldenhanceourunderstandingof malignantprogression.Here,wediscussexosomalrolesin malignization,angiogenesis,andtumormicroenvironment(TME) modificationinOSCCprogressionandthepost-treatment response(Fig. 1).

Malignization.Accordingtotranscriptomeanalysis,headand necksquamouscellcarcinoma(HNSCC)-associatedexosomesplay asigniﬁcantroleinvariousprocessesthroughoutcancerdevelopment.31 AsthemostcommonmalignancytypesinHNSCCs,OSCCs exhibitvariousexosomalalterations.Fromtheoutset,theinjection ofOSCC-tumor-derivedexosomesacceleratedthemalignancy progressionofprecancerouslesionsinmurinemodels,32 andthis phenomenonwasattributedtoexosomalmiR-10bthroughAKT signaling.33 Thesameinitiationofmalignancyalsooccursin recurrentOSCCswithincreasedserumexosomallongnon-coding RNA(lncRNA)-CCDC144NL-AS1andMAGI2-AS3viathePI3K-AKTmTORpathway34 (Fig. 1a).Uponthemanifestationoftheseearly malignancysymptoms,thesubsequentprogressionofOSCCs ensuesthroughdiverseexosomalalterations.

Angiogenesis.Inducingthevasculatureisanimportanthallmark ofcancer,35 andtheexosomesacceleratethisprocessthrough severalpathways(Fig. 1b).Phosphataseandtensinhomolog deletedonchromosometen(PTEN)isatumor-suppressingfactor, anditsexpressionisdownregulatedinOSCCs.36 ExosomalmiR130b-3pderivedfromOSCCscellsandmiR-23b-3pfromsalivary adenoidcysticcarcinomas(SACCs)cellsnegativelyregulatePTEN expression,promotingmigrationandangiogenesisinHUVECs.37 38 miR-221sandmiR-210-3palsoregulatedHUVECsangiogenesis throughthePI3K/AKTpathway.39,40 Besidescancercells,the mesenchymalstemcells(MSCs)inoralcarcinomascansecrete angiogenesis-stimulativeexosomes.OSCC–MSC-derivedexosomal matrixmetalloproteinases1(MMP1)signiﬁcantlyenhancesthe functionofHUVECs.41

TMEmodiﬁcation.Inadditiontotumorcells,TMEexertsa dominantinﬂuenceonthedevelopmentofOSCCs,involving

Malignization Angiogenesis

miR-10b, lncRNAs...

PI3K-AKT-mTOR

TME modifictaion c

PI3K/AKT pathway

miR-130b-3p miR-23b-3p

miR-221s miR-210-3p

Epithelial cells migration and angiogenesis

M1 M2 polarization polarization

THBS-1 (p38, Akt and SAPK/ JNK signaling)

miR-382-5p,miR-146b-5p

d Post-treatment reaction

miR-21, miR-24... STAT3, PTEN pathways miR-196a

miR-30a inhibitor of miR-155

Enhance EMT UCA1, ERHSP-90,CMTM6,miR-29a-3p,(PD-L1)stress Cytotoxicity

circ_0069313(PD-L1) immune escaping

Fig.1 ExosomesinOSCCs.ExosomesplaycrucialrolesinseveralkeyprocessesthroughOSCCsprogression. a AdditionofexosomalmiR-10band increasedlevelofseveralexosomallncRNAs(targetingPI3K-AKT-mTORpathway)leadtoinitiationofOSCCs. b OSCCcellsderivedexosomalmiR130b-3pandmiR-23b-3ppromotedangiogenesisthroughdownregulatingPTEN,whilemiR-221sandmiR-210-3pactivatedthroughPI3K/AKT pathway.MSCs-derivedMMP1playedthesameroleinangiogenesis. c DuringtheprogressionofOSCCs,TMEexhibitsintricateinteractionswith tumors.ExosomesderivedfromOSCCcellspromoteddifferentiationofCAFs,whileCAFssecretedexosomalmiR-382-5pandmiR-146b-5pto enhancetumordevelopment.Inimmuneregulations,cancer-derivedexosomalTHBS-1inducedpolarizationofTAMstowardM1type.Exosomal UCA1,miR-29a-3p,CMTM6,HSP-90andPD-L1(underERstress)leadTAMstoM2type.BothM1andM2macrophagescontributetothe progressionofmalignancy.Tcellswerealsomodulatedbycancercellsthroughexosomalcirc_0069313,targetingPD-L1.Inaddition,Tregsplayed acrucialroleinfacilitatingimmuneevasioninOSCCs. d ExosomescanregulatetreatmentresponseofOSCCtochemotherapyandradiotherapy

intricateinteractionswithexosomes(Fig. 1c).TheTMEis composedoftheextracellularmatrix(ECM)andrelevantcells, includingcancer-associated ﬁbroblasts(CAFs),endothelialcells, andimmunecells.42 InOSCCs,tumor-derivedexosomesinduce

CAFdifferentiation.43 Inturn,theCAFshavereceivedgreat attentionfortheirsigniﬁcanceinpromotingtumordevelopment throughintracellularcommunications,primarilyviaexosome secretion.44 InOSCCs,theCAF-derivedmiR-382-5p45 andmiR-

146b-5p46 exosomesareupregulated,leadingtotheinvasionof cancercellsandmetastasis.Inaddition,themiR-34a-5p-devoid exosomesfromCAFspromotemalignancybytargetingtheAXL(a componentofthereceptortyrosinekinase)ofcancercells.47

ByanalyzingthegenesrelatedtoEVformationintheHNSCCs samplesandtheirimpactoncellularbehaviors,otherscientists havefoundasigniﬁcantcorrelationbetweenEVsandimmune modiﬁcation(T/Bcells,macrophage,andneutrophils)whena malignancyoccurs.48 Thetumor-associatedmacrophages(TAMs) playacrucialroleasimmunecellswithintheTME.Generally,the TAMsmainlypolarizeintotwostates theM1andM2subtypes.49 Accordingtotheconventionalperspective,theM1macrophages performanti-tumorfunctionsbydirectcytotoxicityorantibodydependentcell-mediatedcytotoxicity(ADCC),whiletheM2 macrophagespromotecancerprogressionandmetastasisby secretingcytokinesandrelatedmolecules,suchasILs,epithelial growthfactors(EGFs),andMMPs.50 However,theinvolvementof M1macrophagesintheprocessofmalignancyinvasionhasbeen recentlyobserved,suggestingthattheirpresencemaycontribute tomoreaggressivemalignization,butnotahighersurvivalrate.51

InOSCCs,theTAMshavecomplexinteractionswiththetumor cells.Theexosomaltransforminggrowthfactorbeta(TGF-β) derivedfromHNSCCscellscanpromoteangiogenesisbyboth interactingwithepithelialcellsandmodulatingTAMschemotaxis forpro-angiogenicfunctions.52 OSCC-derivedexosomalthrombospondin1(THBS-1)activatesanM1-likemacrophagethrough p38,Akt,andSAPK/JNKsignaling,whichpromotescancer progression.53 M1-likeTAMsenhancetheOSCC epithelial–mesenchymaltransition(EMT)andcancerstemcell formationthroughtheIL-6/Jak/signaltransducerandtheactivator oftranscription3(Stat3)/THBS-1axis.54

Furthermore,oralcancercellsarecloselyassociatedwiththe conventionaloncogenicM2macrophagesthroughmultiplepathways.50 OSCCcancerstemcell-derivedexosomespolarizeTAMs intoM2macrophagesbytheurothelialcarcinoma-associated1 (UCA1)secretiontargetingLAMC2-PI3K/AKTsignalingpathway. Theseexosomesalsosuppressanti-tumorimmunity,including CD4+ Tcellsactivationandinterferon-γ (IFN-γ)production.55 In addition,OSCCcancercellssecreteexosome-enclosedcargos, inducingM2macrophages.MiR-29a-3p,amemberofthemiR-29 familythatsigniﬁcantlyincreasesinOSCCs,56 exertseffectsonM2 polarizationthroughthesuppressorofcytokinesignaling1 (SOCS1)/signaltransductionandthetranscriptionalactivator (STAT)pathway.57 TheCKLF-likeMARVELtransmembrane domain-containing6(CMTM6)58 andheatshockprotein-90 (HSP-90)59 areimportantproteinicinﬂuencersofM2macrophage conversion,indicatingnovelcrosstalkbetweencancercellsand immune-modulation.ERstress,afunctionalproteinaceous responsethatreactstocellulareventslikeoncogenesis,drives OSCCcellstosecreteexosomalprogrammeddeathligand1(PDL1)andactivatetheM2polarizationofTAMs.60

BesidesTAMs,Tcellsarealsoimportantimmuneregulatorsin OSCCs.T-effector(Teff)cellsandT-regulatory(Treg)cellsarethe twomainclassiﬁcationsofTcells.61 Teffcellsprimarilyexecute killingfunctionstowardpathogensor,anomaly,self-antigens.Treg cellsmodulateover-functioningTeffcellsandmaintainimmune homeostasis.However,intheoncogenesisprocess,Tregcellsmay leadtoimmuneevasion.61 InOSCCs,exosomalcircularRNA circ_0069313couldincreasethePD-L1expressionincancercells withmiR-325-3pspongingandinteractwithTregcells.Thus,the Teffcellsaresuppressed,whiletheTregcellsareactivated, resultingintheimmuneescapingofmalignancy.62

Besidesresistance,theproﬁleofexosomesinOSCCsaltersin responsetotreatment.Aftersurgeryand/orchemo-radiotherapies,monitoringimmune-relatedexosomalproteins(suchasPDL1)couldaccuratelyindicatethetreatmentreactionand recurrencepossibility.73,74 Melatonin,ahormonesecretedbythe pinealgland,playscrucialrolesinmultiplephysiological processes.75 Therecentresearchhashighlighteditspotential functioninOSCCtherapyasanadjuvant,owingtoitsabilityto inhibittumorprogressionbymodulatingkeyregulators,suchas MMP-9,p53,andepidermalgrowthfactorreceptor(EGFR),aswell asenhancingimmunefunctions.76,77 TheexpressionofOSCCcellderivedexosomes(miR-21andmiR-155)undergoesalterations followingmelatoninapplication,indicatingtheirpotentialfor assessingthetreatmentresponseandpredictingtheprognosis.78 Inconclusion,exosomesexhibitvariouscharacteristicsduring thedevelopmentandprognosisofOSCCs.Byimprovingour comprehensionofthesealterations,wecanapproachabetter understandingoftheenigmasurroundingOSCCoccurrenceand conductfurtherinvestigationsontherapeuticinterventions.

EARLYDIAGNOSTICMETHODSUTILIZINGEXOSOMESINORAL DISEASES

Accordingtotheabove,itisconvincingthatexosomesplaya crucialroleintheprogressionoforaldiseases.Ourparticular emphasisliesinexosomalapplicationforearlydiagnosisdueto thepotentialexacerbationofpatients’ sufferingcausedby delayeddetection.Incontrasttothemoreapparentbiomarkers, suchasinflammatorymoleculesofperiodontitisandgene expressionchangesinOSCCs,exploringthefulladvantagesof exosomesrepresentsapromisingavenue.Liquidbiopsyisan emergingdiseasediagnosticmethodthatprimarilyinvolvesthe isolationandevaluationof fluidentities,suchasDNAs/RNAs, proteins,andextracellularvesiclesinhumansaliva,blood,and urine.79 Comparedwiththeotherpatternsofbiomarkers, exosomesprotecttheircargos,remarkablyenhancingthe accuracyandpracticabilityofdetection.80 Inthe fieldoforal medicine,exosome-associatedliquidbiopsyhasdemonstrated valueinthediagnosisofmultiplediseases.81 Theidentificationof novelbiomarkersandreliabledetectivetechniquesarepotential researchprospects.

Biomarkersforperiodontitis

Thetraditionaldiagnosticcriteriaforperiodontitisprimarilyrelyon clinicalsymptoms,suchastheperiodontalpocketdepthandthe pathologicallossofthealveolarbone.However,thepossibilitiesof “overdiagnosis” or “underdiagnosis” remainunsolved,asthese clinicalvariablesmayfailtoaccuratelypredictthedisease

Post-treatmentreaction.Thetraditionalnon-surgicaltreatments forOSCCsincludechemotherapyandradiotherapy.63 However, resistanceandunwillingtreatmentresponseswillimpactthe prognosisandmortality.Therefore,itisimperativetoidentify precisemarkersthatcaneffectivelyassesstheefficacyofthese therapies.Chemoresistanceoccursinnearlyallanti-tumordrugs, andtheunderlyingmechanismcanbeintrinsic(genemutations) oracquired(TME,epigeneticalteration).64 Accordingtorecent studies,theexosomesplayacrucialroleinchemoresistanceof OSCCsthroughseveralpathways65 (Fig. 1d).ExosomalmicroRNAs (suchasmiR-21andmiR-24)derivedfromOSCCcelllines contributetochemoresistancebytargetingmultiplesignaling molecules,includingSTAT3andPTEN.65,66 Macrophage-derived exosomesandCAF-derivedexosomalmiR-196acanalsoreduce drugsensitivity.67,68 Inaddition,theexosomescaninfluence chemoresistancebymodulatingthedrugefflux,vesicularpH,antiapoptoticsignaling,DNAdamagerepair(DDR),aswellasthe EMT.65 Inreverse,thedeliveryofexosomalmiR-30aandthe inhibitorofexosomalmiR-155showedtheabilitytoenhancethe sensitivityofcisplatin-resistantOSCCs,69,70 highlightingtheir potentialroleinimprovingcancertherapy.Cancercells’ reactions towardradiotherapyarealsoassociatedwithexosomes(Fig. 1d), mainlythroughmodulatingDDR,celldeathsignals,andthe EMT.71 InHNSCCradiation-resistancecases,thefunctionsof tumor-promotingexosomesarestrengthenedafterradiation.72

progressionandtreatmentresponse.82 Theidentiﬁcationof exosomalbiomarkershasthepotentialtoenhanceourunderstandingoftheintricatebiologicalmechanismsunderlying periodontitisprogressionandfacilitatethedevelopmentof advancedclinicalmanagementstrategiesforpatients.Generally, theconcentrationofEVsfromtheGCFofperiodontitispatientsis evidentlyhigherwhencomparedtothatofhealthysamples.83

Nucleicacids,asprominentconstituentsofexosomalcargos, canprovidevaluablediagnosticinsights.InadditiontoDNA/RNAs thatdirectlyencodeproteins,non-codingRNAsplayimportant rolesinregulatingcellulareventsthroughdiversepathways,such asgenesilencingandpost-translationalmodiﬁcation.84 MiRNAs aresmall,non-codingRNAsthatmodulatemRNAexpressionby formingRNA-inducedsilencingcomplexes(RISCs)ordirectly bindingtomRNAsthroughbasepairing.85

AccordingtoKamaletal.,86 1995salivaryexosomalmiRNAsand 333plasmaexosomesweresigniﬁcantlyalteredintheirperiodontitissamples.Amongthesepotentialbiomarkers,serum exosomalmiR-let-7d,miR-126-3,miR-199a-3,andsalivaryexosomalmiR-125a-3werenotablefordistinguishingthediseasestatus andcorrelationwiththeclinicalstages.86 Inaddition,researchers reportedthatserumexosomalmiR-1304-3p,miR-200c-3p,small nucleolarRNASNORD57,SNODB1771,87 salivaryexosomalmiR223-3p,14 OsxmRNA88 andGCFexosomalmiR-122689 were downregulatedinperiodontitis.However,salivaryexosomalmiR140-5p,miR-146a-5,miR-628-5p,90 miR-381-3p,91 tumornecrosis factor-alpha(TNF-α),88 andPD-1mRNAsexhibitedhigherexpressionlevelsintheperiodontitissamples.92 Asforproteins,the levelsofthetetraspaninsCD9andCD81weredecreasedinthe salivaryexosomesofperiodontitis,whichisassociatedwithan inﬂammatoryreaction.93 Thelevelsofimmune-relatedproteins, suchascomplementcomponents(C6,C8A,andC8B)and chemokines,wereincreasedinthesalivaryexosomesofyoung severeperiodontitispatients,94 implyingthatimmunechangesare responsibleforthispathologicalalteration.

Biomarkersfororalcancers

Scalpelbiopsiesandhistologicalexaminationshavelongbeen regardedasthegoldenstandardformalignancyresearch.95 However,oralcancers,particularlyOSCCs,usuallydevelop imperceptibly,withminimalclinicalmanifestationsintheearly stages.Therefore,bythetimethesymptomshavebeenidentiﬁed, themalignancymayhavealreadyappeared,leadingtoapoor prognosis.96 Inthemeantime,thepursuitofnon-invasive detectionmethodshasbecomeaprevailingtrendinthemedical ﬁeld.Thesestatusesemphasizethenecessityfortheapplicationof exosomalbiomarkersinOSCCdiagnosis.Comparedwiththe samplesfromhealthycontrols,theexosomesfromOSCCpatients showedanincreasedconcentrationandlargerdiameter,with higherCD63andlowerCD9/CD81expressionlevels,revealing basicevidencefordistinguishment.97 However,themostcomprehensiveandconvincibledistinctionsarethecargosofthe exosomes.Wehavesummarizedthesebiomarkersandthe relevantresearchinTable 1.

InOSCCs,thealteredsecretionofexosomalmiRNAscansignal thediseasestatus.Basedonthestatisticalevidence,thelevelsof serum/plasmaexosomalmiR-19a/27b/20a/28-3p/200c/151-3p/ 223/20b,98 miR-130a,99 miR-155,miR-21,100 miR-3168,miR-125a5p,miR-451a,andmiR-16-2-3p,101 andsalivaryexosomalmiR-4865p,miR-486-3p,miR-24-3p,102 miR-1307-5p,103 miR-200a,andmiR134104 weresigniﬁcantlyelevatedinthepatientswithOSCCs, performingpotentialoncogenicroles.However,theserum exosomalmiR-370/139-5p/let-7e/30c,98 miR-126,100 andsalivary exosomalmiR-10b-5p105 106 expressionlevelswerereduced,as theyaretumorsuppressors.Throughtheinvitroculturingof OSCCcelllines,miR-365andothermiRNAsareproducedin exosomesaberrently107 andcanbeusedtoassesshuman papillomavirus(HPV)involvement.101 Tissue-derivedexosomal

circRNA_047733canbeusedtoindicatethelymphnode metastasis(LNM)outcomesofOSCCcaseswithsatisfying speciﬁcityandsensitivity.108 Theinvivoapplicationofthese biomarkersnecessitatesfurtherresearch.CirRNAsarebacksplicingformedRNAsthatinﬂuenceproteintranslationthrough interactingwithmiRNAs,RNA-bindingproteins,andRNAPol.109 Serumexosomalcirc_0000199showedahigherlevelinOSCC patientsandisassociatedwiththeTumorNodeMetastasis(TNM) stageandprognosis.110

Regardingtheproteincargosoftheexosomes,thereare notabledifferencesbetweentheOSCCpatientsandhealthy individuals.TGFβ,awell-knowncancerbiomarker,providesmuch morediagnosticandprognosticinformationforOSCCsinan exosomalformthaninasolubleform.111 Theleveloftheseraand salivaryexosomalmarkerAlixincreasessigniﬁcantlyinOSCC patients,butthesensitivityinearlycancer(stageI)detectionisnot satisfying.Moreover,exoAlixbehavesdifferentlyinseraandsaliva inastage-dependentmanner;onlyserumexoAlixpresents prognosisinformation.112 Inadditiontosingle-exosomalprotein biomarkerdetection,Lietal.113 demonstratedtheadvantagesof combinedexosomalC-reactiveprotein(CRP),vonWillebrand factor(VWF),andleucine-richalpha-2-glycoprotein(LRG)inthe determiningspeciﬁcityandsensitivityofearlyOSCCsdiagnosis. Theserumexosomalplateletfactor4variant(PF4V1),C-X-Cmotif chemokine(CXCL7),coagulationfactorXIII,A(F13A1)and ApolipoproteinA-I(ApoA1)notonlyenablediscrimination betweentheOSCCcasesandhealthycontrols,butalsoprovide informationonthelymphnodemetastasisstatus.Thecombinationofthesefourbiomarkerscouldalsoincreasethe preciseness.114

Biomarkersforotheroraldiseases

Exosomalbiomarkershavealsobeendiscoveredinotheroral diseases.Viralinfectioushand,foot,andmouthdisease(HFMD) mostlyaffectsyoungpatientsunder5yearsoldwithherpes.115 In theseraofpatientswithHFMD(boththemildandseveretypes), thereisanelevatedlevelofexosomalmiR-16-5p,whilethemiR671-5pandmiR-150-3plevelsweredecreased.116 Thesestatistical datademonstratedthesatisfyingsensitivityandspeciﬁcityof exosomesasbiomarkersinHFMDdiagnosis.

Orallichenplanus(OLP)isanimmune-relatedmucosadisease thatiswellrecognizedasapotentiallymalignantdisorder.117 Therefore,itsearlydiagnosiswouldinhibittheprogressiontoward oralcancersandbenefitthepatients’ prognosis.Theexosomal alterationin fluidshasprovidedvaluableinformationregarding OLPdetection.Basedontheevidencefromthepolymerasechain reaction(PCR)technique,theserumexosomalmiR-34a-5p118 and salivaryexosomalmiR-4484119 weresignificantlyupregulatedin theOLPsamples.Furthermore,thehumancytomegalovirus (HCMV)-encodedmiR-UL59manifestedahigherlevelintheOLP samples,120 suggestingtheunderlyingconnectionsbetween HCMVinfectionandtheunclearetiologyofOLP.

Asanautoimmunediseaserelatedtothesalivaryglands, Sjögren’ssyndromecausesalterationsinvariouscontentsof patients’ ﬂuids.Since2010,scientistshavereportedthepotentialof exosomalmiRNAbiomarkersusedfordiagnosingSjögren’s syndrome.121 Novelsequencingevidencehasrevealedthatthe levelsofexosomalcircRNAscirc-IQGAP2andcirc-ZC3H6increased intheserumsamplesfromprimarySjögren’ssyndromepatients.122 Inaddition,inmurinemodels,thelevelsofserumexosomal miRNA-127-3p,miRNA-409-3p,miRNA-410-3p,miRNA-541-5p,and miRNA-540-5pwereupregulated.123 These ﬁndingsdeservefurther researchontheunderlyingmechanismviahumanstudies.

Analysistechniqueforexosomes Priortoourevaluationofexosomalbiomarkers,theprimaryfocus wasthedevelopmentofefﬁcientandprecisedetectiontechniques.Theseparationandquantitativeanalysisofexosomesserve

Table1. Exosomalcargosasbiomarkersfororalcancerdiagnosis

TissueSampleExo-biomarkerAlternationStatisticscaleMechanismOtherinformationRef

Tongue SCCs

PlasmamiR-19a/27b/20a/ 28-3p/200c/151-3p/ 223/20b

miR-370/139-5p/let7e/30c Downregulated

OSCCsPlasmamiR-130aUpregulated184patients 196controls N/AAssociatedwithpoor prognosis (advancedTNMstages/ poorlydifferentiation)

OSCCsSerummiR-155/21Upregulated35patients 11controls Downregulatingtumor suppressorsPTENandBcl-6 N/A

miR-126DownregulatedInhibitingoncogeneEGFL7 expression LowmiR-126wasassociated withpoorprognosis

HNSCCsSerummiR-3168/125a-5p/ 451a/16-2-3p Upregulated22patients 10controls (withbenign neoplasm)

OOCsSalivamiR-486-5pUpregulated25patients 25controls N/AHigherlevelinstageII

miR-10b-5pDownregulatedN/A

OSCCsSalivamiR-24-3pUpregulated49patients 14controls Targetingcircadiangene-

OSCCsSalivamiR-1307-5pUpregulated12patients 7controls Suppressingonco-related genesTHOP1,EHF,RNF4, GET4andRNF114

OSCCsSerumcirc_0000199Upregulated108patients 50controls AssociatedwithmiR-145-5p andmiR29b-3p

OSCCsSerum/ saliva AlixUpregulatedSerum 29patients 21controls Saliva 23patients 20controls

OSCCsSerumCombinedCRP,VWF andLRG Upregulated40patients 20controls N/ACombinedbiomarkersshow greatersensitivityand speciﬁcitythanindividual ones.

OSCCsSerumPF4V1,CXCL7, F13A1andApoA1 Downregulated (F13A1 upregulated) 10patients (noLNM) 10patients (withLNM) 10controls

N/APF4V-tumordifferentiation level

PF4V1/F13A1-positivenode number ApoA1-smokingand drinking

SCC squamouscellcarcinoma, OSCC oralsquamouscellcarcinoma, HNSCC headandnecksquamouscellcarcinoma, OOC oralandoropharyngealcancer, miR micro-RNA, IL interleukin, circRNA circularRNA, TGF-β transforminggrowthfactorbeta, Alix programmedcelldeath6-interactingprotein(PDCD6IP), CRP C-reactiveprotein, VWF vonWillebrandfactor, LRG leucine-richalpha-2-glycoprotein, PF4V1 plateletfactor4variant, CXCL7 C-X-Cmotifchemokine, F13A1 coagulationfactorXIII,A, ApoA1 apolipoproteinA-I, PTEN chromosometen, PER1 Period1

ascrucialstepsinclinicalapplications.124 Centrifugationisthe conventionalmethodusedforexosomeseparation.Toimprove efﬁciency,differentialcentrifugationisthemostcommonlyusedand practicaltechnique.Thisapproachallowsfortheisolationofnucleic acidcargosatfractionsof0.3×103 and2.0×103,whileproteincargos appearatdifferentfractions;Alixpredominantlyappearsatthe 160.0×103 fraction,andHSP70exhibitsanevendistributionacrossa widerangefrom0.3×103 to160.0×103124 125 (Fig. 2a).

Therequirementsforexosomalquantitativeanalysisvary dependingonspecificobjectives,suchasparticleenumeration, proteinquantification,RNAquantification,etc.124 Consequently,

therelevantmethodologiesincludenanoparticletrackinganalysis (NTA)and flowcytometryforparticleenumeration,bicinchoninic acid(BCA)assayingandPAGE-SDSstainingforproteinquantification.124 Aimingatenhancedefficiency,otherresearchershave comeupwithmorepracticalandprecisemethodsfororal exosomalquantitativeanalysis.SurfaceenhancementRaman spectroscopy(SERS)isamodifiedformofRamanspectroscopy (theinelasticscatteringoflight),whichshowsanamplified vibrationalRamanspectrumwhenthetestingsampleisinclose proximitytoaplasmonicnanostructuredsurface(Fig. 2b).Based ontheanalysisofsalivaexosomes,SERSexhibitsexceptional

Differential centrifugation

Modified raman spectroscopy

Modified mass spectrometry with CD63 capturing (1) (2) & (Nucleic acids)

Raman shift/cm–1

SERS spectra differences between HNSCC cancer and control groups127

Au nanoparticle with labeling DNA modification

modified electrode Cholesterol modified primer DNA and padlock DNA

Salivary exosomes

CD63 capturing exosome

Ag nanoparticle with CD63 modification

Fig.2 Schematicillustrationofexosomaldetectiontechniques. a Differentialcentrifugationcanisolateexosomecargosatdifferentfractions. b SurfaceenhancementRamanspectroscopy(SERS)demonstratesdistinctRamanspectrabetweensalivasamplesfromHNSCCandhealthy controlgroups. c ModifiedmassspectrometrywithCD63capturingallowsforquantitativeanalysisofexosomes. c-1Detectionofexosomal signalisachievedthroughcholesterol-basedrollingcircleamplificationandgold-nanoparticle-labeledDNA. c-2Redbloodcellmembrane (RBCM)-modifiedelectrodecanproduceelectrochemicalsignalsofexosomeswithAunanoparticles

sensitivityandspecificityindistinguishingmalignantandnormal ones,enablingtheearlydiagnosisofheadandneckcancers.126 Chengetal.introducedanewtechniqueforinductivelycoupled plasmamassspectrometry,performingexcellentlyinoralexosomesquantitativeanalysis.127 Theycapturedexosomeswiththe CD63antibodyanddetectedasignalwithcholesterol-based rollingcircleamplificationandgold-nanoparticle-labeledDNA (Fig. 2c-1).AlsoassociatedwithCD63capturing,redbloodcell membrane(RBCM)-modifiedelectrodecouldproduceelectrochemicalsignalsonceconfrontingexosomes,showinggreatprecision insalivaexosomesdetection128 (Fig. 2c-2).

Althoughsignificantprogresshasbeenmadeintheidentificationofexosomalbiomarkersfordiagnosingoraldiseases,certain limitationsstillexist.Themajorityofstudieslacksufficient evidenceduetoinadequatesamplecollectionandclassifications forexosomalbiomarkers.Thesedeficienciesposechallengesin standardizingtheirclinicalapplications.Furthermore,thetechniquesusedforisolatingandanalyzingexosomesfromhuman fluidsareimpracticalformostbasicmedicalinstitutions,anda validconsensushasnotyetbeenreached.Toaddresstheseissues, itisimperativetofurtherdeveloptechnicalcapabilitiesandgain deeperinsightsintotheroleofexosomesintheprogressionof oraldiseases,whichwillundoubtedlyinspiremorereliable diagnosticstandards.

EXOSOMESINTARGETEDTHERAPYFORORALDISEASES

Overrecentyears,exosomeshavebeenextensivelyinvestigated inthetreatmentofmultipleoraldiseases.Theprimaryresearch areasencompasstheutilizationofengineeredexosomesasan innovativedrugdeliverytoolwithspeciﬁccargosandtheclinical applicationofexosomesderivedfromorofacialstemcellsintissue regeneration.Inthissession,wesummarizethecurrentresearch onexosomaltherapiestowardinfections,oralcancers,andthe regenerationofpulpandbone.

Engineeredexosomes

Anti-infection.Infectiouspathogensplayasignificantroleas causativeagentsandriskfactorsinvariousoraldiseases.Recent studieshaveunveiledthepotentialoftheapplicationofexosomes asinnovativetherapiesagainstmicrobialinfections.Aninfection withEnterovirus71(EV-71)servesastheprimarytriggerforHFMD, leadingtoanelevatedlevelofexosomesinthepatients’ sera samples.116 Consequently,theutilizationoftheexosomeinhibitor GW4869couldeffectivelyreduceinfectiousactivities. 129 Exosomal miR-155exhibitsacomparableantiviraleffectbytargetingthe phosphatidylinositolclathrinassemblyprotein(PICALM).130 Asthe firstinhabitantmicrobesintheoralcavity,streptococciprominentlyinfluenceoralhealthstatus,wheredysbacteriosismaylead tocariesandotheroralinflammatory-relateddiseases.The

Table2. Exosomalcargosexhibitinganti-tumoreffectonOSCCs

Exo-cargoOriginFunctionMechanismResearchmodelRef

miR-1294OSCCtissueInhibitingOSCCcells proliferationandmigration

miR-101-3pBonemarrowmesenchymal stemcells(BMSCs)

miR-6887-5pEldecalcitol(ED71)-induced OSCCcells

miR-34aEngineeredexosomesfrom HEK-293TcellswithmiR-34a loading

circRNAGDP dissociation inhibitor2 (circGDI2)

Engineeredexosomesfrom OSCCcellswithcircGDI2 loading

LncRNALBX1-AS1ImmunoglobulinkappaJ region(RBPJ)overexpressed macrophages

InhibitingOSCCoccurrence andprogression

InhibitingOSCCcells proliferation

InhibitingOSCCcells proliferation,migration,and invasion

InhibitingOSCCcells proliferation,migration, invasionandglycolysis

InhibitingOSCCcells proliferation,andinvasion

exosomesseparatedfromhoneycontainantimicrobialagentsthat havemorepotenteffectson Streptococcusmutans incomparison tothoseoftheotherstrains.131

Anti-cancer.Traditionalchemo-/immunotherapiesforOSCCsare oftennon-speciﬁctothemalignancyandincompatiblewiththe hosttissue.However,exosomes,whichactasphysiological “packages” betweencells,mayofferasolutiontotheseissues.132 TheapplicationofengineeredexosomesinOSCCtreatmentcan disruptvariousprocessesinvolvedinoncogenesisandtumor microenvironmentmodulation,therebyeffectivelyinhibiting cancerprogression.Accordingtorecentstudies,manycargosof exosomesattenuatetheoncogenesisofOSCCsthroughcomplex signalingpathways133–138 (Table 2).

Engineeredexosomessecretedfromdiversecelltypesexert inhibitoryeffectsonOSCCdevelopmentthroughadministrating differentdrugsormolecules.Macrophage-derivedexosomal miR144/451(tumorsuppressive)connectedwithchitosannanoparticlesexhibitananti-tumoreffectinOSCCs.139 Theexosomes secretedbymenstrualstemcellsperformedanti-angiogenesisin OSCCs.140 Inadditiontohost-derivedexosomes,otherresearchers havealsoexploredalternativesourcesoftherapeuticexosomes forOSCCtreatment.Milkexosomesarewidelyrecognizedfortheir exceptionalresilienceinacidicconditionswithinthedigestive systemandtheirabilitytotraversephysiologicalbarriers.141 The engineeredexosomesofmilkcombinedwithdoxorubicinandan anthraceneendoperoxidederivativedemonstrateremarkable efﬁcacyineradicatingOSCCscells,142 showinggreatpotentialin clinicalapplications.

Noveldeliverysystembasedonengineeredexosomes.Besidesthe dentaltissue-derivedandnaturaloriginalexosomesmentioned above,engineeredexosomeshavedemonstratedgreatvalueasa noveldeliverysystem.Theprocedureofexosomeengineering mainlyconsistsofcargoencapsulationandsurfacemodification, witheachstepencompassingvariousmethodologiesandindications.143 Exosomalcargopackingcouldbeinducedinsituby interactionsbetweenthecargosanddonorcellcomponentsor invitroafterexosomepurification.132 Generally,theinvitro methodsshowmore flexibilityandpracticalityinapplications, includingelectroporation,incubation,sonication,extrusion,etc.80 InOSCCtreatment,Epstein–BarrVirusInduced 3(EBI3)transfected fibroblastswereelectroporatedwithanti-tumorsmallinterfering RNAs(siRNAs),theproductiveengineeredexosomesofwhich significantlytargetingOSCCscellsbydiminishingtheir

Targetingoncogenec-MycOSCCcelllines

TargetingcollagentypeXalpha1chain (COL10A1)

TargetingHeparin-bindingprotein17/ ﬁbroblastgrowthfactor–bindingprotein1(HBp17/FGFBP-1)

TargetingspecialAT-richsequencebindingprotein2(SATB2)

RegulatingmiR-424-5pandsuppressorof cancercellinvasion(SCAI)

OSCCcelllines Nudemice 135

OSCCcelllines Nudemice

OSCCcells-HN6

OSCCcelllines BALB/cmice

RegulatingmiR-182-5p/FOXO3pathwayOSCCcelllines Nudemice

133

proliferations.144 TheincubationofmiRNA-34awithHEK-293T cellsalsoacquireseffectiveexosomesagainstOSCCs.137

Surfacemodiﬁcationisanotherimportantstepinexosome engineering,aimingatenhancingexosomaltargetingtoward certainreceptors.Multiplecomponentscanbeaddedtothe exosomalmembrane,suchasproteins,antigens,antibodies,and DNA/RNAaptamers.132 Therelevantresearchhasprovedthatthe surface-modiﬁedexosomeswithpeptidescancrossblood–brain barrierandtreatcerebralischemia.145,146 Althoughsimilar researchonoralmedicineisunderway.However,exosomemimeticnanoparticleshavereceivedgreatattentioninoralcancer treatmentfortheirconvenienceinsurfaceengineering.147,148 Thesebiomimeticparticlescouldresemblemembranestructures ofmultiplehostcells(bloodandstemcells)andescapefrom immuneclearance.148

Dentalstemcell-derivedexosomesinregenerativetherapy Dentalstemcells(DSCs)refertoagroupofprimitivecellsderived fromdentaltissue,withthepotentialtoproliferateand differentiate.DSCsaregenerallyclassiﬁedintodentalpulpstem cells(DPSCs),stemcellsfromhumanexfoliateddeciduousteeth (SHED),stemcellsfromapicalpapilla(SCAPs),PDLSCs,dental folliclecells(DFCs),andoralmesenchymalstemcells(OMSCs), basedontheirdistincthistologicorigin.149–151 Theapplicationof DSCsinoralregenerationtreatmenthaslongbeenembraced,and stemcell-derivedexosomeshaverecentlyshowngreatpromise forhealingdentaldefectsandorofacialtissues.

Pulpregeneration .Duetoinfectiousortraumaticetiologies, pulpaland/orapicaldiseasesinvariablyresultintheirreversible impairmentofblood,neural,andnutrientsuppliestothenatural teeth.152 Despitethewell-establishedefﬁcacyofrootcanal treatment(RCT),thequestforachievingbiologicalpulpregenerationwithcompletephysiologicalfunctionalityremainsceaseless. Thereareaseriesofcrucialstepsinachievingdentalpulp regenerationandformingaphysiologicalpulpodentinalcomplex, forinstance,differentiationfromMSCstofunctionaldentalpulp cells(DPCs),thepromotionofangiogenesis,andthefacilitationof neuralreconstruction. 153 Suchasstemcellsfrompulptissue, DPSC-derivedexosomespositivelystimulatedthedifferentiation ofstemcellstowardDPCsthroughtheP38/MAPK154 and miR 150 Tlr4pathways.155 Meanwhile,Schwanncellsarerecruited toenhanceneurogenesiswiththepresenceofDPSC-derived exosomes,particularlyundertheconditionoflipopolysaccharide (LPS)stimulation.156 LPSalsopromotesangiogenesisviaDPSC-

derivedexosomes,157–159 whichsimultaneouslycouldbe enhancedbyhypoxiawithhigherleveloflysyloxidase-like2 (LOXL2).160,161 Comparedwithanormalstatus,DPSCsunder odontogenicdifferentiationconditionsecretemoreeffective exosomes,forinstance,thelevelsofmiR-27a-5pareelevated, whichinducesDPSCdifferentiation.154,162 Furthermore,the evidencesuggestedthatyoungerdonorsofDPSCsgavebetter performancetoexosomalabilityinpulpregeneration,163 as exosomalmiR-26asecretedbyaggregatingstemcellsfrom deciduousteeth(SHED)stronglypromotestheangiogenesisof HUVECsinpulptissuethroughTGF-β/Smad2/3signaling.164 Moreover,theexosomesderivedfromdentalpulptissue(DPT) exhibitsuperiorefficacyinmodulatingSCAPsforpulpregenerationcomparedtothatoftheDPSCs,whichisattributedthe “cellhomingtechnique“ 165 SCAPscanalsoreleaseexosomes,facilitatingananti-inflammatoryeffectonpulpitisduringTregconversion andthedentinogenicdifferentiationofMSCs,166,167 which demonstrategreatpotentialaspulpregenerativetherapies. BesidestheDSCs,thereareseveralotherstemcellsthatsecrete functionalexosomesinpulpregeneration.Embryonicstemcell (ESC)-derivedexosomespromoteDPCsmaturationthroughCD73 (atypeofnucleotidase)-mediatedAKT/ERKpathwayactivation.168 Furthermore,theexosomesfromumbilicalcordmesenchymal stemcells(UCMSCs)showedagreateffectoninflammatory alleviationafterapulpinjury.169 Andplatelet-sourcedexosomes alsohavethepotentialforpulpregenerationwiththrombin activation.170

InadditiontoenhancingthedifferentiationofPDLSCs,thereare alsoothermethodsusedtopromoteorofacialboneregeneration, suchasthedirectactivationofosteoblastsandtheinductionof BMSCosteogenesis.PDLSC-derivedexosomesarecapableof inducingosteoblastactivation,192 whileosteogenic-inducedand differentiatedPDLSCscanaccelerateBMSCdifferentiationtoward osteoblastsviasignificantexosomalmiRNAsalteration,targeting variousosteogenic-relatedsignalingpathways,suchastheMAPK andAMPKpathways.193 Inaddition,DPSC-derivedexosomes inducedjawboneregenerationinvivo,194 andSHED-derived exosomesarecapableofpromotingnaïveBMSCs’ differentiation intoosteoblasts.195–197 WhileSHED-derivedexosomescanpromoteDPSCs’ osteogenesisbyregulatingthemitochondrial transcriptionfactorA(TFAM).198 Tostrengthenthebonerepair effect,BMSCscanbeinnovativelyengineeredwithoverexpressed bonemorphogeneticprotein2(BMP2)ormiR-26acargo.These functionalmodificationssignificantlyimproveboneregeneration throughtheexosomes,targetingtheBMP2-associatedsignaling cascade/mTORpathway.199,200 Moreover,immunemodifications performedbyengineeredexosomesalsopromoteboneregeneration.Dendriticcell-derivedexosomesloadedwithIL-10and TGF-β caninhibitimmune-relatedboneresorptionandsuppress boneloss.201 TheexosomessecretedbyM2macrophagescanbe engineeredwithmelatonin,exhibitinganti-inflammatorybehavior andrescuingPDLSCpotencyindifferentiation.202 Besidesperiodontitis,therearealsootheroraldiseases characterizedbybonedestructionthatnecessitateexosomal regenerativetherapies.Osteoarthritis(OA)isacommonbone degenerativediseasethatmainlyoccursinjointswithcartilage degradation.203 Thetemporomandibularjoint(TMJ),asthekey jointenablingmandiblemovement,significantlyinfluences chewingandpronouncingfunctions.Therefore,temporomandibularjointosteoarthritis(TMJOA)causesseverepainandinconvenienceamongpatients.204 Similartothemechanismoforofacial boneregeneration,theaimsofexosomaltherapiesforTMJOAare reducingtheinflammatoryresponseandinducingchondrocyte differentiation.205 TheSHED-derivedexosomalmiR-100-5pdownregulatestheinflammatoryfactors(IL-6,IL-8,andMMP1)inTMJOA bytargetingmTORsignaling.206 Moreover,MSC-derivedexosomes enhancedtheoverallcartilageregeneration,andthismechanism isassociatedwiththeadenosineactivationoftheAKT,ERK,and AMPKsignalingpathways.205

Salivaryglandrevitalization .Exosomalregenerativetherapies havealsobeendevelopedtotreatotheroraldiseases.The dysfunctionofthesalivaryglandmayoccurinSjogren’s syndrome,menopause,diabetes,orafterradiotherapyforOSCC patients.Invivostudiesofexosomaltherapiesforsalivarygland recoveryhavebeenconductedusingmurinemodels.TheDPSCderivedexosomesrescuedsalivaryglandepithelialcellsthrough theG-proteincoupledestrogenreceptor(GPER)-mediatedcAMP/ PKA/CREBpathwayinSjogren’ssyndrome.207 Andtonsilmesenchymalstemcell(T-MSC)exosomescontributetotheregainingof salivaryglandfunctionafteranovariectomy,resemblingthe menopauseperiod.208 TheapplicationofBMSC-derivedexosomes couldreducethesalivaryglandcomplicationsindiabetesviathe TGF-β/Smad3pathway,209 whiletheexosomesfromthesalivary glandperformedasimilareffectviaanunclearmechanism.210 Urine-derivedstemcells(USCs)underhypoxiastimulisecrete exosomestorepairthesalivaryglandafterradiotherapyviathe Wnt3a/GSK3β pathway.211

Skinregenerationbasedonanti-agingeffect.Skinsenescenceisa progressiveprocess,withthedecliningproliferationofcells,

Orofacialboneregeneration.Theconventionaltherapeutic sequenceforperiodontitisencompassesplaquecontrol,reevaluation,andsurgicalintervention.Regenerativesurgeryhas emergedasapioneeringapproachinclinicalpractice,aimingto restoreperiodontaltissueandregainfunctions.171 Alongside variousbiofilmandbonegraftmaterials,recentstudieshave highlightedthepotentialofexosomalagentsfororofacialbone reconstruction.172 Controllingtheanomalyinflammatorychanges inperiodontalcellsandthemicroenvironmentisthepremiseof periodontitisregenerativetreatment.11 Exhibitingperiodontal anti-inflammatoryeffect,gingivalmesenchymalstemcell (GMSC)-secretedexosomestargetNF-κBsignalingandWnt5ain aperiodontalmicroenvironment173,174 andmodulatemacrophage polarizationinhigh-lipid-level175 orTNF-α pre-conditioncircumstances.176 Asimilarmacrophagetransformationalsooccurswith chitosanhydrogel-engineeredDPSC-derivedexosomesviamiR1246.177 Inaddition,MSC-derivedexosomalmiR-1246178 and PDLSC-derivedexosomalmiR-155-5p179/miR-205-5p180 inhibit inflammationbybalancingtheTh17/Tregratio. Alveolarbonelossisatypicalmanifestationofperiodontitis. Howtopromotebonerepairandregenerationisakeyissuein periodontitismanagement.PDLSCsareagroupofstemcells residingintheperiodontium.Theavailableevidencestrongly supportsthatPDLSCspossessarobustself-renewalcapacityand multipotentialdifferentiationabilities.Inperiodontalregeneration, PDLSCscandifferentiateinto fibroblasts,osteoblasts,cementoblasts,etc.181 Duringtheseprocesses,variousstemcellsbecome thesourcesforexosomes,facilitatingPDLSCproliferationand differentiation.Bonemarrowmesenchymalstromalcell(BMSC)derivedexosomeshavebeenappliedwithhydrogelinvivo,182 andSHED-derivedexosomesweretestedinvitro,183 bothofwhich showedapromisingeffectonPDLSCsproliferation,migration,and differentiation.DFC-derivedexosomesexhibitedahigherefficiencywithLPSstimulationthroughanROS-mediatedantioxidant mechanisminPDLSCs.184,185 UCMSC-andPDLSC-derivedexosomesareabletoactivatePDLSCs’ functionseveninhigh-glucoselevelcircumstances.186–188 TheapplicationofMSCsenhances periodontalligament(PDL)cellactivationafterimpairmentvia exosomesthroughtheAKT/ERKpathway.189 Inaddition,adiposederivedstem/stromalcells(ADSCs)andDFCsexosomes performedaperiodontalhealingfunctioninmurineperiodontitis modelswithnewlyformedPDLandalveolarbone.190,191 Nevertheless,thereisapaucityofdatarelatedtotheunderlying mechanismsandapplicationsinhumanmodels.

reducingECM,anddecreasingrepairabilityresultinginskin dysfunction.Bothintrinsicandextrinsicfactors(smoking/ultravioletlight)couldinﬂuencetheskinsenescenceprocedure.212 This issuehasgarneredsigniﬁcantpublicattention,particularlyinthe orofacialarea,duetoitsprofoundimpactonpatients’ esthetic appearanceandfunctionalneeds.Novelresearchhasprovedthe potentialapplicationsofstemcell-derivedexosomesinskin regenerationbasedontheiranti-agingeffects.

Thedermis,thelayerofskinundertheepidermis,mainly consistsoftheECM,whichisregulatedbydermal fibroblasts.Asa long-livedcelltype,dermal fibroblastscanbeusedtoindicatethe skinsenescentstatusthroughaccumulateddamageandrepair.213 Inducedpluripotentstemcell(iPSC)-derivedexosomesexhibit significantanti-agingeffectsondermal fibroblasts,which manifestsasthedownregulatedlevelofsenescence-associatedβ-galactosidase(SA-β-Gal)andMMP1/3andtherestorationof collagentypeI.214 Inagedmurinemodelswithwounds,the exosomesisolatedfromyoungdonorwoundedge fibroblastscan facilitate fibroblastsdifferentiationthroughmiR-125b,inhibiting sirtuin7(Sirt7).215 Similarly,trophoblast-derivedexosomescan activatedermal fibroblastsaswell.216

Woundhealingisanintricateprocessincludinganinflammatoryresponse,stemcelldifferentiationandproliferation,ECM modulation,etc.217 SenescentskincellscanperformSASP, inducingvicinalinflammationandpostponingthehealing process.218 Apressureulcer,definedasthelocalizeddamageof skintissueduetoacombinationofshearandfriction,219 commonlyappearsintheorofacialarea.Arecentsurveyhas suggestedthehighincidenceoffacialpressureulcersinpatients withrespiratorydestruction(suchasCOVID-19)duetostaying prone.220 ESC-derivedexosomescanrejuvenateepithelialcells andpromotetheangiogenesisprocessinagedmurinemodelsof pressureulcers.ThismechanismisassociatedwithenrichedmiR200acargoandactivatednuclearfactor-like2(Nrf2)signaling.221

Besidestheagingfactor,theSASPalsoplaysaroleinseveral endocrinediseases,includingdiabetes.222 Diabeticwounds commonlyappearonoralsoftandhardtissues,whichstruggle toheal,leadingtogreatsuffering.223 Therefore,diabeticwound healingrequiresanti-agingtherapiesaswell,andstemcellderivedexosomescanreducetheSASP.ADSC-derivedexosomes accelerateddiabeticwoundhealing,andNrf2overexpression enhancedthiseffect.224 Anovelbiomaterial,oxygen-releasing, antioxidantwounddressing,OxOBand,loadedwithADSC-derived exosomeshasbeenappliedinmurinediabeticmodelsandshown greatperformances.225 Fetalmesenchymalstemcells(fMSC)can alsopromotediabeticwoundhealingthroughexosomes.226 Of note,dentalstemcells,suchasSHED-derivedexosomes,have beenusedfortendonregeneration,withasigniﬁcantanti-aging effectthroughNF-κBinhibition,227 whichhasencouragedusto expandtheapplicationoforaloriginalexosomesintoother ﬁelds.

Scaffoldsforexosomesinoralregenerativetherapy.Inadditionto exploringthenovelsourcesofexosomesfororaltissueregeneration,itiscrucialtoconsiderappropriatescaffoldsthatinteract synergisticallywiththeexosomesinordertooptimizethe therapeuticefficacy.Accordingtopreviousstudiesonpulp regenerativetreatmentwithstemcells,aninjectablehydrogelis convenientasabiomaterialappliedtotherootcanals.Different typesofscaffolds(suchasnaturalcollagen-basedscaffoldsand synthetic/hybridmaterials)withinjectablehydrogelhavebeen investigated.228 Intermsofthescaffoldsloadedwithexosomes, weshouldwidenourscopetomeetthenewdemandsfor exosomalapplications.Generally,DPSC-derivedexosomescan bindtocollagentypeIand fibronectin,therebyconnectingwith thebiomaterialsandpromotingDPSCdifferentiation.154 However, these findingswerenotcompletedwithacertainimplementable biomaterialsystem.Furthermore,thehydrogelsengineeredwith hydroxypropylchitin(HPCH)/chitinwhisker(CW)andthe

hydrogelswith ﬁbrincanbothfacilitateattractionbetweenthe exosomesandMSCs,showinginjectableandbiocompatible behaviors,ultimatelyacceleratingtheexosomes’ effectonpulp regeneration.229,230 Thecontrolledreleasementofexosomesis alsoapromisingprospectforbio-scaffolding.Poly(lactic-coglycolicacid)(PLGA)-basedbiodegradablemicrosphereshave beenrecentlydevelopedtohavecontinuousexosomaleffectson pulpregeneration.231

Inorofacialboneregeneration,thetraditionalscaffoldsmostly focusonmimickingtheextracellularmatrix(ECM)ofnaturalbones, aimingatenhancingMSCadhesionandosteogenicdifferentiation.232 Withafurtherunderstandingoftheroleofexosomesin boneregeneration,newdemandsforbio-scaffoldsareemergingin cell-freetherapies.LyophilizedBMSC-derivedexosomesonhierarchicalmesoporousbioactiveglass(MBG)cansatisfyboth bioactivemaintenanceandthecontinuousreleasementofexosomes.233 Invitroexperimentsalsoprovedthattitaniumnanotubes loadedwithBMP2-stimulatedmacrophage-derivedexosomes upregulateosteogenicmarker(suchasalkalinephosphatase) expression.234 Asabiodegradablepolymerthathasbeenwidely acceptedincontrolleddelivery,235 PLGAhasagreatperformancein exosomalboneregeneration.236 Poly-dopamine(pDA)-modified PLGAcanensurethecontrolledreleaseofexosomesfromadiposederivedstemcells(ADSCs),significantlyenhancingskullbonerepair inmurinemodels.237 Combinedwithmetal-organicframework (MOF),thePLGA/Exo-Mg2+-gallicacid(GA)systemprovidesthe advantagesofADSC-derivedexosomes,Mg2+ andGAintheantiinflammation,angiogenesis,andosteogenicdifferentiationofbone regeneration.238 Moreover,addingVEGFandDPSC-derivedexosomestoaninjectablechitosannanofibrousmicrosphere-based PLGA-poly(ethyleneglycol)(PEG)-PLGAhydrogelstronglypromotes angiogenesisandosteogenesis.239 Inrecentdecades,theproductionofthree-dimensional(3D)scaffoldshasbeenwidelyusedin exosomalregenerativemedicine.240 Three-dimensional-printedsilk fibroin/collagenI/nano-hydroxyapatite(SF/COL-I/nHA)scaffolds loadedwithUCMSC-derivedexosomescouldstimulatealveolar bonedefectrepairinmurinemodels.241 Inaddition,novelscaffolds canalsoenhancecartilageregeneration.Lithium-substituted bioglassceramic(Li-BGC)significantlypromotedtheBMSCderivedexosomaleffectonchondrogenesis.242

BasedontheconvincingevidencethatDSC-derivedexosomes contributetovariousoralregenerativetherapies,moreresearchis underway.TheexpansionofMSC-derivedexosomesfromother tissueoriginsmaybeneﬁtoraltherapies.Andthecrosstalk betweenoralandotherdiseases,suchasgeneralOA243 and TMJOA,shouldbehighlighted.Overall,theapplicationof exosomesisoneofthekeypointsinoralregenerativetreatment. Basic-to-clinictranslationisoneofthefuturefocuses.

CONCLUSIONSANDPROSPECTS

Exosomeshaveemergedasanovelresearchfrontier,witha significantapplicationpotentialacrossdiverse fields.Intherealmof oralmedicine,exosomesholdpromiseasnon-invasivebiomarkers forearlydiseasediagnosisbydistinguishingbetweenpathological andhealthystates,withperiodontitisandOSCCsasthemost convincingexamples.Furthermore,exploringthedistinctcharacteristicsandalterationsinexosomesduringtheprogressionoforal diseasescandeepenourunderstandingoftheirunderlying pathologicalmechanismsandpavethewayfortargetedtherapeutic interventionsandtreatmentefficacy.Stemcell-relatedstudiesand applicationsarecurrentlyattheforefrontofmedicalresearch, particularlyinrelationtoregenerativetreatmentsfororaldiseases. Thisintersectionwithexosomebiologyprovidesavaluable foundationfortherationalutilizationofstemcells(Fig. 3).

Despitetheexistingresearchandapplicationsofexosomesin oralmedicine,therearestillseverallimitations.First,the identiﬁcation,analysis,andsynthesisoforalexosomesarenot

Differential centrifugation

Modified Raman spectroscopy

Emergingrolesofexosomesinoraldiseasesprogression Wangetal.

Cancer

Isolated exosomal cargos

Modified mass spectrometry with CD63 capturing

Stem cells

Cancer Periodontitis or

Clinical application

Clinical trial

Systematic classifiation In vitro and in vivo experiment

Exosome isolation and analysis

Exosomal research pyramid

Fig.3 Research flowforexosomesinoraldiseases.Exosomescanbeobtainedfromhuman fluids,suchassalivacollectedfromtheoralcavity, providingnon-invasivemethodsfordiseasedetection(a).Subsequently,exosomesareisolatedfromdiversesources,separatedandanalyzed bynoveltechniques(e.g.,modifiedcentrifugation)(b, c).Thedistincteffectsofexosomesarethenexaminedthroughinvitroand/orinvivo experiments(d).Thesecomprehensiveresearch findingscontributetothedevelopmentofasystematicunderstandingofexosomes,serving asafoundationforclinicalapplications(e).Finally,withawell-establishedexperimentalbasis,exosomesmeetpracticalclinicalapplicationsin oralmedicine(f)

preciseorconvenientenough.Inaccordancewiththeupdated guidelinesforexosomalresearch244 andthespecificcharacterizationoftheoralcavity(suchastheavailabilityofsaliva),itis imperativetodevelopmoreadvancedtechniquesthatwill undoubtedlyexpandtheapplicationsofexosomesinoral diseases.Reducingthetechniquebarriersisalsoimportantfor thepromotionofexosomesinreal-worldmedicine,especiallyin large-scaleproductionandstablestorage-to-transportationstrategies.Exosomalbiomimeticmaterialsmightalsobepromising, combiningtheinherentadvantagesofnaturalexosomeswith industrialsynthesistechniques.Meetingthesediversedemands necessitatescollaborativeeffortsacrossthebiomedical field,while weshouldraisespecificviewsbasedonoraldiseasesproperties. Second,thereisstilllackofclearcatalogoftheexosomesinoral diseases.Thoughthecurrentstudiesinvolveexosomesinvarious aspectsoforalmedicine,theyarenotyetsystematic.While exosomes’ classificationprimarilyreliesontheirorigin,itisworth consideringalternativeapproaches,suchastheirtargetingspecific signalingpathwaysorexploringtheirdistincteffectsoncellular functions.Withmoreinsightsintotheabundantexosomalroles throughoutoraldiseaseprogressionandtreatment,weoughtto findgeneralpathsforthefutureexplorationofnovelexosomes. Theseavenuesnecessitateadeeperunderstandingofboththe exosomesthemselvesandtheunderlyingmechanismsinvolvedin oraldiseases.Third,clinicaltrialsonexosomesinoraldiseasesare stillscarce.Accordingtoarelevantanalysis,theclinicaltrialson exosomeshavemainlyfallenundertherespiratoryresearch category,suchasbiomarkersforlungcancerandtherapiesfor SARSCoV-2pneumonia.245 Theprimarychallengesinconducting clinicaltrialsonexosomesfororaldiseasespertaintoestablishing standardizedproductioncriteriaanddeterminingtheprecise dosagesforspecificindications.Therefore,futurestudiesshould prioritizequalitycontrolmeasuresandexpandtheexperimental modelstomoreadvancedanimals.Inaddition,investigatingthe

crosstalkbetweenoraldiseasesandothersystemicdiseases focusingtheexosome’scommunicationscouldprovidevaluable insightsforfutureresearch.

Insummary,whilethecurrentresearchonexosomesinoral medicinehasyieldedsigniﬁcantachievements,thereisasubstantial amountofworktobeconducted.Futurestudiesshouldnotonly focusonthephysiologicalandpathologicalmolecularmechanisms associatedwithexosomes,butalsoemphasizethefeasibilityof relevantclinicaltranslation,enhancingtheexplorationandapplication ofefﬁcientandeffectiveexosomaltherapies.

ACKNOWLEDGEMENTS

ThisworkwassupportedbytheNationalNaturalScienceFoundationofChinaGrants (82370945,82171001,82222015and82370915).ResearchFundingfromWestChina School/HospitalofStomatologySichuanUniversity(RCDWJS2023-1).Figureswere createdwithBioRender.com.

AUTHORCONTRIBUTIONS

J.W.andY.F.organizedthemanuscript.J.W.wrotethedraft.J.J.,C.Z.andY.F. reviewedandeditedthemanuscript.Allauthorshavereadandapprovedthearticle

ADDITIONALINFORMATION

Competinginterests: Theauthorsdeclarenocompetinginterests.

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