Properties of Experimental Dental Composites Containing Antibacterial Silver-Releasing Filler

27 Pages·2017·4.36 MB·English

materials
Article
Properties of Experimental Dental Composites
Containing Antibacterial Silver-Releasing Filler
RobertStencel1,JacekKasperski2,WojciechPakieła3,AnnaMertas4,Elz˙bietaBobela4,
IzabelaBarszczewska-Rybarek5andGrzegorzChladek3,*
1 PrivatePractice,CenterofDentistryandImplantology,ul.Karpin´skiego3,41-500Chorzów,Poland;
[email protected]
2 DepartmentofProstheticDentistry,SchoolofMedicinewiththeDivisionofDentistryinZabrze,
MedicalUniversityofSilesia,pl.Akademicki17,41-902Bytom,Poland;[email protected]
3 FacultyofMechanicalEngineering,InstituteofEngineeringMaterialsandBiomaterials,SilesianUniversity
ofTechnology,ul.Konarskiego18a,44-100Gliwice,Poland;[email protected]
4 ChairandDepartmentofMicrobiologyandImmunology,SchoolofMedicinewiththeDivisionofDentistry
inZabrze,MedicalUniversityofSilesiainKatowice,ul.Jordana19,41-808Zabrze,Poland;
[email protected](A.M.);[email protected](E.B.)
5 DepartmentofPhysicalChemistryandTechnologyofPolymers,SilesianUniversityofTechnology,
44-100Gliwice,Poland;[email protected]
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:20May2018;Accepted:11June2018;Published:18June2018
Abstract: Secondary caries is one of the important issues related to using dental composite
restorations. Effectivepreventionofcariogenicbacteriasurvivalmayreducethisproblem. Theaim
ofthisstudywastoevaluatetheantibacterialactivityandphysicalpropertiesofcompositematerials
withsilversodiumhydrogenzirconiumphosphate(SSHZP).Theantibacterialfillerwasintroduced
atconcentrationsof1%,4%,7%,10%,13%,and16%(w/w)intomodelcompositematerialconsisting
ofmethacrylatemonomersandsilanizedglassandsilicafillers. Theinvitroreductioninthenumber
of viable cariogenic bacteria Streptococcus mutans ATCC 33535 colonies, Vickers microhardness,
compressive strength, diametral tensile strength, flexural strength, flexural modulus, sorption,
solubility,degreeofconversion,andcolorstabilitywereinvestigated. Anincreaseinantimicrobial
filler concentration resulted in a statistically significant reduction in bacteria. There were no
statisticallysignificantdifferencescausedbytheintroductionofthefillerincompressivestrength,
diametraltensilestrength,flexuralmodulus,andsolubility. Statisticallysignificantchangesindegree
ofconversion,flexuralstrength,hardness(decrease),solubility(increase),andincolorwereregistered.
AfavorablecombinationofantibacterialpropertiesandotherpropertieswasachievedatSSHZP
concentrationsfrom4%to13%. Thesecompositesexhibitedpropertiessimilartothecontrolmaterial
andenhancedinvitroantimicrobialefficiency.
Keywords: dental composites; antibacterial properties; silver; mechanical properties; degree of
conversion;sorption;solubility;colorstability
1. Introduction
Worldwide, around 2.4 billion people (33% of the population) suffer from dental caries in
permanentteeth,andthepercentageofthischronicdiseaseincreasedbetween2005and2015by14%[1].
Moreover, in some countries like Poland, more than 90% of the adult population has experienced
dentalcariesandusedentalfillingsordentures[2]. Thesefactsillustratetheprogressiveextentofthe
demandfordentalmaterialsandtheroleofconstantdevelopmentinthisspecificfieldofmaterial
science. Dentalcaries,butsometimesalsodentaltraumaorextensivewearcaused,e.g.,bybruxism,
Materials2018,11,1031;doi:10.3390/ma11061031 www.mdpi.com/journal/materials
Materials2018,11,1031 2of27
mayleadtothelossofhardtissuesoftheteeth. Oneofthestrategiesallowingreconstructionofthe
teethstructureisusingdirectrestorativematerials,whichareshapedintraorallytocreaterestorations
directlyinteethcavities[3]. Currently,themostcommonofthemarephotopolymerizableresin-based
composites, introducedfewdecadesagoasasubstituteforamalgams[4]. Thistypeofmaterialis
alsoconsideredtobethemostprospective,whichhasresultedinagrowingnumberofnewproducts
onthemarketandnumerousinvestigationsinthisarea. Incomparisonwithotherdirectrestorative
materials, composites show optimal esthetic properties, which are related to possibilities of color
matching(translucency,shades),satisfyingcolorstabilityandpolishability[5]. Compositesarealso
reasonablyeasytouseandneedlessinvasivepreparationtechniquesthanamalgams[6],whichshould
be considered as additional clinical advantages. As a result of many years of evolution, modern
composites show good mechanical and physical properties [7], with wear rates similar to human
enamel[8]aswellassuitablebiocompatibility[9]. Nevertheless,useofresincompositesmaystilllead
tohigherfailureratesincomparisontoamalgams[10,11].Thetwomostfrequentreasonsforcomposite
failuresarefracturesandsecondarycaries[12,13]. Pereira-Cencietal.[14],intheirextensivereview,
concluded that secondary caries is the cause of up to 55% of resin composite filling replacements.
It is defined as “positively diagnosed carious lesion, which occurs at the margins of an existing
restoration” [15]. However, currently, it is commonly accepted that it is a primary carious lesion
ofteethatthemarginofafilling,butitoccursaftersometimefromplacingtherestoration[15,16],
incontrasttotheremainingcaries,whicharecausedbyincompleteeliminationofinfectedtoothtissues
duringcavitypreparation[15]. Secondarycariesisoftenlinkedtothepresenceofmicroleakagecaused
byvariousfactors[17–19],whichmaybethereasonfortheoccurrenceofliquids,chemicalsubstances,
andfinallybacteriabetweenthetoothandtherestoration[20,21]. Regardlessofthedoubtsaboutthe
etiologyofcariesaftertheplacementoffillings,itisrecognizedasaseriousandwidespreadclinical
problem. Moreover,compositesaccumulatemorebiofilmandplaquethanotherdirectrestorative
materials[22].Forthisreason,itisbelievedthattheperfectresincompositefillingshouldnotonlyhave
suitablemechanicalandestheticpropertiesbutalsooughttopossessantibacterialpropertiestoavoid
colonizationofthetooth/restorationinterfacebypathogenicbacteria,suchasStreptococcusmutans
(S.mutans)[23,24].
Diverse research with different additives has been carried out to develop effective
antibacterial composites. Numerous experiments have focused on resins containing
polymerizableantibacterialadditives,suchasquaternaryammoniumdimethacrylate(QADM)[25],
12-methacryloyloxydodecylpyridiniumbromide(MDPB)[26],dimethylaminohexadecylmethacrylate
(DMAHDM) [27], dimethyl-hexadecyl-methacryloxyethyl-ammonium iodide (DHMAI) [28],
ordimethylaminododecylanddimethylaminohexadecylmethacrylates[29]. Otherorganicmaterials
includingquaternaryammoniumpolyethylenimine(PEI)nanoparticles[30],chlorhexidine[31,32],
triclosan [33], chitosan [34], and benzalkonium chloride and acrylic acid [35] were also tested
with varying degrees of success. The use of different experimental fillers is another important
strategy for developing antimicrobial composites. Tavassoli Hojati et al. [36], Kasraei et al. [37],
and Aydin Sevinç et al. [38] reported the reduction of cariogenic bacteria after incorporation of
zinc oxide nanoparticles, probably due to the mechanism of production of active oxygen species,
such as H O or the possible leaching of Zn2+ ions. Khvostenko et al. [39] used bioactive glass
2 2
(65% SiO , 31% CaO, 4% P O ) and obtained a 61% reduction of S. mutans penetration of the gap
2 2 5
depth under laboratory conditions, which suggests that the release of ions from glass into the
gap may help control the local chemistry by creating an antimicrobial environment that reduces
biofilm propagation. Łukomska-Szyman´ska et al. [20] noted that composites additionally filled
withcalciumfluoridehadshownasignificantreductionofS.mutansandL.acidophilus,whichwas
probablyrelatedwithcreatinghydrofluoricacidthatcanpenetratethebacterialmembrane,generate
acidification of cytoplasm, and inhibit enzymes. Sodagar et al. [40] modified the commercially
available orthodontic composite with titanium dioxide nanoparticles and proved inhibition of
S.mutansandS.sanguinisgrowth. Themostwidelytestedmaterialsinpreviousyearswerethose
Materials2018,11,1031 3of27
containingsilver. Niuetal.[41]successfullyappliedtetrapod-likezincoxidewhiskerstoincrease
antibacterialresistance. Chatzistavrouetal.[42]confirmedasignificantreductionofS.mutansfor
Ag-doped bioactive glass and additional bioactivity of tested materials. Ai et al. [43] investigated
compositeresinreinforcedwithsilvernanoparticle-ladenhydroxyapatitenanowires,wherenanowires
wereusedasreinforcementandnanosilverasanantimicrobialagent. Thereductionofmicroorganisms
wasnoted,however,onlywhentheexperimentalfillerwasaddedintothematrixanditsconcentration
was limited to 10%, so those interesting results needs confirmation in follow-up experiments on
materials with typical reinforcing fillers. Łukomska-Szyman´ska et al. [44], reported a viability
of S. mutans from 48% to 87% in comparison to control samples on the surface of experimental
compositeswiththeadditionofsilverparticlesaloneandcombinedtitaniumdioxide,silicadioxide,
and zirconium dioxide nanoparticles or microparticles. Kasraei et al. [37] and Azarsina et al. [45]
modified commercially available composites with silver nanoparticles and noted a reduction of
bacterialcolonies.However,ambertobrowndiscolorationofmaterialswithnanosilverhasbeennoted,
whichisalimitationforanestheticmaterial[37,44,45]. Also,theinhibitoryeffectagainstS.mutans
of resin composites with silver-containing inorganic particles like silica gel have been confirmed,
whichtheauthorslinkednotwithsilverionreleasebutwiththepresenceofactiveoxygen,including
hydroxylradicals,createdbythecatalyticactionofsilverduringphotoactivationorcontactwithwater
atpolarsurfaces[46]. Additionally,simultaneouseffectsofsilvernanoparticleswithhydroxyapatite
nanoparticles[47]orantimicrobialmonomers[27,48]werealsoinvestigated.
Silver sodium hydrogen zirconium phosphate (SSHZP) is a silver-releasing ceramic.
Thissubmicron-sizedantimicrobialmaterialiswhiteandstable,soasopposedtosilvernanoparticles,
itshouldnotcausethetypicalinitialamberorbrowndiscolorationduetotheplasmoneffect[49],
whichisproblematicinthecaseofdentalmaterials. However,thequestionoffurthercolorchanges
relatedwithsilverionreleaseduringcontactwithawetenvironmentanditsoxidationremainsopen.
So far, SSHZP has been reported as an additive into a polymethyl methacrylate (PMMA)
denturebasematerial[50]andapolydimethylsiloxane-basedsoftdenturelining[51]. SSHZPwas
also previously investigated as an antimicrobial additive into chitosan and alginate fibers [52,53].
Moreover,itisincorporatedintosomecurrentlyavailablealginateandcarboxymethylcellulosewound
dressings[54,55].Inthisstudy,wereporttheuseofSSHZPasantibacterialfillerforadistinctlydifferent
material—aexperimentaldirectrestorativephotopolymerizableresin-basedcomposite,reinforcedwith
variedfillertypesathighconcentrations. Therefore,theaimofthepresentedworkwastoinvestigate
theimpactoftheproposedfiller(SSHZP),introducedintoresin-basedcompositesintendedasdirect
restorativematerials,foritsantimicrobialeffectiveness,mechanicalproperties,degreeofconversion,
sorption,solubility,andcolorchanges. Ourhypothesiswasthatcompositesadditionallyfilledwith
SSHZPwouldshowantimicrobialeffectivenessagainstcariogenicbacteriaandsuitablepropertiesfor
dentalrestorativematerials.
2. MaterialsandMethods
2.1. MaterialsPreparation
Thematrixconsistedofthreemixedmonomers: bisphenolAglycidylmethacrylate(bis-GMA),
urethane-dimethacrylate (UDMA), and triethylene glycol dimethacrylate (TEGDMA) at a weight
ratioof42:38:20,respectively(allpurchasedformSigma-Aldrich,St. Louis,MO,USA).Additionally,
0.4% (w/w) of camphorquinone (CQ, Sigma-Aldrich, St. Louis, MO, USA) as the photosensitizer
and 1% (w/w) of N,N-dimethylaminoethyl methacrylate (DMAEMA) as a photoaccelerator (both
Sigma-Aldrich, St. Louis, MO, USA) were introduced. The reinforcing fillers were two silanized
bariumborosilicateglassfillers(Esschem, Linwood, PA,USA),withameanparticlesizedeclared
by the manufacturer of 2 µm (G1) or 0.7 µm (G2), and silanized silica nanofiller Aerosil R7200
(AR) (Evonic Industries, Essen, Germany), used at a weight ratio of 50:35:15, respectively. Silver
sodiumhydrogenzirconiumphosphatecontainingapproximately10%ofsilver(w/w),withmolecular
Materials2018,11,1031 4of27
formulaAg Na H Zr (PO ) [56](MillikenChemical,Spartanburg,SC,USA)wasusedasan
0.46 0.29 0.25 2 4 3
antimicrobialfiller. TheSSHZPwascompoundedatconcentrationsof1%,4%,7%,10%,13%,and16%
(w/w),andthemassesnecessarytopreparethecompositeswerecalculatedaccordingtotheequation:
c × m
m = SSHZP MRF (1)
SSHZP 1− c
SSHZP
wherem wastheSSZHPg;c wastheSSZHPconcentration,%(w/w);andm wasthe
SSHZP SSHZP MRF
matrixwithreinforcingfillermass(alwaysconstant).
The fillers were compounded into a matrix in 50 mL glass Griffin form beakers at room
temperature in the following order: SSHZP, G1, G2, and AR as the last one. All composites
werepreparedinstandardizedportionsbasedonthesamemassesofmatrixandreinforcingfillers.
ThecompositionsofstandardizedportionsoftestedmaterialsarelistedinTable1. Theintroducing
processwascarriedoutgraduallyinstandardportionsof1g(SSHZP,G1,G2)or0.5g(AR).Forthe
lowestconcentrationofSSHZP,orwhenthelastportionofparticularfillerswasadded,theywere
smaller. Compoundingwaseffectedbymultiplespreadingsandmixingsofmaterialswithastainless
steelspatulaonthewallofthebeakertoapplyshearforces. Thesubsequentdosesoffillerswereadded
whenahomogeneousconsistencyforthepreviousdosewasachieved. Theprocessofcompounding
for one material took about 2.5–4 h; the longer time was needed for materials with higher filler
concentrationsduetotheirincreasingviscosity. Theobtainedcompositionswereplacedunderthe
pressure of 80 mbar for 25 min in a modified vacuum stirrer (Twister evolution, Renfert GmbH,
Hilzingen, Germany). AllmaterialswerepolymerizedwithaDY400-4LEDlamp(DenjoyDental,
Changsha,China),power5W,intensity1400–2000mW/cm2,opticalwavelength450–470nm.
Table1. Compositionsofinvestigatedmaterialswiththemassesofcomponentsneededtoprepare
standardportions.
Code Matrix,g Matrix,%(w/w) RF,g RF,%(w/w) SSHZP,g SSHZP,%(w/w) TF,%(w/w)
Control 15.00 35.00 27.86 65.00 0 0 65.00
AC1 15.00 34.35 27.86 64.65 0.43 1 65.35
AC4 15.00 33.60 27.86 62.40 1.76 4 66.40
AC7 15.00 32.55 27.86 60.45 3.22 7 67.45
AC10 15.00 31.50 27.86 58.50 4.76 10 68.50
AC13 15.00 30.45 27.86 56.55 6.40 13 69.55
AC16 15.00 29.40 27.86 54.60 8.16 16 70.60
AC—antibacterial composite, RF—reinforcing fillers, SSHZP—silver sodium hydrogen zirconium phosphate,
TF—totalconcentrationofcompoundedfillers.
2.2. ScanningElectronMicroscopy(SEM)Investigations
Fillerswereaddedto99.8%ethanol,ultrasonicallyhomogenized,anddroppedoncarbontape.
Polymerizedsamplesforcompositemorphologyobservationsmeasured10×2×2mm. Twotypes
of specimens were used. The first type was subjected to the standard procedure which involved
wet-grindingandpolishingusingdiamondpastes.Theothertypewasimmersedinliquidnitrogenand
broken. Compositesamplesafterpolishingwerealsoetchedwithorthophosphoricacid. Allsamples
weresputteredwithgold. ObservationswereperformedusingaZeissSUPRA35scanningelectron
microscope(Zeiss,Oberkochen,Germany)atacceleratingvoltagesfrom3kVto20kV.
2.3. AntibacterialTest
Specimensmeasured11mmindiameterand2mminthicknessandwerepreparedinTeflon
molds.Themoldwasplacedatamicroscopeslidecoveredwith50µmthickpolyesterfoil.Thematerial
wasplacedintothemoldandcoveredwiththefoilandmicroscopeslide. Then,theuppermicroscope
slidewasmanuallypressedandtakenaway. Whenthesamplewaspolymerized,thepolyesterfoilwas
removed. Themoldswithsampleswerewet-groundsequentiallywithP800-andP1200-gritabrasive
Materials2018,11,1031 5of27
paperstoremoveexcessofmaterialandtostandardizethesurface. Next,thesampleswererinsed
withdistilledwaterandpushedoutofthemolds.
The invitro reduction of bacteria was examined according to the previously described
method [51,57,58] with some modifications. The standard strain of bacterium Streptococcus mutans
ATCC 33535 was used. Sterilized samples of composites were immersed individually in 2 mL
of bacterial suspensions in tryptone water, which contained approximately 1.5 × 105 CFU/mL
(CFU—colonyformingunits)ofS.mutans. Asuspensionofbacteriaintryptonewaterwastestedasa
positivecontrol. Puretryptonewaterwastestedasanegativecontrol. Incubationwascarriedoutina
shakingincubatorfor17hat37◦C.Afterincubation,20µLofsuspensionwasseededontoColumbia
agar(bioMerieux,Marcyl’Etoille,France)with5%sheepbloodplates. Theculturedplateswerefinally
incubatedat37◦Cfor24h,andthenumbersofbacterialcolonieswerecounted. Therelativereduction
inthenumberofviablebacteriacolonies(RB)wascalculatedaccordingtotheequation:
V −V
RB = c t ×100% (2)
V
c
whereV wasthenumberofviablemicroorganismcoloniesofthepositivecontrol(BLANK)andV
c t
wasthenumberofviablemicroorganismcoloniesofthetestspecimen.
2.4. CompressiveStrength
Compressive strength was examined according to the method presented by Mota et al. [59],
withsomenecessaryspecificationsconcerningsamplepreparation. Cylindricalspecimens(3mmin
diameterand6mminheight)werepreparedasdescribedforthemicrobiologicaltest. However,due
totheirheight, polymerizationwascarriedoutatthetopandatthebottombeforetheremovalof
thepolyesterfoil. Furthermore,afterremovingthemfromthemold,thesampleswerecuredonfour
lateralsurfaces,accordingtotherecommendationofGalvãoetal.[60]. Tensampleswereprepared
fromeachcomposite. Thesampleswereconditionedindistilledwaterat37±1◦Cfor24h. Testswere
conductedusingauniversaltestingmachine(ZwickZ020,ZwickGmbH&Com,Ulm,Germany)ata
cross-headspeedof0.5mm/min. Compressivestrengthwascalculatedaccordingtotheequation:
F
σ = (3)
cs
A
where σ was the compressive strength, MPa; F was force at fracture, N; and A was the initial
cs
cross-sectionalareaofspecimen,mm2.
2.5. DiametralTensileStrength
The samples for the diametral tensile strength (DTS) tests (6 mm in diameter and 3 mm in
height) [61] were prepared with a method similar to the microbiological test, but irradiation was
carried out at the top and at the bottom before removing the polyester foil. Ten samples were
prepared from each composite. The samples were conditioned in distilled water at 37 ± 1 ◦C for
24h[61]. Compressiveloadwasappliedonthelateralsurfaceofthesamplesatacross-headspeed
of0.5mm/min[20]usingauniversaltestingmachineZwickZ2.5. TheDTSvalueswerecalculated
accordingtotheequation:
2F
DTS = (4)
πdh
whereDTSwastheultimatediametraltensilestrength,MPa;Fwastheforceatfracture,N;dwasthe
diameter,mm;andhwasthethickness,mm.
2.6. FlexuralStrength
Three-point bending tests were carried out using a universal testing machine Zwick Z2.5 in
accordance with the ISO 4049 standard [62], with specifications concerning sample preparation.
Specimens measuring 25 × 2 × 2 mm were prepared using silicone (Zetalabor Platinum 85Touch,
Materials2018,11,1031 6of27
ZhrmackSpA,BadiaPolesine,Italy)moldsplacedinastainless-steelframe. Materialswerepacked
intoamoldandpolymerizedbyamethodsimilartotheprevioustest,butfiveoverlappingirradiations
werecarriedout,startingfromthecenterofthesample. Aftercuring,samplesweretakenoutofthe
mold,theexcessofmaterialwascutoffwithascalpel,andthespecimenswerethenwet-groundwith
P800-andP1200-gritabrasivepapers. Tensampleswerepreparedfromeachcomposite. Thesamples
werestoredindistilledwaterat37 ± 1◦Cfor24h. Thetestwasperformedatacross-headspeed
of0.75mm/minandthedistancebetweenthesupportswas20mm. Flexuralstrengthandflexural
moduluswerecalculatedaccordingtotheequations:
3Pl
σ = (5)
fl 2bh2
P l3
E = 1 (6)
4bh3δ
where σ was flexural strength, MPa; E was flexural modulus, GPa; l was distance between the
fl
supports,mm;bandhwerethespecimenwidthandheight,mm;Pwasmaximalforce,N;P was
1
theloadatchosenpointattheelasticregionofthestress-strainplot,kN;andδwasthedeflectionat
P ,mm.
1
2.7. VickersHardness
Vickers microhardness was measured on specimens like for DTS, however, samples after
wet-grindingwerealsopolishedwith6-µmand3-µmdiamondsuspensions(StruersGmbH,Willich,
Germany). Threesamplesweremadefromeachcomposite. Thesampleswerestoredindistilledwater
at37±1◦Cfor24h.Hardnesswasmeasured10timesforeachspecimenatrandomlychosenlocations
usingthemicrohardnesstester(Future-TechFM-700,Future-TechCorp,Tokyo,Japan)ata100-gload
andaloadingtimeof15s[63]. Vickershardnesswascalculatedaccordingtotheequation:
1.8544×F
E = (7)
d2
whereFwastheload,N,anddwastheaveragelengthofthediagonalleftbytheindenter,mm.
2.8. DegreeofConversion
Thedegreeofconversion(DC)wasdeterminedusingthemethoddescribedbyAtiraetal.[64]
withmodificationsmadeduringsamplepreparation. Specimens,measuring5mmindiameterand
2mminheight,werepreparedinTeflonmoldsaspreviouslydescribed,butirradiationwascarriedout
onlyatthetop. Thesampleswereremovedfromthemoldsanddriedindesiccatorswithfreshlydried
silicagelat37±1◦Cfor24h. SpectrawererecordedbyaFouriertransforminfraredspectroscopy
(FTIR)spectrophotometer(PerkinElmerSpectrumTwo,PerkinElmer,Waltham,MA,USA),equipped
with an attenuated total reflectance (ATR) crystal. The absorption intensity of selected peaks was
measured in the range of 1800–1500 cm−1 and recorded with 128 scans at a resolution of 1 cm−1.
TheDCwascalculatedfromthedecreaseoftheabsorptionbandat1637cm−1,referringtotheC=C
stretchingvibration(A )inrelationtothepeakat1608cm−1,andassignedtothearomaticstretching
C=C
vibrations(A )inaccordancewiththeequation[65]:
Ar
(AC=C/AAr)aftercuring
DC(%) = (1− )×100 (8)
(AC=C/AAr)beforecuring
2.9. SorptionandSolubility
Thespecimensmeasuring15mmindiameterand1mminheightwerepreparedusingTeflon
molds[66]andpolymerizedatnineoverlappingirradiationzonesinaccordancewiththemethod
describedintheISOstandard[62]. Aftercuring,theyweregroundwithP1200-gritabrasivepaperto
removeexcessmaterialwithpotentiallypoorlypolymerizedlayers[67]andtostandardizethesurface.
Materials2018,11,1031 7of27
Then, the samples were removed from the molds. Five test samples of each material were made.
ThemeasurementofsorptionandsolubilitywasperformedinaccordancewithISO4049. Thesamples
weredriedinsidedesiccatorswithfreshlydriedsilicagelinadryerat37±1◦Candweigheddaily
(AS110/C/2,Radwag,Radom,Poland)withanaccuracyof0.1mg. Whenthechangesinmasswere
nohigherthan0.1mg,themassvalueswererecordedasm ,andthethicknessanddiameterwere
1
measuredwithadigitalcaliperwithanaccuracyof0.1mm. Eachsamplewasplacedin10mLof
distilled water for 7 days at 37 ± 1 ◦C. After storing, the samples were removed from water with
tweezers,driedfromvisiblemoisturewithfilterpaper,keptatroomtemperaturefor15s,andweighed
(m massvaluesweredenoted). Thedryingprocesswasrepeatedasdescribedabove,andstablemass
2
wasdenotedasm . Sorptionandsolubilitywerecalculatedusingequations:
3
m −m
w = 2 3 (9)
sp
V
m −m
w = 1 3 (10)
sl
V
wherew wassorption,w wassolubility,m wastheinitialmassofdriedsample,µg;m wasthe
sp sl l 2
massafterstoring,µg,andm wasthemassaftertheseconddrying,µg;andVwasthevolumeofthe
3
sample,mm3.
2.10. ColorChangeMeasurement
Toevaluatethecolorchanges,thespecimensmeasuring7mmindiameterand3mminthickness
werepreparedinTeflonmolds. Themoldwasplacedonamicroscopeslide. Thematerialwasplaced
intothemold,coveredwithpolyesterfoilandfinallywithsecondmicroscopeslide. Then,theupper
microscopeslidewasmanuallypressedandtakenaway. Theformpreparedinthiswaywasinverted
(theslidewasontop,foilonthebottom). Thiswasimportanttodobecauseduringpolymerization,
theelasticfoilallowedthematerialtomoveduetopolymerizationshrinkage(typicalmeniscuswas
formed), while the working surface of the composite in contact with the slide adhered to it and
remainedflat. Thecuredsamplewaspushedoutofthemold. Fivesampleswerepreparedfromeach
material. Afterpreparation,sampleswerestoredindryanddarkconditionsat37◦Cfor24handnext
wereimmersedin10mLofdistilledwaterindarknessat37±1◦C.Distilledwaterwasreplacedafter
thesecondandfourthday. Colormeasurementswereobtained24hafterpolymerization(baseline)
andafter7daysofimmersion. Aspectrophotometer(CM2600d,KonicaMinolta,Takyo,Japan)was
usedtorecordtheCIEL*a*b*parameterswithaD65illuminantonawhiteceramictile. TheCIELab
systemiscomposedofthreeaxes: L*isthelightnessfrom0(black)to100(white),a*representsthered
(+a*value)—green(−a*value)axis,andb*representstheblue(−b*value)—yellow(+b*value)axis.
Thecolorchange(∆E*)wascalculatedusingtheequation[68]:
(cid:113)
∆E∗ = (∆L∗)2+ (∆a∗)2+ (∆b∗)2 (11)
where∆L*=L −L ;∆a*=a −a ;and∆b*=b −b .
(7days) (baseline) (7days) (baseline) (7days) (baseline)
2.11. StatisticalAnalysis
Statistical analysis of the results was done with the use of the Statistica software (software
version 13.1, TIBCO Software Inc., Palo Alto, CA, USA). The distributions of the residuals were
tested with the Shapiro–Wilk test, and the equality of variances was tested with the Levene test.
When the distribution of the residuals was normal and the variances were equal, the one-way or
two-wayANOVAwithTukeyHSDposthoctestswereused(α=0.05),otherwisethenonparametric
Kruskal–Wallis test (α = 0.05) was used. Regression analysis was performed to determine the
correlationbetweenDCandhardness(α=0.05).
Materials2018,11,1031 8of27
3. Results
Materials 2018, 11, x FOR PEER REVIEW 8 of 26
3.1. ScanningElectronMicroscopyInvestigations
3.1. Scanning Electron Microscopy Investigations
Figure1presentsthemorphologiesoftheusedfillers.Forbothglassfillers(Figure1a,b),numerous
particles sFhigouwree d1 aprmesuencths stmhea mlleorrp(shtoalrotginiegs forfo tmhe5 u0snedm f)ilolerrsl.a rFgoerr b(outph tgola8ssµ fmill)erssi z(eFigthuaren 1tha,eb)m, ean
size dneucmlaerreodusb pyartthicelems sahnouwfeadc tau mreurch(2 smµmallearn (dsta0r.t7inµg mfro).mT 5h0 enmsh) aopr elasrgoefr t(huep tpoa 8r tµicmle) ssizwee trheanir trheeg ular.
Nanompeaarnti csliezea gdgercelagraetdio bnys mthee amsuarniunfgacutupretor 5(20 µnmm wanedr e0n.7o µtemd).f oTrhsei lischaapfielsle rof( Fthigeu prear1ticc)l.esF owreSrSe HZP
irregular. Nanoparticle aggregations measuring up to 50 nm were noted for silica filler (Figure 1c).
particles measured approximately from 100 nm to 500 nm (Figure 1d) but also larger structures,
For SSHZP particles measured approximately from 100 nm to 500 nm (Figure 1d) but also larger
consistingofparticlesconnectedtoeachother,wereobserved(Figure1e).
structures, consisting of particles connected to each other, were observed (Figure 1e).

(a) (b)

(c) (d)

(e)
Figure 1. Scanning electron microscopy images presenting the morphologies of used fillers: glass
Figure1.Scanningelectronmicroscopyimagespresentingthemorphologiesofusedfillers:glassfillers
fillers with a mean particle size of 0.7 μm (a); 2 μm (b); silica nanofiller (c); and silver sodium
withameanparticlesizeof0.7µm(a); 2µm(b); silicananofiller(c); andsilversodiumhydrogen
hydrogen zirconium phosphate (d,e).
zirconiumphosphate(d,e).
Materials2018,11,1031 9of27
Materials 2018, 11, x FOR PEER REVIEW 9 of 26
SEMSiEmMa gimesagiellsu isllturasttriantgintgh tehem moroprphhoollooggiieess ooff ccoommppoosistiet ererienifnofrocerdce wditwh igthlasgsl aasnsd asnildicas ifliilclearsfi llers
areparrees epnretesednitendF iing uFirgeu2rea ,2ba.,bT. hTehem moroprphhoollooggiieess ooff mmaatteerriaialsl swwithit hadadditdioitniaoln aanltiabnatcitbearicatle friillaelr fialrlee rare
presepnrteesdeninteFdi ginu Freig2ucr–ef .2cG–of.o Gdodoids tdriibsturitbiountioonf soifl iscialicnaa nnaonpoapratrictilcelsesb ebtewtweeeenng gllaassss ssuubbmmiiccrrooppaarrttiiccleless and
microapndar tmiciclerospinarttihcelesm iant rtihxe wmaastroixb sweravs eodbs(eFrivgeudr e(F2ibg)u.rLe a2rbg)e. Laagrggree gagagtiroengastoiofnAs Rofw AeRre wneorte dneotte cted.
detected. The SSHZP was also well distributed up to the highest concentrations. Single particles
TheSSHZPwasalsowelldistributeduptothehighestconcentrations. Singleparticleswereclearly
were clearly visible, however, clusters measuring up to 2 µm were also noted. Observations for
visible,however,clustersmeasuringupto2µmwerealsonoted. Observationsforfrozen-brokenbut
frozen-broken but not etched samples (Figure 2e,f) showed good contact between the particles and
notetchedsamples(Figure2e,f)showedgoodcontactbetweentheparticlesandthematrix.
the matrix.

(a) (b)

(c) (d)

(e) (f)
Figure 2. Representative SEM images presenting the morphologies of the cured base composite
Figure 2. Representative SEM images presenting the morphologies of the cured base composite
compounded with: reinforcing fillers (a,b); addition of 7% (c) and 16% (c–f) of silver sodium
compoundedwith:reinforcingfillers(a,b);additionof7%(c)and16%(c–f)ofsilversodiumhydrogen
hydrogen zirconium phosphate; (a–d)—wet-ground, polished, etched samples (e,f)—frozen-broken
zirconium phosphate; (a–d)—wet-ground, polished, etched samples (e,f)—frozen-broken but not

etchedsamples,blackarrows(c,d)indicatethegapsbetweenSSZHPandmatrixafteretching.
Materials 2018, 11, x FOR PEER REVIEW 10 of 26
but not etched samples, black arrows (c,d) indicate the gaps between SSZHP and matrix after
Materiealtsch20in18g,.1 1,1031 10of27
3.2. Antibacterial Test
3.2. AntibacterialTest
The achieved results of the antibacterial tests are listed in Table 2. Introducing the SSZHP into
the cTohmepaocshiiteevse dhardes ua ltssigonfitfhiceaanntt iebfafcetcetr i(apl t=e s0ts.0a0r0e2l)i sotend tihneT arebdleu2c.tiIonntr oodf uSc.i nmgutthaenSs ScZoHloPniienst.o Fthoer
cmoamteproiasli twesithhaoduta asnitginmiificcraonbtiaelf ffielcletr(,p R=B 0v.a0l0u0e2s) wonerteh ceormedpuacrtaibolne otof Sth.em puotasintsivceo cloonniterso.l.F Coormmpaotesritieasl
wwiitthho fuiltlearn ctiomncicernotbriaatliofinllse fr,roRmB v1a%lu teos 4w%e rsehocowmedp aRraBb mleetdoiathnes pfroosmiti v4e3.c8o%n ttroo 7l.0C.1o%m, paonsdi ttehsowseit vhafilulleesr
cshonouceldn trbaet icoonnssifdroemred1 %ast odi4ff%ersehnot wif ewdeR tBakme eidnitaon ascfcrooumnt4 3th.8e% obtota7in0.e1d% m, ainndimtahlo asendv amluaexsimshaol uRldB
bvealucoens.s Fidoerr ceodnacesndtrifafteiroennst sitfarwtiengta fkroemin 7to%a, cacllo oubnttaitnheedo RbBta vinaeludems wineirme a1l0a0n%d. maximal RB values.
Forconcentrationsstartingfrom7%,allobtainedRBvalueswere100%.
Table 2. The reduction in the number of viable colonies (RB) of Streptococcus mutans ATCC 33535,
Taaftbelre 127. hT hofe irnecduubcatitoionni nwtihthe cnoummpboesritoefsv siaamblpelceosl.o nies(RB)ofStreptococcusmutansATCC33535,after
17hofincubationwithcompositessamples.
CFU/mL (Vt) ×104 RB, %
cSSZHP, %
MedC FU/mLM(Vat)x ×104 Min Med RB,M%ax Min
cSS0Z HP,% 3.53 3.99 3.13 4.7 15.5 −7.7
Med Max Min Med Max Min
1 2.08 2.89 1.87 43.8 49.6 21.9
0 3.53 3.99 3.13 4.7 15.5 −7.7
4 0.68 0.13 0.00 70.1 93.2 65.7
1 2.08 2.89 1.87 43.8 49.6 21.9
7 4 00.6.080 0.01.300 0.000.00 701.100.0 93.1200.0 65.7100.0
107 00.0.000 0.00.000 0.000.00 1001.000.0 1001.000.0 100.1000.0
131 0 00.0.000 0.00.000 0.000.00 1001.000.0 1001.000.0 100.1000.0
13 0.00 0.00 0.00 100.0 100.0 100.0
16 0.00 0.00 0.00 100.0 100.0 100.0
16 0.00 0.00 0.00 100.0 100.0 100.0
cSSZHP—concentration of silver sodium hydrogen zirconium phosphate; CFU—colony forming units;
cSSZHP—concentration of silver sodium hydrogen zirconium phosphate; CFU—colony forming units;
RRBB——tthhee rreelalatitviveer erdeudcuticotnionin inth tehne unmubmerbeorf ovfi avbilaebblea cbtearcitaercioal ocnoileosn; iMese;d M—emde—dimane,dMiainn,— Mmiinn—immalinviamluael,
Mvaalxu—e,m Maxaixm—almvaalxuiem. al value.
33..33.. CCoommpprreessssiivvee SSttrreennggtthh
TThhee mmeeaann ccoommpprreessssiivvee ssttrreennggtthh vvaalluueess aarree pprreesseenntteedd iinn FFiigguurree 33.. TThhee SSSSHHZZPP ccoonncceennttrraattiioonn ddiidd
nnoott hhaavvee aa ssiiggnniifificcaanntt iinnflfluueennccee oonn tthhee ccoommpprreessssiivvee ssttrreennggtthh ooff tthhee ccoommppoossiitteess ((pp == 00..00552244)).. TThhee mmeeaann
vvaalluueess wweerree ffrroomm 228844 MMPPaa ttoo 330077 MMPPaa..

Figure 3. Mean values and standard deviations of compressive strength.
Figure3.Meanvaluesandstandarddeviationsofcompressivestrength.
3.4. Diametral Tensile Strength