One-pot synthesis of antibacterial monomers with dual biocidal modes

18 Pages·2014·4.16 MB·English

by  Wei Zhang

journal of dentistry 42 (2014) 1078–1095

Available onlineat www.sciencedirect.com

ScienceDirect
journal homepage: www.intl.elsevierhealth.com/journals/jden

One-pot synthesis of antibacterial monomers with

dual biocidal modes

Wei Zhanga,1, Xiao-juan Luoa,1, Li-na Niub,1, Si-ying Liuc,

Wan-chun Zhud, Jeevanie Epasinghee, Liang Chenf, Guo-hua Lig,

Cui Huangc, Jing Maoa,**, David H. Pashleyh, Franklin R. Tayh,*

aDepartmentofStomatology,TongjiHospital,HuazhongUniversityofScienceandTechnology,Wuhan,China

bState Key Laboratory of Military Stomatology, Department of Prosthodontics, School of Stomatology, The Fourth

MilitaryMedicalUniversity,Xi’an,China

cHospitalofStomatology,WuhanUniversity,Wuhan,China

dDepartmentofStomatology,NorthSichuanMedicalCollege,Nanchong,China

ePrincePhilipDentalHospital,TheUniversityofHongKong,HongKongSpecialAdministrativeRegion

fResearchandDevelopment,BiscoInc.,Schaumburg,IL,USA

gDepartmentofStomatology,FuzhouDongfangHospital,Fuzhou,China

hCollegeofDentalMedicine,GeorgiaReagentsUniversity,Augusta,GA,USA

articl e i nfo ab s tract

Articlehistory: Objectives: Thepresentstudyreportedamethodforpreparingablendofantibacterialquaternaryammonium

Received 14 May 2014 silanes and quaternary ammonium methacryloxy silane (QAMS) based on the sol–gel reaction between dimethyl-
Receivedinrevisedform diethoxy silane and two trialkoxysilanes, one with an antibacterial quaternary ammonium functionality and the
otherwithamethacryloxyfunctionality.
27May2014
Methods: Reactionproductsofthesol–gelreactionwerecharacterisedbydirectinfusionmassspectrometry,FTIR
Accepted3June2014
andproton,carbonandsiliconNMR.Thisblendofmonomerswasincorporatedintoanexperimentaluniversal

adhesiveforevaluationofantimicrobialactivityagainstStreptococcusmutansbiofilms,microtensilebondstrength

andcytotoxicty.Retentionofquaternaryammoniumspeciesonpolymerisedadhesive,leachingofthesespecies

Keywords: from the adhesive and the ability of resin–dentine interfaces to inhibit S. mutans biofilms were evaluated over a 3-
monthwater-ageingperiod.
Antibacterial
Results: TheantibacterialadhesiveversionkilledbacteriainS.mutansbiofilmsnotonlythroughthereleaseofnon-
Biofilm
copolymerisable quaternary ammonium silane species(release-killing), but also via immobilised quaternary
Dualbiocidalmodes
ammonium methacryloxy silane that are copolymerised with adhesive resin comonomers (contact-killing). Con-
Interface tact-killingwasretainedafterwater-ageing.TheQAMS-containinguniversaladhesivehassimilartensilebond

Universaladhesive strengthasthecontrolandtwocommerciallyavailableuniversaladhesives,whenitwasusedforbondingtodentine

intheetch-and-rinsemodeandself-etchingmode.Incorporationoftheantimicrobialquaternaryammonium

speciesblenddidnotadverselyaffectthecytotoxicityoftheuniversaladhesiveformulation.

Conclusions: Insteadofusingquaternaryammoniumdimethacrylatesandnanosilver,analternativebimodal

antimicrobialstrategyforformulatingantimicrobialuniversaldentineadhesivesisachievedusingtheone-pot

sol–gelsynthesisscheme.

ClinicalSignificance: TheQAMScontaininguniversaldentineadhesiveswithdualantimicrobialactivityisapro-

misingmaterialaimedatpreventingsecondcariesandprolongingthelongevityofresincompositerestorations.

#2014ElsevierLtd.Allrightsreserved.

* Correspondingauthorat:DepartmentofEndodontics,CollegeofDentalMedicine,GeorgiaReagentsUniversity,Augusta,GA,USA.

Tel.:[phone];fax:[phone].

** Correspondingauthor.

E-mailaddresses:[email protected](J.Mao),[email protected](F.R.Tay).

1 Theseauthorscontributedequallytothiswork.

http://dx.doi.org/10.1016/j.jdent.2014.06.001
0300-5712/#2014ElsevierLtd.Allrightsreserved.
journal of dentistry 42 (2014) 1078–1095 1079

trialkoxysilane to another trisialkoxysilane containing the
1. Introduction
designatedorganicfunctionality.14Thesol–gelsynthesisroute

isahighlyeffectivemethodforthesynthesisofcrystallineand

Antimicrobialpolymercoatingsrepresentaclassofbiocides amorphoussilicaandorganosilicatesundermildconditions.15

that has become increasingly important as alternatives to Byintroducingorganicfunctionalitiesintotheinorganicsilica/

topically applied biocidal solutions and aerosols.1 These silicate matrices, the properties of the sol–gel derived

antibacterialpolymersmaybebroadlyclassifiedintobiocide- materials may be tailored at the molecular level according

releasing polymers and contact-killing biocidal polymers.2 to different requirements.16,17 An example of this facile

Althoughimpregnationofreleasablebiocidessuchassilver synthesis scheme is the use of tetraethoxysilane (TEOS) for

nanoparticles or antibiotics into polymers represents a couplingSIQACto3-(trimethoxysilyl)propylmethacrylate(3-

highly effective strategy for rapid killing of microbes, the MPTS).18Anantimicrobialquaternaryammoniummethacry-

graduallydecreasinglevelofthereleasedbiocidemayresult loxysilane(QAMS)moleculeisformed,whichcanbedissolved

in sub-inhibitory biocidal concentrations in the vicinity of in low concentration in methyl methacrylate to produce a

thecoatings,withthepotentialfordevelopingantimicrobial methacrylateresincomonomerblend.Whenthiscomonomer

resistance. To circumvent these problems, polymers have blendwasmixedwithpoly(methyl)methacrylatepowder,an

beendesignedwithantimicrobialfunctionalgroupsthatare antimicrobial orthodontic acrylic was produced that has

covalentlyimmobilisedonthematerialsurface.3Aconceiv- retained in vitro contact-killing effects against bacteria

ableadvantageofthisalternativestrategyisthatantimicro- commonly encountered in dental caries, as well as fungus

bialefficiencyislesslikelytodeterioratebythewearofthe presentinoralcandidiasis,forupto3months.19

polymercoating.Apotentialdisadvantageofthealternative In sol–gel synthesis of silica and silicate, matrix-forming

strategy, however, is that unless the killed microbes are precursorssuchasTEOSandtetramethoxysilane(TMOS)are

periodically removed, their accumulation over the coating preferredasanchoringunitsbecausetheyhavefourfunction-

surfacewillreducetheantimicrobialefficiencyofthecoating algroups undergoing hydrolysisand condensation tocreate

over time. Attempts to incorporate the two antibacterial three-dimensionalsilica/silicatenetworks.Nevertheless,the

strategiesintoasinglesystemhaverecentlybeenreported use of tetrafunctional alkoxysilane as the precursor renders

by various authors.4–6 These dual-functional polymeric the so-formed QAMS molecule insoluble in most dimetha-

coatingsdemonstratedveryhighinitialantimicrobialeffec- crylatesthatareemployedintheformulationofdentalresin

tiveness due to the leaching of the non-polymerisable compositesandadhesives.AlthoughtheTEOS-derivedQAMS

biocidalagent,followedbysignificantsustainedantimicro- molecule is soluble in methyl methacrylate and ethanol,

bial activity contributed by the immobilised contact-killing methyl methacrylate is a potent contact allergen and its

biocidal polymer after exhaustion of the leachable biocidal applicationasacomonomerisdentineadhesivesisrestrict-

agent. ed.20 The limited solubility of the TEOS-derived QAMS

Oneoftheearliestexamplesofnon-leachingorganosili- molecule in solvents such as methanol, ethanol or acetone

con quaternary ammonium compounds capable of killing also poses problems in dentine bonding. Evaporation of the

microorganismsoncontactwasreportedintheearly1970s. non-methacrylate solvent can result in phase separation of

Research scientists from Dow Corning Corporation found the dimethacrylate components, thereby jeopardising the

that surface bound 3-(trimethoxysilyl)-propyldimethylalkyl bondstrengthoftheadhesivecomonomerblendtodentinein

ammonium chloride possessed the most effective bacteri- thepresenceofwateroriginatingfromthedentinaltubules.21–

cidalandfungicidalpropertieswhenitsquaternaryammo- 23Intheformulationofdentineadhesives,itiscustomaryto

nium alkyl chain consisted of 18 carbons.7 After extensive employ resin monomers that are soluble in 2-hydroxyethyl

toxicological testing, Dow Corning applied to the U.S. methacrylate(HEMA),becausethelatterismisciblewithwater

EnvironmentalProtectionAgencyforindustrialregistration andmostdimethacrylatemonomers;comonomerswhichare

of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium dissolvedinHEMAdonotundergophaseseparationfollowing

chloride (SiQAC).8 The free SiQAC (aka Dow Corning 5700) removalofwaterduringthebondingprocedure.24

won an IR-100 award for one of the best products to be To circumvent the problem of limited solubility of

commercialised in 1977. Being an antimicrobial quaternary TEOS-derivedQAMSinHEMA,theauthors haveinventeda

ammonium silane (QAS), SIQAC had since been applied as new sol–gel synthesis method for preparing antibacterial

coatings on garment fabrics and the surface of medical QAMS,byreplacingthetetrafunctionalTEOSanchoringunit

prostheses.9–13ItisgenerallybelievedthatSIQACandother withadifunctionalanchoringunit,dimethyldiethoxysilane

surface-immobilisedquaternary ammoniumor phosphoni- (DMDES).DifunctionalalkoxidessuchasDMDEScannotact

umcompoundskillbacteriabycontactasaresultofboththe asnetworkformersduetoinsufficientfunctionalitiestoform

penetrationandinterruptionofthebacterialmembraneby three-dimensionalnetworks.25Thepresenceofnon-hydro-

theirlongalkylchains,andtheincreasedosmoticpressure lysableorganicgroups(i.e.methylinsteadofethoxy)inthe

betweenthebacterialcytoplasmandthelow-ionicstrength synthesis mixture reduces the cross-linking ability of the

surroundings.1 silicatenetwork.26Thistendencytoformlinearchainsmay

Because SiQAC does not possess functional groups that alsobeusedtoprovidesomeflexibilitytothepolymermatrix

enableittocopolymerisewithotherorganofunctionalmono- network27,whentheresultantDMDES-derivedQAMSmono-

mers,agenericcouplingschemehasrecentlybeendeveloped meriscopolymerisedwithotherdimethacrylatesindentine

using the sol–gel synthesis route. This scheme uses a adhesive formulations. Because one-pot sol–gel synthesis

tetralkoxysilaneastheanchoringunitforcouplingtheSIQAC involvingmultiplealkoxysilanesusuallygeneratesaseriesof
1080 journal of dentistry 42 (2014) 1078–1095

moleculeswithdifferentmolecularmassesinsteadofasingle massspectrometer(ThermoFisherScientific,Waltham,MA,

molecule, theDEDMS-derived QAMS produced bythe reac- USA) with electrospray ionisation (ESI) in the positive ion

tion of the dialkoxysilane with two trialkoxysilanes under mode.Theconditionsfordirectinfusionanalysiswere:mass-

alkaline condition was first characterised in the present to-chargerange(150–2000m/z);capillaryvoltage(28.84V)and

study. Universal dentine adhesives, which are capable of ESIspraycurrent(19mA).Theanalytesolutionwascontinu-

bondingtoenamelanddentineintheetch-and-rinseorthe ously introduced by the ESI needle to the ion source in the

self-etchingmode,representthelatestgenerationofdentine spraychamberwhichwasahighvoltageregion.Theneedle

adhesivesthatarewell-receivedbycliniciansbecauseoftheir was placed inside a larger capillary through which nitrogen

multimodebondingcapability,simplicityanduser-friendli- gaswaspumpedtonebulisethesolution.Uponnebulisation,

ness.28–30 Thus, the one-pot synthesised DMDES-derived theanalytemoleculeswereionisedbycations,predominantly

QAMSmixturewasdissolvedinHEMAandincorporatedinto H+ionsfromthesolution.Thechargeddropletspassedacross

ahydrophilicdimethacrylatecomonomerblendtocreatean an electric field between the electrodes at a higher voltage

experimental,antibacterialuniversaldentineadhesive.The relativetotheheatedcapillary.Theionswereelectrostatically

antibacterialpropertyofthepolymeriseduniversaladhesive guided and drifted towards the relatively high negative

wasexaminedusingStreptococcusmutansbiofilmswhichare voltage. In doing so, the ions entered the heated capillary

commonly found in initial dental caries. The ability of this and proceeded into the analyser region. Data processing

antibacterialuniversaladhesiveforbondingtocrowndentine involvedmultiplescanningofthemassrange(200–1200m/z)

wasinvestigated,withfurtherexaminationoftheantibacte- for about 3min to obtain a mass-to-charge ratio (m/z)

rialpotentialoftheresin–dentineinterfaceafter3monthsof spectrumwithrelativesignalintensityforeachiondetected.

water-ageing. For the antibacterial universal adhesive, the

hypothesestestedwere:(i)incorporationofDMDES-derived 2.2.2. Attenuatedtotalreflection-Fouriertransforminfrared

QAMS into an experimental universal adhesive does not spectroscopy(ATR-FTIR)

compromise the bond strength of the adhesive to dentine A Nicolet 6700 FTIR spectrophotometer (Thermo Fisher

whenitisusedintheetch-and-rinsemodeortheself-etching Scientific) with an ATR set-up was used for analysis. As no

mode;and(ii)inclusionofablendofquaternaryammonium photoinitiatorwasusedinthematerialpreparedforATR-FTIR,

species with and without methacryloxy functional groups, theoverallIRspectracollectedbetween4000and400cm(cid:2)1at

createdbyone-potsol–gelprocessingwithintheexperimen- 4cm(cid:2)1werenormalisedandsuperimposedwithrespecttothe

tal universal adhesive, resultsin both initial release-killing aliphaticC Cbandofthemethacryloxygroupsat1636cm(cid:2)1.

and retained contact-killing activity in the resin–dentine

interfaceagainstS.mutansbiofilmsafter3monthsofwater- 2.2.3. Nuclearmagneticresonancespectroscopy(NMR)

ageing. For proton (1H) NMR, the QAMS sample was dissolved in

CD OD (Sigma–Aldrich) to 10% (m/v) concentration before
3
testing.Spectrawereacquiredfrom15to(cid:2)2ppmwitha458

2. Materials and methods pulseangleof6msusingaVarianMercuryNMRspectrometer

(Varian Inc., Palo Alto, CA, USA). All chemical shifts were

2.1. SynthesisofDMDES-derivedQAMS referenced manually using the resonance frequency of

CD OD.Forcarbon(13C)NMR,theQAMSsamplewassimilarly
3
Dimethyldiethoxylsilane(DMDES),3-(trimethoxysilyl)-propyl- dissolvedinCD ODto10%(m/v)concentrationbeforetesting.
3
dimethyloctadecyl ammonium chloride (SiQAC) and 3-(tri- Spectra were acquired from 230 to (cid:2)20ppm with an 80–908

methoxysilyl)propyl methacrylate (3-MPTS) were obtained pulseangleof12ms.Compositepulsedecouplingwasusedto

from Sigma–Aldrich (St Louis, MN, USA) and used without removeprotoncoupling.Forsilicon(29Si)NMR,thewater-and

further purification. The QAMS monomer mixture was alcohol-depletedQAMSwasexaminedinthesolidstateusing

prepared by adding DMDES, SiQAC, and 3-MPTS in a 1:1:1 29SiCrossPolarisation-MagicAngleSpinning(CP-MAS)NMR.

molarratio.BasicMilliQwater(pH10,preparedusing0.01N Analysiswasperformedwitha270MHzspectrometer(JEOL,

NaOH) was added to the QAMS mixture, using a water-to- Tokyo,Japan)equippedwitha7mmMASprobe.Spectrawere

precursormolarratioof4(12molesofwaterto48molesof acquired using a MAS frequency of 4kHz, with a 458 pulse

reactants), as catalyst for the sol–gelreaction and to ensure angleof5ms.Chemicalshiftswerereferencedtotetramethyl-

optimalhydrolysisofthedialkoxysilaneandtrialkoxysilanes. silaneat(cid:3)0.3ppm.

The mixture was stirred for 6h at ambient temperature for

completion of the hydrolysis–condensation reactions, fol- 2.3. PreparationofQAMS-containinguniversaldentine

lowed by vacuum-stripping for 24h to remove the sol–gel adhesive

reactionby-products(i.e.waterandalcohols)fromtherubbery

organicallymodifiedsilicatecondensate. Auniversaladhesivewaspreparedcontaining20wt%HEMA,

andwiththeother80wt%consistingofproprietaryamounts

2.2. CharacterisationofDMDES-derivedQAMS of10-methacryloyloxydecyldihydrogenphosphate(10-MDP),

bisphenolAdiglycidylethermethacrylate(bis-GMA),ethanol,

2.2.1. Direct-infusionmassspectrometry(DIMS) water, camphorquinone and a proprietary tertiary amine

The water- and alcohol-depleted QAMS was dissolved in (courtesy of Bisco Inc., Schamburg, IL, USA). This was

methanolataconcentrationof35%(w/v).Directinfusionmass employed as the non-antibacterial, ‘‘control version’’ of the

spectrometry analysis was performed using a Finnigan LTQ universaladhesive(Lot728-95a).
journal of dentistry 42 (2014) 1078–1095 1081

Forpreparingtheantibacterialuniversaladhesive,35wt% centrifuging at 4000rpm for 10min. The cell pellet was

ofthevacuum-strippedDMDES-derivedQAMSsolidwasfirst washed three times with sterile phosphate buffered saline

dissolvedinHEMAtoproduceaHEMA-QAMSsolution.Sixtrial (PBS,0.01M,pH7.3),re-suspendedin100mLofthegrowth

versionsoftheuniversaladhesivewereinitiallypreparedby medium, and further adjusted with growth medium to a

replacingthe20wt%ofHEMAinthe‘‘controlversion’’with(a) concentrationof107CFU/mL.

14wt%HEMAand6wt%HEMA-QAMS,(b)12wt%HEMAand Theadhesive-coateddentinedisksfromthecontrolgroup

8wt% HEMA-QAMS; (c) 10wt% HEMA and 10wt% HEMA- and the QAMS-containing experimental group (N=6) were

QAMS; (d) 8wt% HEMA and 12wt% HEMA-QAMS; (e) 6wt% disinfectedunderultravioletlight(wavelength=200–290nm,

HEMAand14wt%HEMA-QAMS;and(f)20wt%HEMA-QAMS. irradiance=30mW/cm2,light-to-targetdistance=60cm)for

The other 80wt% of the composition in these trial versions 2h before transferring to the specimen holder of an oral

was the same as the ‘‘control version’’. These 6 trial biofilm reactor. The biofilm reactor consisted of a reactor

formulations were used for bonding to dentine in the etch- vessel and a specimen holder with 18 recessed holders for

and-rinsemodeandthebondedspecimensweresubjectedto insertion of substrate disks over which bacteria biofilms

microtensile bond testing (described below). Because there couldbegrown.18Thesixdisksfromeachgroupwereaffixed

werenodifferencesinmicrotensilebondstrengthamongthe tothesampleportsofthespecimenholder,andincubatedin

six versions (data not shown), the version with the highest pooledsterilesalivafor1hat378Ctocreateasalivarypellicle

concentrationofHEMA-QAMS(i.e.20wt%)waschosenasthe on the surface. Sterile pooled saliva was obtained by

‘‘antibacterialexperimentalversion’’oftheuniversaladhesive collectingwholesalivafromthreehealthyvolunteerswithout

(Lot728-95b).TheamountofDMDES-derivedQAMSpresentin stimulation(flowrate0.25mL/min;pH7.3).Thepooledsaliva

this‘‘antibacterialexperimentalversion’’was7wt%. was centrifuged at 2000rpm for 15min to remove cellular

debris,oralmicroorganismsandparticles.Thesupernatant

2.4. Adhesive-coateddentinedisksforevaluationof wasmixedwithdithiothreitol(Sigma–Aldrich,2.5mmol/L)to

antimicrobialactivity reducesalivaryproteinaggregation,andcentrifugedagainat

2000rpmfor15minat48C.Thesupernatantwasthenfilter-

Caries-free extracted human wisdom teeth were obtained sterilised through sterile 0.22mm disposable membrane

with the patients’ consent under a protocol that was filters(NalgeNumcInternational,Rochester,NY),andstored

approvedbytheHumanAssuranceCommitteeoftheGeorgia at208Cuntiluse.

Regents University. These teeth were stored in 0.9% NaCl The specimen holder with the pellicle-containing, adhe-

containing 0.02% sodium azide to prevent bacteria growth. sive-coated dentine disks from the two groups was then

Foreachtooth,mid-coronaldentinealongtheocclusalplane transferredtothereactorvesselwhichwassubsequentlyfilled

was exposed by removing the occlusal enamel with a with S. mutans cell suspension (107CFU/mL). The assembly

diamond blade (Isomet, Buehler Ltd., Lake Bluff, IL, USA) was first incubated for 90min at 378C in an orbital shaker

under copious water cooling. The exposed dentine was incubator at 50rpm, to develop the adhesion phase of the

ground with 600-grit silicon carbide paper for 30s under respective biofilm. Following the adhesion phase, the speci-

watercoolingpriortobonding. men holder was removed, rinsed carefully with 100mL of

Forgrowingofbiofilms,thedentinediskswerebondedin sterile PBS (0.01M, pH 7.3), and transferred aseptically to a

theetch-and-rinsemode,usingeitherthecontrolversionor new, sterile reactor vessel, which held the disks on a fixed

the experimental version of the universal adhesive (N=10). stage in 200mL of the growth medium. The assembly was

Eachdentinesurfacewasetchedwith32%phosphoricacidgel placedoveranorbitalincubator(378C;50rpm),andconnected

(Uni-Etch,Bisco,Inc.)for15sandrinsedwithwaterfor20s. toseveralvessels(nutrient,sucrosesolution,andwaste)and

Twocoatsoftherespectivecontrolorexperimentaluniversal toaninfusionpump,tocompletetheinvitroartificialmouth

adhesive were applied to the visibly moist acid-etched system. A desiredflow rate was established before allowing

dentine, with 15s of adhesive agitation for each coat. The biofilm formation to proceed for 24h. The flow rate was

adhesive was air-dried for 5s, and light-activated for 10s adjustedaccordingtothechemostatmodeatadilutionrateof

usingalight-curingunit(XL3000,3MESPE,St.Paul,MN,USA). 0.10h(cid:2)1. S. mutans biofilms were grown under anaerobic

Priortotheuseoftheseadhesive-coateddentinedisksfor conditions (5% carbon dioxide, 10% hydrogen and 85%

growingbiofilms,thediskswereimmersedinsteriledeionised nitrogen).Attheendofthe24-hgrowthperiod,thespecimen

waterandagitatedat200cyclesperminforonemintoremove holderwiththeadhesive-coateddentinediscswasaseptically

unpolymerised monomers within the surface air-inhibition removed and immersed in 100mL of sterile PBS to remove

layer. non-adherent bacteria cells. The adhesive-coated dentine

diskswithadherentbiofilmswerethenretrievedforfurther

2.5. Antibacterialassay investigation.

2.5.1. Bacterialcultureandbiofilmpreparation 2.5.2. Confocallaserscanningmicroscopy(CLSM)

S. mutans (ATCC 35668; American Type Culture Collection, Biofilms grown on top of the adhesive-coated dentine disks

Manassas, VA, USA) was used for the formation of single- fromthecontrolandexperimentaluniversaladhesivegroups
species bio film s. The bac teria w ere cu ltured in B rain Heart were stai nedwith aLI VE/DEAD1Ba cLightTMB acterialV iability

Infusion(BHI)broth(Difco,Becton-DickinsonandCo.,Sparks, Kit (Molecular Probes, Eugene, OR, USA). Live bacteria were

MD, USA) supplemented with 50mM sucrose (pH 7.3). stained with SYTO-9 to produce green fluorescence, and

Bacteria cells were harvested from 24-h fresh culture by bacteria with compromised membranes were stained with
1082 journal of dentistry 42 (2014) 1078–1095

propidiumiodide(PI)toproduceredfluorescence.SYTO-9and TEM. For each adhesive version, bonding was performed in

PI wereactivated at wavelengths of 488 and 568nm respec- both the etch-and-rinse mode and the self-etching mode

tively,andimagedusingaCLSM(LSM510META,Zeiss,Jena, (N=2).Bondingproceduresdescribedintheprevioussection

Germany)at40(cid:4)magnification.Foreachofthesixadhesive- wereusedfordentinebondingintheetch-and-rinsemode.For

coateddentinedisksineachgroup,twoimagestacks(Z-stack) bonding in the self-etching mode, the control or the

were obtained at locations that were characteristic of each experimental universal adhesive was used to prime the

biofilmonthatdisc.Images(fieldsize:212mm(cid:4)212mm)were dentine surface twicewithout rinsing, with 15s of adhesive

acquired at a Z-step of 2mm (i.e. distance between two agitationforeachadhesivecoat.Theadhesivewasair-dried

adjacentimagesofastack),beginningfromthebottomofthe for5s,andlight-activatedfor10s.

biofilmthatwasincontactwiththeadhesivesurface,tothe A2-mmthickstripwassectionedfromthecentreportion

topofthebiofilm.Imagesfromeachstackwereanalysedusing ofeachadhesive-coateddentinedisc.Eachstrip,containing

imageanalysissoftware(BioImageLv2.1;FacultyofOdontol- the adhesive-dentine interface, was fixed in modified

ogy, Malmo¨ University, Malmo¨, Sweden). The biovolume of Karnovsky’sfixative(2.5%glutaraldehydeand2%formalde-

interest,representingthefirst24mmofeachZ-stack(i.e.1st– hydein0.1Mcacodylatebuffer)for4h,rinsedincacodylate

13thimages),wasanalysedforthepercentagedistributionof buffer, post-fixed in 1% osmium tetroxide for 1h, dehy-

liveanddeadbacteria.Aconstantbiofilmthicknessmodelfor drated in an ascending ethanol series, substituted with

CLSManalysiswasemployedtoeliminatetheeffectofvaried propyleneoxideandembeddedinepoxyresin,accordingto

biofilmthickness. the TEM embedding protocol previously reported by Tay

et al.31 Non-demineralized, 90–100nm thick sections were

2.5.3. Colonyformingunit(CFU)counts prepared and examined unstained using a transmission

After biofilm formation, two of the six adhesive-coated electron microscope (JEM-1230, Tokyo, Japan) at 110kV.

dentine disks from each group were placed in a microtube

containing1mLofPBSandvortexedfor2mintodetachthe 2.7. Microtensilebondstrengthevaluation

biofilm.Ten-foldserialdilutionsweregeneratedinsterilePBS

(0.01mM, pH 7.3), and each dilution was plated (50mL The control universal adhesive (without DMDES-derived

aliquots) onto brain heart infusion agar plates. The plates QAMS)andtheexperimentaluniversaladhesive(containing

were incubated at 378C for 48h in an anaerobic chamber. 7% DMDES-derived QAMS) were used for bonding to mid-

Afterincubation,theCFUsofS.mutansperadhesive-coated coronal dentine in the etch-and-rinse mode or the self-

disc were calculated manually. Five replicates were per- etchingmodeaspreviouslydescribed.Forcomparison,two

formedforeachadhesive-coateddentinediscineachgroup commercially available universal adhesives, All-Bond Uni-

(N=10).StatisticalcomparisonoftheCFUdataderivedfrom versal (Bisco, Inc.) and Scotchbond Universal Adhesive (3M

the two groups was performed using Student’s t-test at ESPE)wereusedintheirrespectiveetch-and-rinsemodeand

a=0.05,followinglogarithmictransformationtorenderthe self-etching mode for bonding to mid-coronal dentine,

data suitable for the application of parametric statistical according to the instructions recommended by the respec-

methods. tive manufacturer. Six teeth were used for each adhesive

andeachbondingmode(N=6).Followingadhesiveapplica-

2.5.4. XTTassay tion and light-curing, four 1-mm increments of a resin

Theotherfourbiofilm-containingdisksfromeachgroupwere composite (AP-X; Kuraray Co. Ltd., Tokyo, Japan) were

transferred into separate microtubes containing 4mL of incrementally placed over the cured adhesive and light-

sterile PBS (0.01mM, pH 7.3), avoiding disturbances to the activatedindividuallyfor40s.Thebondedteethwerestored

biofilms. Fifty microlitres of 1mg/mL solutionof 2,3-bis-(2- in 100% humidity at 378C for 24h. The resin-bonded

methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxa- specimenswereverticallysectionedinto0.9mmthickslabs.

nilide (XTT; Sigma–Aldrich) and 4mL of 1mM menadione The centre slab was saved for evaluation of antibacterial

(Sigma–Aldrich) were then added to each microtube. The activity after water-ageing. The two adjacent slabs were

solutions were mixed gently, covered with aluminium foil sectioned into 0.9mm(cid:4)0.9mm beams; the two longest

andincubatedfor5hat378C.Afterincubation,thesolution beams from each slab were used for microtensile bond

was transferred to a new microtube and centrifuged at strengthtesting.Thus,foreachadhesiveandeachbonding

4000rpmfor10minat48C.Thesupernatantwasplacedina mode, twenty-four beams (two beams per slab(cid:4)two slabs

96-wellplateandreadat492nmusingaspectrophotometer per tooth(cid:4)six teeth) were used for microtensile testing

(Victor,R&Dsystems,Minnesota,USA).Threereadingswere (N=24). Each beam was stressed to failure under a load

takenforeachadhesive-coateddentinediscfromeachgroup appliedwithauniversaltestingmachine(VitrodyneV1000,

(N=12).StatisticalcomparisonoftheCFUdataderivedfrom Chatillon, Greensboro, NC, USA) at a crosshead speed of

the two groups was performed using Student’s t-test at 1mm/min. Beams that failed during specimen preparation

a=0.05. wererecordedasnullbondstrength.Statisticalcomparison

was performed individually for each bonding mode, with

2.6. Transmissionelectronmicroscopy(TEM) dentinebeamusedasthestatisticalunit. Becausethedata

setswerenormallydistributedandexhibitedequalvariance,

The ultrastructure of the resin-dentine interface of dentine statistical comparisons for each bonding mode were

specimens bonded with the control or the experimental performed using one-factor ANOVA and Tukey multiple

version of the universal adhesive was characterised using comparison testat a=0.05.
journal of dentistry 42 (2014) 1078–1095 1083

2.8. Remnantandleachablequaternaryammonium agitated gently for 10min. Absorbance of the mixture was

speciesafterwater-ageing recordedusingtheUV–visspectrophotometerat603nm.The

molarconcentrationsofN+leachateatdifferenttimeperiods

2.8.1. Sodiumfluoresceinbindingassay werereadfromastandardcurvewhichwasestablishedfrom

Theamountofquaternaryammoniumspeciesretainedonthe theabsorptionvaluesofknownconcentrationsofSiQAC(from

surface of the polymerised experimental universal adhesive 0to1.451mM).

(i.e.theonecontaining7wt%DMDES-derivedQAMS)aftera

12-week period of water-ageing was assessed using the 2.9. Retainedantibacterialactivityafter3monthsof

fluorescein staining method.32 This method was based on waterageing

theabilityofasinglefluoresceinmoleculetobindtotheN+

chargeofaquaternaryammoniummolecule.Adhesivedisks Thecentrecomposite-adhesive-dentineslabs(savedfromthe

(10mm diameter(cid:4)2mm thick) containing 7wt% DMDES- microtensiletestingexperiment)intheexperimentaluniver-

derivedQAMswerepreparedbyevaporatingthesolventfrom sal adhesive group were aged in sterile deionised water at

the experimental universal adhesive prior to light-curing 378C for 3 months. Specimens bonded using the etch-and-

(N=8). Eight examinations were conducted during the 12- rinsemodeandtheself-etchingmodewereusedforageing.

weekwater-ageingperiod:baseline,1st,2nd,4th,6th,8th,10th Afterageing,eachslabwasusedtogrow24-hS.mutansbiofilm

and 12th week. Except for the baseline, each disc was inthemannerpreviouslydescribed.Thebiofilmswerestained

immersed in 2mL of sterile deionised water in a separate with BacLightTM stain and imaged with CLSM at 40(cid:4)

scintillation vial at 378C under continuously shaking. At magnificationtodeterminethepresenceofretainedantibac-

differenttimeperiods,eachdiscwasretrievedfromtheageing terial activity along the resin-dentine interface after water-

solutionandplacedintoanindividualvialcontaining2mLofa ageing.

10mg/mL stock solution of fluorescein sodium salt (Sigma–

Aldrich). Specimens were left to stand in the dark at room 2.10. Cytotoxicityevaluation

temperaturefor10min.Unboundfluoresceinwasremovedby

rinsing the specimens with deionised water for five times. Dentine adhesive components applied to vital exposed

BoundfluoresceinfromtheQAMS-containingadhesivedisks dentinemaydiffuseintothepulp.Thismayadverselyaffect

was extracted by ultrasonicating each specimen in a vial thesurvivalofprimaryodontoblastsorodontoblast-likecells

containing 1.8mL of a 0.1wt% cetyltrimethylammonium differentiatedfrompulpalstemcells,andtheabilityofthese

chloride stock solution (Sigma–Aldrich) for 20min. An cells to produce tertiary reactionary or reparative dentine.34

additional 0.2mL of 100mM of PBS (pH 8.0) was added to Thus,aratodontoblast-likecelllinederivedfromtheapical

eachvial.Anabsorbancespectrumoftheboundfluorescein papilla(MDPC-23)wasemployedforcomparingthecytotoxic-

wasobtainedusingaUV–visspectrophotometer(160AUV-Vis ityofthecontrolandexperimentalversionsoftheuniversal

spectrometer, Shimadzu, Kyoto, Japan) at 501nm. The adhesive.35 The two adhesives were vacuum-stripped to

extinctioncoefficientoffluoresceininthesolutionwastaken remove the solvent, placed in pre-sterilised Teflon moulds,

tobe77mM(cid:2)1cm(cid:2)1.Thenumberofnanomolesoffluorescein covered with per-sterilised Mylar sheets, and light-cured to

inthe2mLsolutionwascalculatedfromacalibrationgraph prepare5-mmdiameterand2-mmthickadhesivedisksthat

relatingfluoresceinconcentrationandabsorbance,andthen weredevoidofair-inhibitionlayers.Untreatedcellswereused

divided by the surface area of the specimen to generate a asthenegativecontrol.Disksofsimilardimensionstothetest

specificN+chargedensityvalue(nM/cm2). adhesives and prepared from zinc oxide–eugenol based

Intermediate Restorative Material (IRM; Dentsply Caulk,

2.8.2. Bromophenolblueassay Milford, DE, USA) were assigned as the positive control. All

Becausethefluoresceinbindingassaydidnotaccountforthe setmaterialsweresterilisedwithultravioletlight(conditions

amount of QAMS released into solution as an unbound identicaltothosementionedabove)for2hbeforetesting.The

component,theamountofleachedQAMSintheagedsolution MDPC-23cellswereplatedingrowthmediumandincubated

wasdeterminedusingthebromophenolblueassay.33Bromo- at378Cinahumidified5%CO atmospherefor24huntilthey
2
phenolblueisadyewhichformsacomplexwithquaternary achieved70–80%confluency.Thegrowthmediumconsistedof

ammoniumcompounds,andresultsinashiftofl from593 Dulbecco modified Eagle medium (Lonza, Wakersville, MD,
max
to 603nm due to complex formation. Additional adhesive USA)and10%foetalbovineserum(InvitrogenCorp.,Carlsbad,

disks (10.0mm diameter(cid:4)2.0mm thick) containing 7wt% CA,USA)supplementedwith2mmol/LL-glutamineand100U/

DMDES-derivedQAMswerepreparedinthemannerreported mLpenicillin/streptomycin.

intheprevioussection(N=8).Sevenperiodswereexamined:

1st,2nd,4th,6th,8th,10thand12thweek.Thediskswerefirst 2.10.1. Cellviability

stored in 2mL of sterile deionised water in individual An XTT Cell Viability Assay Kit (Biotium Inc., Hayward, CA,

scintillationvialsat378C.Afterretrievingtheeluentcontain- USA) was used to determine cell viability based on the

ing the leached QAMS at each time period, the disks were cleavageoftheyellowtetrazoliumsaltXTTbymitochondrial

placedinfreshdeionisedwaterforcontinuedageinguntilthe enzymesinmetabolicallyactivecellstoformasolubleorange

nextcollectionperiod.Foreachtimeperiod,a0.5mLaliquotof formazan product. Disks from the different groups were

the eluent was mixed with 1.5mL of bromophenol blue placedindividuallyintranswellinsertswitha3-mmporesize

solution(0.001wt%;bufferedwithsodiumcarbonatetopH7.0 (BDFalcon,FranklinLakes,NJ,USA)topreventdirectcontact

to avoid absorption changes owing to pH fluctuations), and ofthecells.Thetranswellinsertswereplacedinsidea24-well
1084 journal of dentistry 42 (2014) 1078–1095

plate with each well covered by an equal volume (2mL) of (pH 8.0) for 5min, centrifuged and re-suspended at 1(cid:4)104

growthmedium.Thediskswereexposedtotheplatedcellsfor cells/mL in 1(cid:4) binding buffer (Biotium Inc). The cells were

3 days, without further change in culture medium, before stained with fluorescein isothiocyanate (FITC)–annexin V

testingformitochondrialdehydrogenaseactivity.Production (AnV; l /l =492/514nm, green fluorescence) and ethi-
abs em
offormazanbythemetabolicallyactivecellswasquantifiedby diumhomodimer-III (Etd;l /l =528/617nm, red fluores-
abs em
measuring its absorbance at 492nm. The absorbance of the cence) and incubated for 15min in the dark. The stained

control was adjusted to 100%, with which the relative cells were subjected to fluorescence-activated cell sorting

dehydrogenase activities of the three other groups were (FACS)usingaFACSCaliburflowcytometer(BDBiosciences,

compared (N=10). Data derived from the control and SanJose,CA,USA)todeterminethepercentagedistribution

experimentalversionsoftheuniversaladhesivewerestatisti- ofvital(AnV/Etdnegative),earlyapoptotic(AnVpositive,Etd

cally compared using Student’s t-test at a=0.05. The IRM negative), late apoptotic (secondary necrosis; AnV/Etd

positive control group was excluded to increase the robust- positive), and necrotic (AnV negative, Etd positive) cell

nessofthestatisticalanalysis. populations.

2.10.2. Apoptosisevaluationbyflowcytometry
3. Results
After MDPC-23 cells were exposed to test materials for 3

days, the cells were washed with PBS to remove dead cell

debrisandremaininggrowthmedium,detachedwith0.05% Fig. 1A represents the sol–gel reaction scheme using

trypsin–0.05mMethylenediaminetetra-aceticacidsolution dimethyldiethoxysilane as the anchoring unit for the two

Fig.1–(A)ReactionschemeforsynthesisingDMDES-derivedQAMSusingdimethyldiethoxysilaneastheanchoringunit.

(B)Majorquaternaryammoniumcontainingcomponentmolecules(theoreticalmass)producedbythesol–gelreaction

correspondingtothemajorm/z(mass-to-chargeratio)peaksofthepositiveionsdetectedbydirectinfusionmass

spectrometry.Themoleculethatcanbecopolymerisedwiththeadhesivemethacrylatecomonomerblendisindicatedbya

boxarounditsmethacryloxygroup.(C)Superimpositionoftheinfraredspectraobtainedfrom0to3hduringsol–gel

reactionbetweenthedialkoxysilaneandtrialkoxysilanemixture,illustratingthehydrolysiskineticsofthereaction.

(D)Superimpositionoftheinfraredspectraobtainedfrom4to6hduringsol–gelreactionbetweenthedialkoxysilaneand

trialkoxysilanemixture,illustratingthecondensationkineticsofthereaction.
journal of dentistry 42 (2014) 1078–1095 1085

trialkoxysilanes,withayieldof65.9wt%followingvacuum whenpeaksthatwereindicativeofthecondensationreaction

strippingofthewaterandalcoholreactionby-products.This began to appear. Condensation in base-catalysed sol–gel

organically modified silicate condensate was found to be reactions proceed with the need for pH adjustment; the

soluble in HEMA, with the maximum concentration of the hydrolysis kinetics and condensation kinetics were pre-

condensate in HEMA being 35wt%. The major linear sented separately only to facilitate interpretation. The

molecular components present in the condensate that condensation reaction that occurred from 4 to 6h was

contained quaternary ammonium species, as analysed by representedbytheincreaseofthemajorlinearsiloxanepeak

directinfusionmassspectrometryintheformofpositiveions at (cid:5)1100cm(cid:2)1, which corresponds to the symmetrical

after electrospray ionisation, are shown in Fig. 1B. For the stretchingmodeoftheSi–O–Silinkage(Fig.1D).36,37

quaternary ammonium linear species, two of them repre- Proton NMR and carbon NMR spectra of the CD OD-
3
sentedthecondensationbetweenSIQACandDMDES(iandii solubilised,vacuum-strippedreactionproductsresultingfrom

in Fig. 1B), while the other 3 species (iii–v in Fig. 1B) thesol–gelreactionareshowninFig.2AandB,respectively.

represented the condensation products of the three alkox- Spectral data for 1H NMR, d (ppm): d=6.08 HCH C(CO); 5.60

ysilanes.Anunknownchemical(measuredm/z298.99)was HCH C(CO);4.79–OCH2–;4.12CH3–O–Si;3.54–3.29N(CH ) CH –
32 2
also detected. The co-existence of quaternary ammonium CH ;3.16–3.13N(CH ) ;1.92CH C(CO) CH ;1.39CH CH Si;1.28–
2 32 3 2 2 2
silanespecieswith(vinFig.1B)andwithout(i–ivinFig.1B)the 1.22 CH (CH ) ; 0.89 CH -(CH ) ; 0.74–0.72 Si–CH –; 0.17–0.14
3 216 3 217 2
methacryloxy functional group was also identified using CH –Si.Spectraldatafor13CNMR,d(ppm):d=168.4CO(–O–)(C);
3
DIMS. During the sol–gel reaction, the hydrolysis and 137.6 C(CH )(CO)(CH ); 126.1 CH C; 67.5 –OCH (CH –); 65.5–
3 2 2 2 2
condensation processes were monitored by ATR-FTIR. 64.6–NCH (CH –);51.2–43.3CH –O–Si;33.0–23.7(CH ) CH ;18.7
2 2 3 216 3
Fig. 1C and D shows the kinetics of hydrolysis and CH –C CH ; 14.7 CH –(CH ) ;10.8–9.5 Si–CH CH ; 1.5–0.9
3 2 3 217 2 2
condensationofdialkoxysilaneandtrialkoxysilanemixture, Si(CH ) . Solid-state silicon CP-MAS NMR spectrum of the
32
respectively,byreal-timeATR-FTIR.Hydrolysiswasreflected rubbery solid produced after vacuum stripping revealed the

by the decrease in the peak height of the Si–O–C band at presence of two series of peaks that are characteristic of

1082cm(cid:2)1, increases of the alcohol peaks (CH OH and siloxaneswithtrifunctional(Tseries)anddifunctionalstruc-
3
C H OH)between(cid:5)1016–1032cm(cid:2)1,andtheincreaseinthe tural units (Dseries).38 Deconvolution of these peaks further
2 5
silanol (SiOH) peak at 912cm(cid:2)1 (Fig. 1C).36 The extent of identifiedthepresenceofT andT peaksintheTseries,andD
3 2 2
hydrolysis, although not complete, slowed down after 3h, andD peaksintheDseries(Fig.2C).Spectraldatafor1H!29Si
1
Fig.2–(A)1HNMRspectrumofwater-andalcohol-depletedcondensateoftheDMDES-derivedQAMS.(B)13CNMRspectrum

ofwater-andalcohol-depletedcondensateoftheDMDES-derivedQAMS.(C)1H!29SiCPMASNMRspectrumofwater-and

alcohol-depletedcondensateoftheDMDES-derivedQAMSshowingD ,D ,T andT species.
1 2 2 3
1086 journal of dentistry 42 (2014) 1078–1095

Fig.3–Confocallaserscanningmicroscopyimages(2Doverlayprojections)ofBacLight-stained24-hStreptococcusmutans

biofilms(live-green;dead-red)grownonpolymeriseduniversaladhesivedisks.(A)Controluniversaladhesivewithout

DMDES-derived-QAMS.(B)Antibacterialuniversaladhesivecontaining7wt%ofDMDES-derived-QAMS.Thechartbeneath

eachimageindicatesthecorrespondingbiomassofliveanddeadbacteriaasafunctionofthebiofilmlevel(Zstep=2mm).

(C)Colonyformingunit(CFU)cellviabilitycountsand(D)XTTcellmetabolismassayofS.mutansgrownonuniversal

adhesive-coateddentinediskspreparedusingthecontroluniversaladhesive(0wt%ofDMDES-derivedQAMS)and

antibacterialuniversaladhesive(7wt%ofDMDES-derivedQAMS).(Forinterpretationofthereferencestocolourinthis

figurelegend,thereaderisreferredtothewebversionofthisarticle.)

CP-MASNMR,d(ppm):d=(cid:2)12.8D (–SiO)Si(OH)(CH ) ;(cid:2)19.2D identification of the T siloxane structural unit provides
1 32 2 3
(BBSiO) Si(CH ) ; (cid:2)59.3 T (BBSiO) Si(OH)R; (cid:2)68.3 T (BBSiO) SiR. evidence of some degree of 3-D network formation
2 32 2 2 3 3
T3:T2:D2:D1=100:45.2:36:2:7.0. Although the presence of in the reaction products that are attributed to the two

theDseriesisindicativeoftheexistenceoflinearmolecules, trialkoxysilanes.39
journal of dentistry 42 (2014) 1078–1095 1087

Fig. 3A and B shows stacked confocal images (top and remained relatively stable until the end of three months.

bottomviews)andthedistributionoflive/deadbacteriainS. Fig. 4F shows the molar concentrations of N+ species in the

mutans biofilms grown on the surfaces of representative leachate derived from the QAMS-containing experimental

adhesive-coated dentine disks derived from the control universal adhesive disks during the 3-month water-ageing

universal adhesive group and the antibacterial experimental period.AburstreleaseofQAMscouldbeseenduringthefirst

universal adhesive group, respectively. For each Z-stack, the month.Thereafter,leachingofN+specieswasnegligibleuntil

‘‘topview’’ofthebiofilmrepresentsthe12thlayeroftheZ-stack theendofthethirdmonth.

whichis24mmfromtheadhesivesurface,whilethe‘‘bottom Fig.5Aisaschematicillustratingtheexperimentalsetup

view’’ of the biofilm represents the 1st layer of the Z-stack, employedforexaminingthegrowthofS.mutansbiofilmalong

whichis2mmfromtheadhesivesurface.Livebacteriathatwere a slice of the sectioned resin-dentine interface. After a 3-
stained on ly by th e S YTO-9 co mponen t of the LIV E/DE AD1 m onth w ater -ageing p eriod, merged CLSM image s of

BacLightTM staining kit exhibited green fluorescence, while BacLightTM-stained24-hbiofilmsshowednovisuallyapparent

deadbacteriawithdamagedmembraneswerestainedbyboth killofthebacteriapresentinS.mutansbiofilmsgrownonthe

SYTO-9andpropidiumiodidecomponentsandexhibitedred resin-dentine interface created by the application of the

fluorescence.40Therelativeareadistributionsofliveanddead controlversionof the universaladhesive in either theetch-

bacteria from the biomass at each level of a Z-stack are and-rinsemode(Fig.5B)ortheself-etchingmode(Fig.5D).In

summarisedinthelineplotsbelowtheconfocalimages.From theslicecreatedbytheuseoftheexperimentalversionofthe

theselineplots,itisapparentthattheexperimentaluniversal universaladhesiveintheetch-and-rinsemode(Fig.5C),ared

adhesive that contained 7wt% DMDES-derived QAMS pos- fluorescencebandcouldbeseenalongtheadhesive-dentine

sessedbothinitiallycontact-killingandrelease-killingcapabil- interface that is indicative of the presence of dead bacteria

itypriortowater-ageing.TheCFUcountsoflivebacteriafromS. along the base of the biofilm. A small amount of red

mutans that retained the capability to produce colonies on fluorescence could also be identified in the composite-

further culturingare shown inFig.3C.The controluniversal adhesive interface above, which could be attributed to the

adhesive group without DMDES-derived QAMS exhibited blendingoftheair-inhibitionlayerofthecuredadhesivewith

significantlyhigherCFUcountsthantheexperimentaluniver- theresincompositeduringpolymerisationofthecomposite.

sal adhesive group containing DMDES-derived QAMS Thepresenceoflivebacteriacoloniesgrowingontopofdead

(p<0.001).ResultsoftheXTTcellmetabolismassayareshown bacteriainthebiofilmissuggestiveofretainedcontact-killing

inFig.3D.Bacteriaderivedfromthecontroluniversaladhesive potential following depletion of the leachable quaternary

group exhibited significantly higher metabolic activity than ammonium silane components after water-ageing. Similar

thosederivedfromtheexperimentaluniversaladhesivegroup featurescouldbeidentifiedintherepresentativesliceofthe

(p<0.05). resin-dentineinterfacecreatedbytheuseoftheexperimental

Fig.4AandCisrepresentativeTEMimageoftheuseofthe version of the universal adhesive in the self-etching mode

experimental universal adhesive for bonding to dentine in (Fig.5E).

the etch-and-rinse mode or the self-etching mode, respec- TherelativecellularredoxenzymaticactivitiesofMPDC-

tively.Similartothecontroluniversaladhesive(notshown), 23odontoblast-likecellsinresponsetoindirectcontactfrom

application of the experimental universal adhesive in the the control and experimental versions of the universal

etch-and-rinsemodeafterphosphoricacidetchingresulted adhesive are shown in Fig. 6A. There was no significant

in a 5–7mm thick hybrid layer that was devoid of remnant differencebetweentherelativepercentagesofhealthycells

apatitecrystallites(Fig.4A).Whenemployedforbondingto inthecontroluniversaladhesivegroup(86.5(cid:3)3.7%)andin

dentineintheetch-and-rinsemode,therewasnosignificant the experimental universal adhesive group (84.8(cid:3)3.2%)

difference in the microtensile bond strengths among the (p>0.05). Results were semi-quantitative for the apopto-

controlandexperimentalversionsoftheuniversaladhesive, sis/necrosis assay and were not statistically analysed

andthetwocommerciallyavailableuniversaladhesives,All- (Fig.6B).Thepercentageofcellswithintactcellmembranes

BondUniversalandScotchbondUniversal(p=0.313;Fig.4B). (i.e. non-apoptotic and non-necrotic), after exposure to the

Similar to the control universal adhesive (not shown), controluniversaladhesive,was51.0%.Thepercentageofthe

application of the experimental universal adhesive in the same category of vital cells after exposure to the experi-

self-etching mode to dentine resulted in a 300nm thick, mental version of the universal adhesive was 65.6%. The

partiallydemineralisedhybridlayerthatcontainedremnant percentage of vital cells in the negative control (untreated

apatitecrystallites(Fig.4C).Whenemployedforbondingto cells) was 93.5% instead of the expected 100%. This was

dentine in the self-etching mode, there was no significant probablyduetoalterationofmembraneintegrityduringcell

difference in the microtensile bond strengths among the detachment by trypsinisation; trypsin caused disruption of

controlandexperimentalversionsoftheuniversaladhesive, fibronectin–integrin interaction between cells, while EDTA

andthetwocommerciallyavailableuniversaladhesives,All- chelated calcium ions required for promoting fibronectin–

Bond Universal and Scotchbond Universal (p=0.286; integrin interactions. Two-dimensional plots of the distri-

Fig. 4D). bution of sorted vital, early apoptotic, late apoptotic and

Fig. 4E represents the plot of surface charge density/unit necroticcellsareshowninFig.6Cforthecontroluniversal

area of the quaternary ammonium species present on adhesive, Fig. 6D for the experimental universal adhesive,

theQAMS-containinguniversaladhesivesurfaceduringa3- Fig. 6E for the IRM positive control, and Fig. 6F for

monthwater-ageingperiod.Thequantityofremnantquater- the untreated cells that had not been exposed to test

naryammoniumspeciesdecreasedduringthefirstmonthand materials.