Effect
of
layered
double
hydroxide
intercalated
with
fluoride
ions
on
the
physical,
biological
and
release
properties
of
a
dental
composite
resin
Loredana
Tammaro
a,*
,
Vittoria
Vittoria
a,
Anna
Calarco
b,d,
Orsolina
Petillo
b,d,
Francesco
Riccitiello
c,
Gianfranco
Peluso
b,daDepartmentofIndustrialEngineering,UniversityofSalerno,Fisciano,SA,Italy bInstituteofProteinBiochemistry,CNR,Naples,Italy
cConservativeOdontostomatologyandMaxillofacialSurgery,UniversityofNaples,Naples,Italy dInstituteofBiosciencesandBioResources,CNR,Naples,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received9July2013 Receivedinrevisedform 30October2013
Accepted31October2013
Keywords: Dentalmaterials Compositeresin
Layereddoublehydroxide Fluoriderelease
Cellproliferation
a
b
s
t
r
a
c
t
Objectives: Theaimofthisworkwasthepreparationofanewfluoride-releasingdental materialcharacterizedbyareleaseoffluoriderelativelyconstantovertimewithoutany initialtoxicbursteffect.Thistypeofdeliveryisobtainedbyamatrixcontrolledelutionand elicitsthebeneficialeffectofalowamountoffluorideonhumandentalpulpstemcells (hDPSCs)towardsmaturephenotype.
Methods: Themodifiedhydrotalciteintercalatedwithfluorideions(LDH-F),usedasfiller, waspreparedviaionexchangeprocedureandcharacterizedbyX-raydiffractionandFT-IR spectroscopy.TheLDH-Finorganicparticles(0.7,5,10,20wt.%)weremixedwitha photo-activatedBis-GMA/TEGDMA(45/55wt/wt)matrixandnovelvisible-lightcuredcomposites wereprepared.Thedynamicthermo-mechanicalpropertiesweredeterminedbydynamic mechanicalanalyzer.Thereleaseoffluorideionsinphysiologicalsolutionwasdetermined usingaionometer.TotalDNAcontentwasmeasuredbyaPicoGreendsDNAquantification kit toassess theproliferation rateofhDPSCs. Alkaline phosphatase activity(ALP)was measuredinpresenceoffluorideresins.
Results: Incorporationofevensmallmassfractions(e.g.0.7and5wt.%)ofthefluorideLDHin Bis-GMA/TEGDMAdentalresinsignificantly improvedthemechanicalpropertiesofthe pristineresin,inparticularat378C.Theobservedreinforcementincreasesonincreasingthe fillerconcentration.Thereleaseoffluorideionsresultedveryslow,lastingmonths.ALP activitygraduallyincreasedfor28 days inhDPSCs cellgrown, demonstratingthatlow concentrationsoffluoridecontributedtothecelldifferentiation.
Conclusions:The prepared compositescontainingdifferentamount ofhydrotalcite filler showedimprovedmechanicalproperties,slowfluoridereleaseandpromotedhDPSCscell proliferationandcelldifferentiation.
#2013ElsevierLtd.Allrightsreserved.
*Correspondingauthorat:DepartmentofIndustrialEngineering,UniversityofSalerno,ViaGiovanniPaoloII,132,84084Fisciano,SA,Italy. Tel.:+39089964019;fax:+39089964057.
E-mailaddress:ltammaro@unisa.it(L.Tammaro).
Available
online
at
www.sciencedirect.com
ScienceDirect
journalhomepage:www.intl.elsevierhealth.com/journals/jden
0300-5712/$–seefrontmatter#2013ElsevierLtd.Allrightsreserved.
1.
Introduction
Therapidincreaseofinterestinthefieldofbio-hybridmaterials thatexhibitimprovedstructuralandfunctionalpropertiesis more and more attracting researchers from life science, materialsscience,andnanoscience.Concomitantresultsoffer valuableopportunitiesforapplicationsthatinvolvedisciplines dealingwithengineering,biotechnology,medicineand phar-macy,agriculture,nanotechnology,andothers.
Nano-structuredmodification ofboththermoplasticand thermosettingpolymers,usinginorganicfillersdispersedat nanoscale, hasopened up new perspectivesfor multifunc-tional materials. By choosing the appropriate synthetic polymerorresinaswellasthespecificfillers,unprecedented morphologicalcontroldowntothenanoscaleisobtained.The potential for new multifunctional materials lies in the versatilityofboththepolymerandtheinorganicchemistry thatcanbeexploitedinthematerialssynthesis.1–7
Greatattentionhasrecentlyemergedaroundthe compo-sitesinwhichlayered fillersaredispersed ata nanometric levelinapolymericmatrix.Suchcompositespossessunusual properties,verydifferentfromtheirmicroscalecounterparts. In particular Layered Double Hydroxide (LDHs), or hydro-talcite-likecompounds,haveattractedconsiderableattention intherecentdecadefortheirapplicationsinmanyfields.8–11 LDHsconsistofflattwo-dimensionalnetworkcomposedof differenthydroxylayersofbivalentandtrivalentmetalions. IsomorphicsubstitutionofsomebivalentmetalionsofLDHs withtrivalentionsgivesrisetopositiveresidualchargesinthe frameworkwhichiscounterbalancedwithanionsandwater molecules located interstitially. These compounds, also knownas‘‘anionicclays’’,havethegeneralformula[M(II)
(1!x)-)M(III)x(OH)2](Ax/n)mH2O],whereM(II)isadivalentcationsuch
asMg,Ni,Zn,CuorCoandM(III)isatrivalentcationsuchasAl, Cr,FeorGawithAn!ananionofchargensuchas(CO
3)2!,Cl!,
NO3!ororganicanion.
Oneofthestillopenproblemsinpolymerresinsfordental applicationsis the microleakage between the sealantand toothsurfacecausingbacterialinvasionandsecondarycaries. Several types of resin, both filled and unfilled, have been employed as a pit and fissure sealants.12 Over the years, severalresearcheshavebeenconductedonsealantmaterials and methods to improve their properties like retention, marginalintegrity,etc.,buttodaythereisaneedofmaterials forremineralization.Calciumandphosphateionsfromsaliva or other sources (dentifrices, chewing gums, beverages, remineralizing solutions and restorative materials), and fluoridefromtopicalorsystemicsources,havethepotential to re-mineralize the carious lesions or minimize caries development.
The anti-cariogenic property of the fluoride is well documented and involves various mechanisms including thereduction ofthedemineralization,the enhancementof theremineralization,theinterferenceofpellicleandplaque formation and the inhibition of microbial growth and metabolism.13–20
The beneficial effects of the fluoride arise from its incorporation in tooth mineral as fluoroapatite leading to thedecreasedsolubilityofthetoothenamel.21
Inaddition,fluoridecanaffectdentinogenesisthroughthe modulationofproliferationanddifferentiationofDentalPulp StemCells(DPSCs),self-renewablemultipotentstromalcells of the pulp, fundamental for dental tissue regeneration. However,thislasteffectoffluorideisdose-dependent.Only low concentrations of fluoride can positively affect the differentiation of normal human DPCs (hDPSCs) in vitro, whilehighfluorideconcentrationhasinhibitoryeffectsonthe samecells.22
Thiscellbiologicalresponsetodifferentfluorideamounts is ofimportancetothoroughlyinvestigate theinfluence of fluoride-releasingrestorativedentalmaterials(F-RM)onpulp stemcells.
Indeed,thefirstattemptstoproduceF-RM,whichcouldact asafluoridereservoirincreasingthefluoridelevelinsaliva, plaqueanddentalhardtissues,werepromptedbytheconcept thatfluoride-releasefromthematerialsexertedusefuleffects intheclinicalpracticereducingtheincidenceofdentalcaries notonlybyinhibitingmicrobialgrowth,butalsoimproving toothregenerationandreconstructioncapacity.
Butrecently,itwasdemonstratedthatF-RMcouldbetoxic forhDPSCsandthatthedifferencesincytotoxicitybetween the tested F-RM were related to the amount of fluoride released.23–25Itappears,therefore, thatit shouldpaymore attention toimproveF-RM characteristicsinanattemptto controlthereleaseoffluorideavoiding,inparticular,theinitial burstreleasecharacterizedbyhigh,potentiallytoxic,fluoride concentrations.
Releaseoffluorideisundertheinfluenceofsomeinternal variables such as material formulation, filler, and fluoride content.
Inthispaperwepresenttheformulation,preparationand characterizationofnovelvisible-lightcuredcompositesbased on photo-activated Bis-GMA/TEGDMA matrix, containing a filler based on an hydrotalcite-like compound intercalated with fluoride ions. We have obtained a dental resin with improvedphysicalandbiologicalpropertiesand,inaddition, able to release low amountof fluoride ina controlledand tunablewayforalongperiodoftime.
Incontrasttotheconventionalandresin-modified glass-ionomers, our F-RM show to have no initial toxic fluoride ‘burst’effectandlevelsoffluoride releaseremainrelatively constant overtime. This type ofdelivery is obtained by a matrix-controlledelutionandelicitsthebeneficialeffectsof lowamountoffluorideonhDPSCdifferentiation.
Thisstudyrepresentsafirstsuccessfulattempttopreparea fluoride-releasing material based on a commercial dental resin. A forthcoming paper will deal with the degree of conversion and polymerization shrinkage of the prepared compositeresins.
2.
Materials
and
methods
2.1. Preparationoffluoridelayereddoublehydroxide
(LDH-F)
The intercalation of the fluoride anions in the LDH was performed by equilibrating, at room temperature, under stirringandundernitrogenflowfor48h,500mgofLDHin
the nitrate form [Mg0.65Al0.35(OH)2](NO3)0.35"0.68H2O
(LDH-NO3), with 10ml of a CO2-free aqueous solution 0.25mol/
dm3 of fluoride sodium salt, NaF, (F!/NO
3! molar
ra-tio=1.3).26,27 The obtained white solid was separated by centrifugation,washed threetimeswithCO2-freedeionized
waterandfinallydriedatrtoverasaturatedNaClsolution(75% ofrelativehumidity,RH).Thefinalproduct(sampleLDH-F)has theformula[Mg0.65Al0.35(OH)2](F)0.35"0.8H2O.
2.2. DispersionofLDH-Fintodentalmatrix
TheLDH-F(withmassfractionof0.7,5,10and20%)wasadded into a commercial light-activated restorative material (RK) providedbyKerrs.r.l.(Italy),whichconsistedofbisphenol-A glycidyl dimethacrylate (Bis-GMA), tri-ethylene glycol dimethacrylate (TEGDMA), camphorquinone (CQ), ethoxy-latedbisphenolAdimethacrylate(EBPADMA)andglassfillers. Specimen disks 20mm in diameter and 1mm thick, were fabricatedusingsteelmolds.Thecompositeobtained were then cured by photo-polymerization using a visible light curing unit (Optilux 380, distributed through KERR, USA; irradiateddiameter:11mm)withanirradiationtimeof120s. Duringtheexperiment,thelightintensitywasmaintainedat 550mW/cm2. The samples are coded RK-Fx, where x is
thepercentagebyweightoftheinorganicsolidLDH-Finthe resinRK.
2.3. Characterizationandevaluation
2.3.1. X-raypowderdiffraction(XRPD)
XRPDpatternswererecorded,inreflection,withanautomatic Bruker diffractometer (equipped with a continuous scan attachment and a proportional counter), using the nickel filteredCuKaradiation(l=1.54050A˚)andoperatingat40kV and40mA,stepscan0.058of2uand3sofcountingtime.
2.3.2. Fouriertransforminfraredanalysis(FT-IR)
Infrared absorption spectra were obtained by a Bruker spectrophotometer, model Vertex 70, with a resolution of 4cm!1(32scanscollected).
2.3.3. Dynamic-mechanicalanalysis
Dynamic-mechanical properties of the samples were per-formed in triplicate with a dynamic mechanical thermo-analyzer(TAinstrument-DMA2980).Thesamplesweretested byapplyingavariableflexuraldeformationindualcantilever mode.Thedisplacementamplitudewassetto0.1%,whereas themeasurementswereperformedatthefrequencyof1Hz. Therangeoftemperaturewas!50to1508C,scanningrateof 38C/min.
2.3.4. Fluoridereleasestudy
Weigheddisks wereputin contactwithafixed volumeof 50mlofaphysiologicalsalinesolution(NaCl0.90%w/v)under stirringat378C.Afterspecifictimeintervals(afteranhourfor thefirstsixhours,thenevery12h)themeasurementofthe fluorideconcentration(ppm)ofeachsolutionwascarriedout using a ionometer (Thermo Orion Mod 720 Aplus). The measurements were made in triplicate and averaged the values.
2.3.5. Primarycellculture
Human dental pulp stem cells (hDPSCs) were isolated as described previously.28,29 Briefly, human impacted third molarsfrom10adults(18–22yearsofage)werecollectedat Federico II University Dental Hospital (Naples, Italy). The experimental protocol was approved by the Hospital’s InstitutionalReviewBoard(accessionnumber7413),andthe patientsprovidedinformedconsent.Theteethwerecracked opentoremovethepulptissuesgentlywithforceps.Thepulp tissueswerethenmincedanddigestedinasolutionof3mg/ ml collagenasetypeI and 4mg/mldispase (Sigma–Aldrich, Italy)for30minat378C.Thedigestedmixtureswerepassed througha70-mmcellstrainer(Falcon,Italy)toobtain single-cellsuspensions.Cellswereseededontosix-wellplatesand culturedwitha-minimumessentialmedium(a-MEM) supple-mentedwith15%FBS,2mML-glutamine,100mML-ascorbic acid-2-phospate,100U/mlpenicillin-G,100mg/ml streptomy-cin,and0.25mg/mlfungizone(Hyclone,Italy)andmaintained in5%CO2at378C.Colonyformationunitsoffibroblasticcells
werenormallyobservedwithin1–2weeksaftercellseeding and were passaged at 1:3 ratio when they reached #80% confluence changing medium every 2days. Heterogeneous populations of hDPSCs were frozen and stored in liquid nitrogenatpassages0–2.Beforeincubationwithcells,allthe RK-Fxresinsweregassterilizedusingethyleneoxide.Second passage hDPSCs were seeded at a concentration of 1$104cells/welldirectlyontoRK-Fxresins(14mmin diame-ter)placedin24-wellplates(Falcon).Thenon-adherentcells wereremovedbywashingthreetimesinmediumat24h.
2.3.6. hDPSCproliferationassay
Toassesstheproliferationrateofthecells,totalDNAcontent (n=3)wasmeasuredbyaPicoGreendsDNAquantificationkit (Molecular Probes, Italy).Cells were seededat a density of 4$103cells/wellina96-wellplate,andafter24-hincubation,
medium was replacedwith fluoride-medium(fluoride final concentrationsof0.5,1.0,2.0and5.0ppm)forafurther48h. Thencellswerewashedtwicewithasterilephosphatebuffer saline(PBS)solutionandtransferredinto1.5-mlmicrotubes containing1mlofultrapurewater(Eppendorf,Italy).Samples wereincubatedfor1hat378Cinawaterbath,subjectedtoa freeze-thaw cycle and sonicated for 15min before DNA quantification. Then 100ml of PicoGreen working solution wasaddedto100mlofsupernatantsofthesamples.Triplicates were made for each sample or standard. The plate was incubated for 10min in the dark and fluorescence was measuredonamicroplatereader(FluostarOptima,bMGLab technologies)usinganexcitationwavelengthof490nmand an emissionof 520nm.A standard curve was created and sampleDNAvalueswerereadofffromthestandardgraph.
2.3.7. Alkalinephosphataseactivity
Alkaline phosphatase activity is atypical markerfor early odontoblastic differentiation.Toexaminewhetherthe fluo-ride release by the differentresins-induced ALP activity in hDPCs,ALPactivitywasassessedasreportedbyWangetal.30 Briefly,hDPCswereculturedonRK-Fxdisks(RK-F0.7and RK-F10,14mmdiameter)for1,7,14and28days.Then,thecells were scrapedinto cold PBS, sonicated in an ice bath and centrifugedat1500$gfor15min.ALPactivitywasmeasured
in the supernatant using p-nitrophenyl phosphate as a phosphatase substrate and alkaline phosphatase supplied bythekit asastandard. Theabsorbancewas measuredat 405nmandtheamountofALPinthecellswasnormalized againsttotalproteincontent.
2.3.8. Assessmentofextracellularmatrixmineralization
hDPSCcellswereseededonfluoride-releasingresins(RK-F0.7 andRK-F10)atadensityof1$105cells/wellandculturedfor
14days.Calcifiedextracellularmatrix(ECM)wasobservedby alizarinredSstaining(Sigma–Aldrich).Afterculture,thewells werewashedthreetimeswithPBS(pH7.4)andfixedina4% para formaldehyde solution for 20min. Cells werestained witha20mg/mlalizarinredSsolutionin0.1%NH4OHatpH4.2
for20minatroomtemperature.Toquantifymatrix minerali-zation,alizarinredSwassolubilizedin100mmol/l cetylpyr-idiniumchloridefor1h,andtheabsorbanceofthesolution was measured at 570nm. Mean absorbance values were obtainedfrom3independentexperiments.
2.4. Statisticalanalysis
Alldatawerereportedasmeanwithstandarddeviation(n=3). Statistical analysis was evaluated by a Student’s t-test for differences among groups and a value of P<0.05 was consideredstatisticallysignificant.
3.
Results
and
discussion
3.1. Structuralanalysisoffluoridehydrotalcite(LDH-F)
StructuralinformationwasobtainedbyX-rayanalysis(XRPD) and FT-IR absorption spectroscopy. Fig. 1(a) shows the diffraction patterns of the pristine hydrotalcite in nitrate form(LDH-NO3)andtheintercalatedfluorideform(LDH-F).As
aresultoftheNO3/Fexchange,theX-rayreflectionduetothe
interlayerdistance(d003=0.902nm)andpositionedat2u=9.98
(LDH-NO3)moves at muchhigherangles, 2u=11.688,
corre-spondingtoaninterlayerdistanceof0.757nm(LDH-F).The decreaseoftheinterlayerdistancewasduetotheintercalation ofthefluorideanionwithsmallerdimensionthanthenitrate
anion.31 The second and third patterns of LDH-F spectra correspondtothehigherharmonicsoftheinterlayerdistance. All the peaks are sharp, and this indicates an ordered accommodationoftheinorganicanionwithintheinterlayer regions.Moreover,theX-rayreflectionsofthenitrateformare absent,andthisindicatesthatthenitrateionsleftoutofthe exchange were solubilized in the interlayer region of the fluorideintercalate.
Fig.1(b)showstheFT-IRabsorbancespectrafortheLDH-F sample. The intercalation of fluoride ions into LDH is represented by the characteristic band at frequency 1385cm!1.31,32 Adsorption bands around 550 and 680cm!1
canbeattributedtotheMg–OHandAl–OHtranslationmodes, respectively.33Theadsorptionbandaround3460cm!1canbe
attributedtotheOHgroupstretchingduetothepresenceof hydroxylgroupsofLDH,co-intercalatedwatermolecules,or both.34
3.2. IncorporationofLDH-Fintothedentalresin
3.2.1. Structuralinvestigation
Theincorporationanddispersionoftheinorganicsolidinto the dental resin was investigated by X-ray analysis. The diffractogramsoftheLDH-F,thepureresinandthe compo-siteswithLDH-FaredisplayedinFig.2.Thebroadpatternin
Fig.2(b)isattributedtothereflectionofamorphusRK,while thediffractionspectraofRK/LDH-Fcomposites(Fig.2(c)–(f)) showcharacteristicreflectionsofLDH-Fpowdersat2u=11.688 and23.6besidesthebroadreflectionofpristineRK, increas-inglyevidentstartingfromthesampleat5%ofconcentration (RK-F5).Inparticularthebasalpeakat11.688of2uisabsentat lowconcentration;itappearsinthesampleRK-F5andthen increases on increasing the inorganic concentration. This suggeststhattheclayisdelaminatedandwelldispersedupto 5%ofconcentration,whereasathigherconcentrationslarge clay tactoids are present in the sample. The X-ray data thereforeindicateamorphologywithmicro-domainsofLDH-F atconcentrationshigherthan5%.
3.2.2. Mechanicalproperties
Themechanicalpropertieswereinvestigatedinawiderange oftemperaturesbyperformingadynamicmechanical
analy-40 35 30 25 20 15 10 5
(a)
LDH-F LDH-NO3 Inten sit y (a .u .) 2θ 500 1000 1500 2000 2500 3000 3500 4000(b)
554 685 1385 3462 Ab sor ba nce (a .u.) Wavelenght (cm-1) Fig.1–(a):XRDpatternsofLDH-NO3andLDH-F,(b):FT-IRspectraofLDH-F.sis,abletodetecteithertheelasticmodulusandthetandin theinvestigatedrangeoftemperature.Fig.3showstheelastic modulusforthepristineresinandtheresincontaining5,10 and20wt.%ofLDH-F.Thestudyofthemechanicalproperties inawidetemperaturerangedemonstratedthatthevaluesof the elastic modulus of the resins containing the fluoride inorganicsolid(RK-Fx)increasedcomparedwiththeresinRK. Thisincrease, which wasevident aftertheglass transition temperature,wasobservedatdifferenttemperaturesandfor differentcompositions.
Thecomparison ofthe storagemoduliat threedifferent temperatures(08C, 378C,508C)andthe valuesoftheglass transition temperaturesarereported inFig.4(aand b).We observedthatthestoragemoduliofthecompositeresinsare consistentlyhigherthanthepristineresinandtheincreaseis particularly relevant at 378C, the body temperature. The observed reinforcement increases on increasing the filler concentration. As expected, as shown in many composite systems,thedeformationatbreakingofthecompositeresin
was found slightlylower than the pristineresin. However, sincethestressisincreasinglyhigherinthecomposites,the toughnessremainedalmostunchanged.
3.2.3. Releaseproperties
One ofthe main goal ofthis study, beside to improvethe physicalproperties,istoobtainaresinabletoreleasefluoride ions,incontrolledandtuneablewayaccordingtotheexternal environmenttowhichtheresinisexposed.
Theresinwassuspendedinphysiologicalsalinesolution andthereleaseoftheactiveingredientwasmonitoredover time.
Asignificantphenomenonwasobservedwhichconstitutes afurtheradvantageofthesystem:theanchorageoftheactive moleculetotheinorganiclamellarcompoundallowsslower release.Thismakes thesystemmuchmoreefficient.Fig.5, showsthereleaseoffluorideions(inppm)atdifferentinitial concentrationsfromtheRK-F0.7,RK-F5,RK-F10andRK-F20 samples. 40 35 30 25 20 15 10 5 (f) (e) (d) (c) (b) (a) Intensity (a.u.) 2θ
Fig.2–XRDpatternsof:LDH-F(a),RKresin(b),RK-F0.7(c),RK-F5(d),RK-F10(e)andRK-F20(f).
We observed a rapid release at the beginning of the experiment,followedbyareleaselinearlydependingonthe time (days). The first step is independent of the initial concentration,asexpectedforaglassyrigidpolymericmatrix, inwhichthediffusivephenomenaareonlydependentonthe frozenfreevolume.Thesecondpartofthereleasecurve is linear with the release time. It inversely depends on concentration,inthesensethatthehighertheconcentration thelowerthereleasedpartoffluorideions.Thisresultreflects the strong influence of the morphology in the case of composites with lamellar clays. When the clay lamellae, wherethefluorideionsareanchored, aredelaminatedand welldispersedintheresin,thefluorideions,lessshieldedby thelamellae,aremoreavailabletobede-touched fromthe clay and diffuse trough the resin. Increasing the clay concentration,bigtactoidsareformedandthefluorideions arelessavailabletobereachedbythecounterions,detachand diffuse in the resin. Fluctuations between the different concentrationshigherthat5%,arepossiblyduetomorphology fluctuations during the composite preparation. This result indicates the possibility to have a tunable and controlled releaseoffluoridefromRKresins.
It is worth observing that the extrapolated release can occuruptooneyear,withappmconcentrationreleasedeach daythatisfarfromthepossibleadversefluorideeffect.
3.2.4. CellproliferationandALPactivity
HumanDPSCscandifferentiateintovarioustissues,suchas odontoblasts,adipocytes,chondrocytes,andosteoblasts.28,35 In addition, hDPSCs are effective in mineralized tissue formation.36–38
It has been hypothesized that the failure of dental restorationsisdependentonthedegreetowhichthepulpal cellpopulationscansurvive,aswellastheabilityofthesecells todetectandrespondtoinjurytoinitiateanappropriaterepair response.39,40Thus,itisfundamentaltouserestorativedental materialsandprocedurescongruentwiththenatural regen-erativeactivityofteethandwiththeinductionofodontoblast phenotypeinhDPSC.
The first sparks to produce active fluoride-releasing materials,withdefiniteinteractions withthehumanDPSC, originatedfromthefactthatmaterials capableofreleasing fluoridecanexertusefuleffectsintermsofDPSC differentia-tioninodontoblasts.However,ithasbeendemonstratedthat the level of fluoride release was in directcorrelation with cytotoxicactivityofF-RMonhumanDPSCs,whilelowlevelsof releasedfluoridecorrelatedtolowcytotoxiceffectonhuman DPSCs.41
To assess whether fluoride concentration affected the proliferation ofhDPSC,weseededgrowth-arrestedcells (in FBS-deprived basal mediumfor 24h)in0.2%FBSand then measuredcellproliferationbyDNAassayusingafluorimetric dsDNAquantificationkit.Fig.6(a)demonstratedthatcytotoxic concentrationoffluoridewasrangingbetween3and5ppm,a value did not present in the conditioned supernatant of fluoridereleaseresins(RK-F0.7andRK-F10)forallthepoints evaluated. Parallel experiments using the supernatants conditioned with RK-F0.7 or RK-F10 demonstrated that all thesupernatantstesteddidnotshowanysignificant inhibi-tion of proliferation on hDPSC. Conditioned supernatants fromtissueculturepolystyreneandRKwereusedascontrol (datanotshown).
As showed in the previous paragraph there was no substantial difference of fluoride release from RK-F0.7 and RK-F5,sowereportonlytheresultsobtainedonRK-F0.7and RK-F10.
Alkaline phosphatase activity (ALP), an early marker of odontogenic differentiationof pulp cells,was measured in cellsculturedonfluoride-releasingrestorativematerials (RK-Fig.5–ReleaseprofilesoffluorideionsfromRK-F0.7,
RK-F5,RK-F10andRK-F20samplesinphysiologicalsolution asafunctionoftime(day).
20 97 21 51 24 74 27 59 10 15 1349 1566 180 0 62 0 984 118 9 1367 100 1000 10000 RK-F20 RK-F10 RK-F5 RK St or ag e M od ul us [M Pa ] 0 °C 37 °C 50 °C
(a)
Tg temperature 96 98 93 79 0 20 40 60 80 100 120 RK-F20 RK-F10 RK-F5 RK Tem pe rat ur e [° C ](b)
Fig.4–(a)StoragemoduliE0(MPa)at08C,378Cand508Cand(b)glasstransitiontemperatures(8C)forthepristineresinand itscomposites.
F0.7andRK-F10).Cellsculturedonpolystyrene(CTL)andon restorative material without fluoride (RK) were used as control. Fig. 6(b) shows that the ALP activity gradually increased for 28 daysin cells grown on RK-F0.7 or RK-F10 comparedtocellsculturedontotissueculturepolystyreneand RK.Thus,thereleaseoflowamountsoffluoridefromRK-F0.7 orRK-F10wasabletomodulatepositivelyhDPSCs differenti-ation.
3.3. Extracellularmatrixmineralization
Mineralization of hDPSC was obtained via alizarin red S staining. As shown in Fig. 7, we observed no remarkable differencesincalcifiedextracellularmatrix(ECM) mineraliza-tionincellsplatedonRK-F0.7andRK-F10,aswasexpected basedontheresultsoftheALPassay,comparedtocellsplated onRK.
Theseresultssuggestthatbothresinsexhibitequivalent biologic activity as scaffolds supporting hDPSCs, despite differentlevelsoffluoridereleased.
4.
Conclusion
The present study investigated the effects of a modified hydrotalcite (LDH-F) dispersed into a commercial light-activated restorative material on mechanical properties, fluoride release and hDPSCscell proliferation.Goodresults makeLDH-Fapromisingfillerfordentalresin.
r
e
f
e
r
e
n
c
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Fig.7–MineralizationofhDPSCsextracellularmatrixon RK-Fs.AtintervalsduringincubationonRK-Fsresins(15, 20and25days),calciumdepositsweremeasuredby alizarinredSstaining.Cellsculturedontotissueculture polystryreneoroncommerciallight-activatedrestorative materialswithoutfluoride(RK)wereusedascontrols. RK-F0.7andRK-F10werefluoride-releasingrestorative materials.ThebarsrepresentmeansWSD(n=3).
Fig.6–(a):EffectsoffluorideconcentrationonhDPSCscell proliferationafter24and48h.Cellculturedontotissue culturepolystyrenewereusedascontrol(CTL).(b)ALP activityofhDPSCsculturedfor28daysonRK-Fs.Cell culturedontotissueculturepolystyrene(CTL)oron commerciallight-activatedrestorativematerialswithout fluoride(RK)wereusedascontrols.RK-F0.7andRK-F10 werefluoride-releasingrestorativematerials.Thebars representmeansWSD(n=3).*p<0.01whenRK-F0.7 groupiscomparedtoCTLorRKgroup;§p<0.01when
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