Table Of ContentSethCaousetal.BMCOralHealth2013,13:4
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RESEARCH ARTICLE Open Access
Streptococcus mitis Actinomyces
Adhesion of and
oris
in co-culture to machined and anodized
titanium surfaces as affected by atmosphere
and pH
Josefin Seth Caous1,3*, Maria Lövenklev2, Jenny Fäldt3 and Maud Langton2,4
Abstract
Background: With the rising demand for osseointegrated titanium implants for replacing missing teeth, often in
patients witha history of periodontitis, implant-related infections have become an issue ofgrowing concern. Novel
methods for treating and preventing implant-associatedinfections are urgently needed. The aim of this study was
to investigate if different pH, atmosphereand surface propertiescould restrictbacterial adhesion to titanium
surfaces used in dental implants.
™
Methods: Titaniumdiscswith machined or anodized (TiUnite ) surface were incubatedwith a co-culture of
Streptococcus mitis and Actinomyces oris (early colonizers of oral surfaces) atpH 5.0, 7.0 and 9.0 ataerobic or
anaerobic atmosphere. The adhesion was analysed by counting colony forming (CFU) units on agar and by
confocal laser scanning microscopy (CLSM).
Results: The CFUanalysis showed that a pH of 5.0was found to significantly decrease the adhesion of S.mitis,and
anaerobic atmosphere,theadhesion of A. oris.S. mitis was found insignificantly less amounts onthe anodized
surface thanthe machined surface, while A. oris was found inequalamountsonboth surfaces. The CLSM analysis
confirmed the results from theCFU count and provided additional informationon how the two oral commensal
species adhered to the surfaces: mainly in dispersed clusters oriented withthe groves of themachined surface and
thepores of theanodized surface.
Conclusions: Bacterial adhesion by S. mitis and A. oris can be restricted by acidic pH and aerobic atmosphere. The
anodized surface reducedtheadhesion of S.mitis compared to themachined surface; whileA. oris adhered equally
well to thepores of theanodized surface and to thegroovesof themachined surface. It is difficult to transfer these
results directly intoa clinical situation. However, it is worth further investigating these findings from an invitro
perspective, as well as clinically,to gain more knowledge ofthe effects acid pH and aerobic atmosphere have on
initial bacterial adhesion.
Keywords: Bacterial adhesion,Dental implants, Peri-implant disease, Confocal laser scanning microscopy
*Correspondence:[email protected]
1DepartmentofChemicalandBiologicalEngineering,ChalmersUniversityof
Technology,GothenburgSE-40229,Sweden
3NobelBiocareAB,P.O.Box5190,GothenburgSE-40226,Sweden
Fulllistofauthorinformationisavailableattheendofthearticle
©2013SethCaousetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background antimicrobial irrigation solutions and gels have an alka-
Advancesindentalimplantologyduringthepast20years line pH [20,21]. Furthermore, most investigations have
have made it possible to provide a greater number of been performed using a monoculture. Considering the
patientswithsuccessfullyosseointegrateddentalimplants. complexity of the microbial community of the oral cav-
[1,2]. However, with the shift from mainly treating fully ity, the use of two bacteria in a co-culture could provide
edentulouspatientstopatientsmissingoneorafewteeth, additionalimportantinformation.
often due to periodontitis, implant-related bacterial infec- In order to prevent implant-related infections and to
tions have become an issue of increasing concern [3-5]. improve the treatment options, more knowledge about
Bacteria colonizing the periodontal pockets can spread to bacterial adhesion to implant surfaces, e.g. the influence
theperi-implanttissueandexposedimplantpart,andmay of surface properties and environmental conditions, is
initiate an inflammatory response in the peri-implant tis- crucial. The aim of this study was to investigate the ef-
sue [6]. An inflammation in the soft tissue surrounding fect of pH 5.0, pH 7.0 and 9.0 in combination with aer-
the implant can disturb the tight connection between the obic or anaerobic atmosphere, on the initial bacterial
mucosa and the implant abutment. This will enable bac- adhesion of a co-culture of S. mitis and A. oris to both
terial adhesion to the smooth surface of the abutment machinedandanodizedtitaniumsurfaces;andtoanalyse
and, if exposed, to the rough surface of the implant [7,8]. if the characteristics of the implant surface affects the
If not successfully treated, the inflammation may eventu- initial bacterial adherence. S. mitis and A. oris are both
ally lead to degradation of the implant-supporting bone, facultative anaerobes that can survive in an aerobic en-
resultinginlossoftheimplant.Treatmentofperi-implant vironment, albeit they have a greater growth potential
disease today often includes thorough mechanical clean- under anaerobic conditions. Our hypothesis is therefore,
ingoftheimplantsurfaceandtheuseofsystemicantibio- that bacterial adhesion is restricted by an aerobic envir-
tics, however, there is no guarantee of a successful onment and by both acidic and alkaline pH. Further-
outcome[9,10]. more, while rough surfaces usually facilitate bacterial
The initial adherence of bacteria to exposed implant adherence, the anodized surface has a titanium oxide
partswillbeinfluenced byvariationsinthe oralenviron- layer, mainly consistent of the crystalline phase anatase,
ment. The pH and level of oxygen in the oral cavity vary which is known to have antimicrobial properties [22,23].
dependingonanumberoffactors,includingfoodintake, Thus, we further hypothesize that less bacteria should
oral health and location in the mouth, i.e. air exposed adhere to the anodized surface than to the machined ti-
tooth surface vs. the oxygen deprived gingival pocket. tanium surface.
[11]. Oral bacteria, such as Streptococcus mitis and Acti-
nomyces oris (formely naeslundii genotype II), are nor- Methods
mally found in the air-exposed margin of the gingival Titaniumdiscs
crevice, and are continuously flushed by saliva with a Discs of commercially pure titanium (diameter of
™
neutral pH of 7. However, with the increase in bacterial 15 mm) with either a machined or anodized (TiUnite )
load the local environment becomes more acidic as a re- surfacewereused(seeFigure1).Thediscswithmachined
sult of bacterial production of lactic acid [12]. Further- surfacehadathicknessof1.0mmandthediscswithano-
more, inflammation in the implant-surrounding soft dized surface had a thickness of 1.7 mm. All discs were
tissue leads to increased production of the slightly alka- produced, cleaned, sterilized and delivered in ethanol by
line gingival crevice fluid [13], and a deepening of the Nobel Biocare AB. The average surface roughness (S ) of
a
peri-implant pocket [10,14]. With the deepening of the thediscswasmeasuredatNobelBiocareABafterproduc-
gingival pocket, the oxygen saturation of the crevice tion, over a 88 × 76 μm area by interference microscopy
fluid decreases [15]. Many of the studies reported in the using the Mapview EX surface mapping software and a
literature have been focused on the effect of acidification Gaussian high-pass 50 × 50 μm filter. The S value (±
a
on the caries-causing Streptococcus mutans [16,17] ,and standard deviation) was found to be 0.48 ± 0.04 μm for
on its adhesion to hydroxyapatite, a bioceramic similar themachinedsurfaceand1.26±0.09μmfortheanodized
to the mineral component of bone and teeth [18]. An surface, corresponding to values presented in the litera-
acidic pH of 4.5 has been shown to reduce the adhesion ture for equivalent implants [24,25]. The S value was
a
of S. mutans to hydroxyapatite, while a pH of 6.0 has obtainedbycalculatingthearithmeticmeanoftheabso-
been reported to have a similar effect on the ability of A. lute values of the measured surface departures from a
oris to adhere [19]. However, no extended research has meanplanewithinthesamplingarea[26].TheGaussian
been conducted onthe effect ofacidification onbacterial filter was applied in order to separate the roughness
adhesion to the surface of titanium implants and abut- from the waviness and form of the surface [25]. The S
a
ments, nor has the effect of alkaline pH on bacterial ad- was measured for at least 2 locations per triplicate of
hesion been thoroughly investigated, although many eachsurface.
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Figure1Scanningelectronmicroscopy(SEM)imagesofmachined(a)andanodized(b)titaniumsurfaces,providedbyNobelBiocare
AB.Themachinedsurfaceisrelativelysmoothwithdistinctorientationofthesurfaceirregularities(anisotropic)whiletheanodizedsurfaceis
roughwithahomogenousstructure(isotropic).
Preparationofinoculums and anodized surface were placed in a 12-well microtiter
The early colonizers S. mitis (CCUG 27741) and A. oris plate and incubated in 2.0 ml of supplemented BHI
(CCUG 33517), originally derived from human dental broth (pH 7) inoculated with the two bacteria to a con-
plaque (Culture Collection, University of Gothenburg, centration of 106 CFU ml-1 of each bacterium, in single
Sweden), were grown on horse blood agar plates. Over- aswellasinco-culture.Incubationwasperformedonan
night cultures of the two bacteria were incubated separ- orbital shaker (80 revolutions per minute, rpm) at 37°C.
ately for 19 hours in brain heart infusion (BHI) medium, Themeasurementswere repeated twice.
37gl-1(DifcoLaboratories,Detroit,Michigan,USA)sup-
plemented with glucose, 10 g l-1, cysteine hydrochloride, Incubationconditionsforbacterialadhesion
0.5 g l-1 (VWR International, Stockholm, Sweden ), yeast Supplemented BHI broth with a pH of 5.0, 7.0 or 9.0
extract, 5 g l-1 (Difco Laboratories, Detroit, Michigan, was inoculated with S. mitis and A. oris to a concentra-
USA) and inactivated horse serum 1% (Fisher Scientific, tion of 106 CFU ml-1 of each bacterium. Machined and
Göteborg, Sweden). The 50/50 co-culture (106 CFU ml-1) anodized titanium discs were placed in a 12-well micro-
of S. mitis and A. oris, in new supplemented BHI broth, titer plate as described above, covered by 2.0 ml of the
was prepared directly prior to use. Agar plates and over- inoculated BHI broth of different pH and incubated in
night cultures were incubated in an anaerobic jar at 37°C aerobic or anaerobic atmosphere. Incubation was carried
with CO Gen (Oxoid, Malmö, Sweden) to create an at- out at 37°C on an orbital shaker (80 rpm) for 2.0 hours.
2
mosphere rich in carbon dioxide (CO 6%) and poor in The experiments with all combinations of pH, atmos-
2,
oxygen (O , 15%). The additional serum and proteins phere and surface were carried out at least six times, on
2
whereaddedtothemediumtoimitatethegingivalcrevice separate occasions.
fluidandtoformaproteinlayeronthetitaniumsurfaces, The values of pH (5.0 and 9.0) were chosen to repre-
likethepellicleformedonhardoralsurfaces. sent the fluctuations in environmental pH that occur in
the mouth depending on location in the dental plaque,
Bacteriallagphasedurationandinitialadhesion food intakeandoralhealth.ThepHofwholesalivaranges
The lag phase duration (LPD) of S. mitis and A. oris, in from 6.75 to 7.25 [28], and pH 7.0 was therefore used as
mono and co-culture, was measured. Bacteria were culti- reference. The pH of the supplemented BHI broth was
vated in supplemented BHI broth, pH 7.0, at 37°C in a adjustedwithHClorNaOHpriortosterilization(at121°C
modified atmosphere (CO , 6%; O 15%) as described for 15 minutes) and also analysed after sterilization to de-
2 2
above.Sevensampleswerewithdrawnatintervalsthrough- tectifanydeviationfromthespecifiedvaluehadoccurred.
outa4-hourperiodofincubation.Samplesweredilutedas Whenstoredaerobicallyforaminimumof24hoursat5°C
appropriate and spread on agar plates for colony-forming the supplemented BHI broth, reached a saturation of dis-
unit (CFU)counting, after incubationat 37°C in ananaer- solved oxygen of approximately 80%, which was used to
obic jar for two days, as described above. The LPD was represent the aerobic environment of the gingival margin.
determined by fitting the model of Baranyi and Roberts The oxygen deprived environment in the gingival crevice
[27] to the growth curves using the freeware MicroFit 1.0 and mature plaque was simulated by flushing the broth
(InstituteofFoodResearch,Norwich,UK). with nitrogen for two minutes directly prior to use, to re-
The initial adhesion of S. mitis and A. oris, in mono duce theoxygen saturation in thebroth from 80% to 20%.
and co-culture, after 2.0 hours of incubation at pH 7.0 Incubation was performed in a sealed blender bag (VWR
in modified atmosphere (CO , 6%; O 15%) was mea- International, Stockholm, Sweden) equipped with a mem-
2 2
sured. Ethanol-sterilized titanium discs with machined brane through which the air was extracted with a syringe
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andreplacedwithnitrogen.Thiswasdoneinordertopre- inverted microscope (Leica DM IRE2, Leica Micro-
vent the oxygen saturation in the broth to increase during systems, Manheim, Germany) equipped with a glycerol
theextentoftheexperiment.Theoxygensaturationofthe immersionobjectivewithamagnificationof63timesanda
BHI broth was measured at room-temperature with an numerical aperture of 1.3. The fluorochromes indicating
oxygenmeter(Oxi340i)equippedwithanoxygengaselec- live and dead cells were excited by 488 and 594 nm light,
trode(CellOx325)(bothfromWTW,Weilheim,Germany) respectively. The emitted light was collected in the wave-
at three separate occasions, prior to the experiment. The length ranges 500–554 and 620–660 nm, the former
oxygen saturation of the BHI broth was found to decrease (green)indicatingviablecellsandthelatter(red)non-viable
to 20% after 1 minute of nitrogen flushing and did not de- cells.S.mitisandA.oriscouldbevisuallyseparatedbytheir
creasefurtheralthoughflushedforupto20minutes.Thus, differenceincell-andcolony-morphology(cocciandchains
atimeof2minuteswaschosenfortheexperiments. v.s. rod and clusters, respectively). Samples were prepared
and analysed in duplicate on two separate occasions. The
Enumerationofadheredbacteria resolutionofallimageswas0.12μmperpixel.
After incubation, each titanium disc was rinsed in sterile The amount of adhered bacteria on the bottom-side of
distilled water for 10 seconds to remove non-adhered the titanium discs after 2.0 hours of incubation was
W
bacteria. The disc was then placed in a plastic blender analyzed by staining the surfaces with LIVE/DEAD as
bag (VWR International, Stockholm, Sweden) with 42 ml described above and imaging them with a fluorescence
sterile buffered peptone water (0.85% NaCl and 1% Pep- microscope(Axioskop,CarlZeiss,Oberkochen,Germany).
tone) (Difco Laboratories, Becton Dickinson, Stockholm, The surfaces were imaged after stomaching (1 min) to
Sweden) and processed in a stomacher as described by makesurethat adhered bacteriawereremoved satisfactor-
GagnonandSlawson[29]with500strokesperminutefor ily.Thisexaminationwasperformedonduplicatesofeach
60 seconds, in order to mechanically remove the adhered surface, one incubated at pH 5.0 and the other at pH 7.0,
bacteriafromthetitaniumdisc. at37°Candmodifiedatmosphere(CO ,6%;O 15%).
2 2
The peptone water solution containing the detached
bacteria was diluted 1:10, spread on blood agar plates Statisticalanalysis
and incubated as described above for CFU counting. Levene’s test of equality of error variances was used to
The colonies formed by the two bacteria differ in col- establish that the logarithm (base 10) of the number of
ony-morphology, and in respect to size, colour and bacteria adhered to the machined and anodized titanium
shape allowing for the CFU of the two bacteria to be discs (log CFU mm-2) had a homogeneous variance. As
counted separately. The total amount of bacteria on the thiswasshowntobethe case,three-wayanalysisof vari-
discsafter2.0hours ofincubation couldbe calculated by ance(ANOVA)couldbeperformedtostatisticallyanalyse
counting the CFU on agar and divided by the area of the theeffectofpH5.0and9.0incomparisontopH7.0com-
discs exposed to the bacteria. Since the disc was placed bined with aerobic or anaerobic atmosphere on the adhe-
on the bottom of a microtiter well, only very small num- sion of S.mitis and A. oris inco-culture to machined and
bers of bacteria were found to adhere to the underside anodizedtitaniumsurfaces.Theeffectsofsurface,pHand
of the disc (microscopy analysis data not shown in pro- atmosphereonthenumberofadheredS.mitisandA.oris
portion to the top-side and edges. Based on this, the (log CFU mm-1), in co-culture, were analysed. Dunett’s t-
bacteria that adhered to the bottom-side was deemed testwasusedtocomparetheeffectofpH5.0and9.0ver-
not to affect the overall comparison, and only the upper sus pH7.0.As theotherfactors (atmosphereandsurface)
surface and the side of the disc were included when cal- onlyhadtwolevels,noposthoctestwasneeded.Allana-
culating the area. Although, the anodized surface has lysiswasperformedinSPSSStatisticsversion17.0andthe
been suggested to have a 95% larger area than an ideal criterionforstatisticalsignificancewassettoα=0.05(i.e.
flat surface, [24] it was considered to be of more clinical comparisons rendering a p-value smaller than 0.05 are
relevance to calculate the area at mm level, not taking in considered to be of statistical significance). The factors
to account the increase in surface area at μm level of the investigated were found to influence the adhesion of S.
anodizedsurface duetothestructuredsurface. mitis and A. oris differently. However, since the bacteria
were in a co-culture, they could not be considered to be
Microscopytechniques independent of each other, and no statistical analysis of
Titanium discs were incubated with the bacterial co- thedifferencebetweenthetwobacteriawasperformed.
culture as described above , stained with 10 μl LIVE/
DEADW (1% insterile distilled water) (Molecular Probes, Results
Stockholm, Sweden) and incubated in the dark for Bacteriallagphasedurationandadhesion
20 minutes. The bacterial adherence was analysed using The lag phase duration of A. oris was found to be
confocal laser scanning microscopy (CLSM) with an 2.4 hours in both mono and co-culture (data not
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shown). The LPD of S. mitis was found to be 3.0 hours
in monoculture and 2.8 hours in co-culture. After 3–
3.5 hours, coinciding with the log phase of S. mitis, the
lag phase of A. oris in co-culture was found to be dis-
turbed, with adecreaseinthenumberof viable cellsasa
result. Since the aim of the study was to investigate the
initial bacterial adhesion and not the growth on the sur-
face or the competition between the two bacteria in co-
culture, an incubation time of 2.0 hours was chosen for
theexperiments.
The adhesion of S. mitis and A. oris, in co-culture,
incubated for 2.0 hours at optimal conditions (pH 7.0,
37°C, CO : 6%, O : 15%) was measured. S. mitis was
2 2
foundtoadheretothemachinedandanodizedtitanium
surfaces in amounts of 2.3 ± 0.14 and 2.0 ± .039 log
CFUmm-2,andA.orisinamountsof1.9±0.55and1.8
± 0.26 log CFU mm-2, respectively. Since 2.0 hours of
incubationwasfoundtobesufficientfor bacterialadhe-
sion, the continuing experiments were performed with
the same time of incubation, although at different pH
andatmosphere.
Effectsofenvironmentalfactorsonbacterialadhesion
The acidic pH of 5.0 was found to reduce the adhesion
of S. mitis to titanium surfaces by 50% when compared
to pH 7.0 (see Figure 2), whereas pH had no effect on
the adherence of A. oris. Furthermore, the adhesion of
both bacteria was found to be unaffected by incubation
atpH 9.0whencomparedtopH7.0.
Figure 2b shows that the amount of A. oris adhered to
the two titanium surfaces were found to be significantly
higher after incubation in ananaerobic environment than Figure2EffectsofenvironmentalpH(5.0,7.0and9.0)and
in an aerobic environment. The adhesion of S. mitis, on aerobicoranaerobicconditionsontheadhesionofS.mitisand
A.orisinco-cultureafter2.0hours’incubation.Statistically
theotherhand,wasfoundtobeunaffectedbytheaerobic
significantlessadheredS.mitiswasfoundafterincubationatpH5.0
atmosphere, and was found in equal amounts after incu-
thanpH7.0andofA.orisafterincubationataerobicenvironment
bation in both anaerobic and aerobic environments. The comparedtoanaerobicenvironment.
amountofS.mitisfoundontheanodizedsurfacewashalf
of that found on the machined surface (see Figure 3),
which was of statistical significance. A. oris was found to the adhesion to machined titanium was found dependent
adhere in equal amounts on both surfaces. The exact onatmosphere.
numbers for the adhesion as affected by the main factors
arepresentedinTable1. Microscopyanalysis
Inaddition,important(andstatisticallysignificant)inter- From the visual examination of the bottom-side of the
actioneffectswerefoundbetweenpHandsurface.Thead- titanium discs after 2.0 hours’ incubation, it was clear
hesionofS.mitis,inco-culturewithA.oris,isreduced on that some bacteria adhered to this side. However, this
bothsurfacesafterincubationatpH5.0comparedtoincu- amount was small in comparison to that on the upper-
bationatpH7.0.Thisreductionisstatisticallymoresignifi- side, which explains the exclusion of the bottom-side
cantfor theadhesiontomachinedtitaniumthananodized from the analysis. The same result, with a low number
titanium, which had a low adherence of S. mitis also at of remaining bacteria, was found when analysing the
pH7,asshownbyFigure4a.Moreover,aborderlineeffect upper-side of the discs after stomaching (data not
wasfoundfortheinteractionbetweenatmosphereandsur- shown).Theseresultsconfirmthat1minuteofstomach-
face as shown by Figure 4b. The already low amount of S. ingisenough toremoveadhered cells fromtheanodized
mitis found on the anodized surface was further reduced surface as well as the machined. Furthermore, the
by incubation in aerobic conditions while no difference in number of remaining bacteria on the surfaces after
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surface than on the anodized surface, as illustrated by
Figure 5a-b. Equal amounts of the two bacteria were
found on the machined surface after anaerobic incuba-
tion at pH 7 (Figure 5a) while the adhesion of S. mitis to
both surfaces was reduced by incubation at pH 5 irre-
spective of atmosphere (Figure 5c-d). A high viability of
both bacteria was seen after incubation at pH 7.0, with
only few non-viable cells in the clusters and chains. The
amount of non-viable cells of S. mitis was, however,
greater after incubation at pH 5.0 (data not shown) and
the same was found for A. oris after incubation at aer-
obic environment(Figure5d).
Discussion
Our results show that the initial adherence of S. mitis to
Figure3EffectofsurfacepropertiesontheadhesionofS.mitis titanium is significantly reduced by incubation at pH 5.0,
andA.orisinco-culturetotitaniumafter2.0hoursof
whichisinlinewithresultspresentedintheliteraturefor
incubation.StatisticallysignificantlessadheredS.mitiswasfound
the adhesion of S. sanguinis to hydroxyapatite [30,31].
ontheanodizedsurfacethanthemachinedtitaniumsurface.
Thesefindingsareinteresting,andthepotentialofashort
acidic treatment inducing bacterial dispersal is something
stomaching was very low in comparison to the number worthfurtherinvestigation,usingmoremixedspeciescul-
of removed bacteria and was, therefore, deemed not to turesaswellasinclinicalinvestigations.
haveeffect onthestatisticalanalysis. No limiting effect of pH 5.0 could be seen on the ini-
CLSM was performed to obtain more information on tial adhesion of A. oris to titanium in the present study.
the bacterial adhesion to the surfaces and the viability of Horiuchi et al. [32] haveshown that 35-76% of A. oris in
attached bacteria. A. oris was often found as single cells planctonic culture died after acidification at pH 4.0 for
or in small clusters, adhering to the grooves formed by 1 hour and Svensäter et al. [33] found no surviving bac-
the machining process of the machined surface; and teria after 3 hours incubation at pH 3.2 . This indicates
around the structures of the anodized surface, as seen in that A. oris is sensitive to an acidic environment when
Figure 5. S. mitis was also found to adhere to the grown in continuous culture, but that a pH of 5.0 is not
grooves of the machined titanium surface (Figure 6a) low enough to harm attached bacteria or inhibit adhe-
while the structure of the anodized surface seemed to sion. It is known that, when associated to a surface
obstruct the adhesion of this bacterium since the chains [11,34] or in a biofilm [35,36] bacteria are more tolerant
were often found partly detached from the surface to surrounding stress factors, such as antimicrobial
(Figure 6b). The surfaces to the left in Figure 5 are agents or toxic environment. In fact, stress factors may
machined titanium while the right surfaces are anodized lead to increased adhesion and biofilm formation [37-39].
titanium. Moreover, live cells are visualized in green and Additional work is needed in order to define the border
deadcellsare seeninred.Surfaces a-cwere incubatedin line between a pH initiating moderate stress leading to
anaerobic atmosphere, surface d in aerobic atmosphere. increased adhesion and a pH acidic enough to inhibit ad-
Surfaces c-d were incubated at pH 5. In line with the herence/initiatedispersalorkilling.
above described results, higher amounts of the long A tendency toward a higher amount of adhered bac-
chains formed by S. mitis were found on the machined teria on the two surfaces was seen after incubation at
Table1Themeannumberofadheredbacteria(logCFUpermm2±SEM)tothetitaniumdisc,asaffectedbysurface,
pHandatmosphere,arepresentedforeachofS.mitisandA.oris
Surface pH Atmosphere
Bacteria MachinedTi AnodizedTi 5.0 7.0 9.0 Anaerobic Aerobic
S.mitis 1.55±0.12 0.81±0.07 0.68±0.08 1.39±0.14 1.47±0.13 1.22±0.11 1.14±0.12
p 0.001* 0.001* 0.483
A.oris 1.67±0.07 1.81±0.05 1.64±0.08 1.76±0.08 1.84±0.052 1.90±0.05 1.59±0.06
p 0.065 1.106 0.001*
Discsareincubatedwiththebacteriainco-culturehoweverCFUforthetwobacteriaareseparatedbytheirdifferenceincolonymorphology.ThenumberofCFU
asaffectedbysurface,pHandatmospherewasanalyzedwithANOVAandtheresultsforthemainfactorspresentedinthistable.Thep-valuesindicatingsignificant
differencearemarkedwith*.
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Figure4Interactionsbetweensurface,pHandatmosphereaffectsthabacterialadhesion.Thefirstfigure(a)illustratestheeffectofpH
andsurfacepropertiesontheadhesionofS.mitisinco-culturewithA.oris.TheadhesionofS.mitisislessonbothsurfacesafterincubationat
pH5.0than7.0thereductionishoweverstatisticallysignificantlargerfortheadhesiontomachinedtitaniumthananodizedtitanium.Figure(b)
illustratestheeffectofsurfacepropertiesandaerobicoranaerobicenvironmentontheadhesionofS.mitisinco-culturewithA.oris.Theadhesion
totheanodizedsurfacewasfoundtobereducedbyaerobicincubationwhiletheoppositewasfoundfortheadherencetomachinedtitanium,
thisdifferenceishowevernotstatisticallysignificant.
Figure5Confocallaserscanningmicroscopyimagesofmachinedtitanium(a,c)andanodizedtitanium(b,d)surfacesincubatedwitha
co-cultureofA.orisandS.mitis.ThebacteriaarelabeledwithLIVE/DEADW(MolecularProbes)thusgreencolorindicateslivecellsandred
colorindicatesdeadcells.Surfacesa-cwereincubatedanaerobicanddaerobic.Thetwobacteriawerefoundinequalamountsonthemachined
titaniumsurfaceafteranaerobicincubationatpH7(a)whilemostlyA.oriscouldbefoundontheanodizedsurface,afterincubationatthesame
conditions(b).TheadhesionofS.mitistobothsurfaceswasfurtherreducedafterincubationatpH5irrespectiveofatmosphere(c,d).Thenumber
ofnon-viablecells(red)ofA.oriswasfoundtobegreaterafteraerobicincubationthananaerobic(d).
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Figure6ConfocallaserscanningmicroscopyimagesofA.oris(smalldistinctdotsandclusters,indicatedbywhitearrows)andS.mitis
(chains)adheredtomachined(a)andanodized(b)titaniumsurfaces,afteraerobicincubationatpH7.Thebacteriaarelabelledwith
LIVE/DEADW(MolecularProbes)visualizinglivecellsingreenanddeadcellsinread.RedarrowsindicatepartlydetachedchainsofS.mitis.
pH 9.0, indicating a tolerance among oral bacteria to al- surface contact (Figure 6a) promoting a strong adhesion.
kaline environments. This is in accordance to results These results indicate that the surface topography could
presented for root canal bacteria [40]. Apical periodon- be more important than the anatase layer in restricting
titis is commonly treated with mechanical instrumenta- theadhesionofthisbacterium.Thisobservationcoincides
tion combined with disinfectants, such as intra-canal with earlier results showing a decrease in the adhesion of
calcium hydroxide dressings. Although direct contact S. mitis after roughening of the denture surface [43],
with this alkaline chemical should be lethal, alkaline- although contradictory results, with more adhesion on
tolerant organisms are often found in the microflora rough surfaces, been observed in in vitro investigations
after treatment. This indicates that the survival mechan- using other Streptococci as model bacteria for surface at-
isms triggered by alkaline stress could be part of a gen- tachment [44,45] stressing the need of including more
eral adaptive survival response in oral bacteria. Chávez species to the consortium. Moreover, in vivo studies on
de Paz et al. further noted that organisms usually prefer- biofilm formation have shown more adhesion on rough
ring an acidic environment (e.g. some Streptococcus, surfaces as well as a clear influence of surface free energy
Lactobacillus and Fusobacterium nucleatum) survived, whenplacedinsupragingivallocations[44,46].Subgingiv-
and were able to adapt to alkaline stress at pH 10.5 for ally,however,thebiofilmformationismoredependenton
fourhours [40]. theoralhealthofthepatientthanthenatureofthesurface
In the present study, A. oris was found to adhere [46,47]. The complexity of biofilm formation on custo-
equally well to both the rough anodized and smooth mizedtitaniumsurfacesisfurther visualizedbytheinsitu
machined titanium surfaces. This is in contradiction to study of Fröjd et al. (2011) [48] where anodization of the
our hypothesis that the anatase layer of the rough sur- titaniumsurfacewasfoundtopromotebiofilmformation.
face would prevent bacterial adhesion. It is suggested These opposing results are hard to interpret, as there
that the antimicrobial properties of the anatase layer in- are fundamental differences in performance, surface
crease after irradiation with ultraviolet (UV) light [41]. characteristics and production of the anodized titanium
Therefore, additional experimentsare needed in order to surface; as well as variations in the methods used to
establish if UV-treatment will increase the antimicrobial quantifythebacterialload.Nevertheless,theseopposing
properties of the investigated anodized surface, as has results emphasize the importance in these experiments
been shown to be the case for other titanium oxide sur- of carefully designing the mixed bacterial consortia as
faces[42].TheadherenceofS.mitis,however,wasfound wellasusingbothinvitroandinsituanalyticalmethods
to be significantly less on the anodized surface than on to evaluate the attachment of bacteria and biofilm for-
themachinedtitanium surface, andevenfurther reduced mationondentalimplants.
by aerobic incubation. When analysed by CLSM, the ad- The complexity of biofilm formation was also demon-
herence of S. mitis was frequently found to be limited to strated by the interaction effects, which show that it is
the crests of the structures while the sections of the not possible to extrapolate results from one condition to
chain inbetweenwherefloatingfreeinthemedium(seen another.TheresultsshowthatahigheramountofS.mitis
as unfocused cells in Figure 6b, indicated by arrows) adhere to the machined titanium surface than the ano-
which is a sign of poor adhesion. On the machined sur- dized surface after incubation at pH 7.0 but that the re-
face, however, most cells in the chains of S. mitis had duction in adhesion after incubation at pH 5.0 compared
SethCaousetal.BMCOralHealth2013,13:4 Page9of10
http://www.biomedcentral.com/1472-6831/13/4
topH7.0islargerforthemachinedtitaniumsurfacethan Authordetails
theanodizedsurface.ThisindicatesthatanacidicpHcan 1DepartmentofChemicalandBiologicalEngineering,ChalmersUniversityof
Technology,GothenburgSE-40229,Sweden.2SIK,TheSwedishInstitutefor
inhibitadhesionoflargeamountsof bacteriabutthatitis
FoodandBiotechnology,P.O.Box5401,GothenburgSE-40229,Sweden.
not enough to inhibit bacterial adhesion in total. Anaer- 3NobelBiocareAB,P.O.Box5190,GothenburgSE-40226,Sweden.
obicconditionswerefoundtopromoteinitialadhesionof 4DepartmentofFoodScience,SLU-SwedishUniversityofAgricultural
Sciences,POBox7051,UppsalaSE-75645,Sweden.
A. oris whereas S. mitis was found to adhere in equal
amounts during both aerobic and anaerobic incubation. Received:25March2012Accepted:27December2012
However, a trend towards reduced adhesion on the ano- Published:8January2013
dized surface after aerobic incubationwasfound,suggest-
ing that adhesion is affected when challenged both with References
1. JungnerM,LundqvistP,LundgrenS:Oxidizedtitaniumimplants(Nobel
aerobicenvironmentandroughsurfacestructure.
BiocareTiUnite)comparedwithturnedtitaniumimplants(NobelBiocare
Although both A. oris and S. mitis are early colonizers markIII)withrespecttoimplantfailureinagroupofconsecutive
found in similar oral environments, these two bacteria patientstreatedwithearlyfunctionalloadingandtwo-stageprotocol.
ClinOralImplantsRes2005,16(3):308–312.
were found to respond differently to the tested factors.
2. HultinM,GustafssonA,KlingeB:Long-termevaluationofosseointegrated
Thisstronglyemphasisestheimportanceofusingseveral dentalimplantsinthetreatmentofpartlyedentulouspatients.JClin
bacterial species for in vitro as well as in situ studies Periodontol2000,27(2):128–133.
3. PyeAD,LockhartDE,DawsonMP,MurrayCA,SmithAJ:Areviewofdental
and,preferably,morethanonestrainofeachspecies. implantsandinfection.JHospInfect2009,72(2):104–110.
4. SchouS,HolmstrupP,WorthingtonHV,EspositoM:Outcomeofimplant
therapyinpatientswithprevioustoothlossduetoperiodontitis.Clin
Conclusions OralImplantsRes2006,17(Suppl2):104–123.
The adhesion of S. mitis to titanium was restricted by an 5. HultinM,GustafssonA,HallstromH,JohanssonLA,EkfeldtA,KlingeB:
Microbiologicalfindingsandhostresponseinpatientswithperi-
acidic environment and the anodized surface. The adhe- implantitis.ClinOralImplantsRes2002,13(4):349–358.
sion of A. oris, on the other hand, was reduced by aer- 6. NorowskiPAJr,BumgardnerJD:Biomaterialandantibioticstrategiesfor
obic incubation.Thesefindingssuggestbacterialadhesion peri-implantitis:areview.JBiomedMaterResBApplBiomater2009,
88(2):530–543.
tobesensitivetoenvironmentalchanges,indicatingapos-
7. EspositoM,GrusovinMG,KakisisI,CoulthardP,WorthingtonHV:
sibility to restrict bacterial adhesion after implant place- Interventionsforreplacingmissingteeth:treatmentofperimplantitis.
ment in order to promote healing. However, more CochraneDatabaseSystRev2008,16(2):CD004970.
8. YasuyukiM,KunihiroK,KurisseryS,KanavillilN,SatoY,KikuchiY:
bacterial species and longer incubation times need to be
Antibacterialpropertiesofninepuremetals:alaboratorystudyusing
investigated. At the same time, complementary work StaphylococcusaureusandEscherichiacoli.Biofouling:JBioadhesion
should be performed on the effect of changes in environ- BiofilmRes2010,26(7):851–858.
9. RenvertS,Roos-JansakerAM,ClaffeyN:Non-surgicaltreatmentofperi-
mental conditions on the dispersal of adhered bacteria. If
implantmucositisandperi-implantitis:aliteraturereview.JClin
the strength in adhesion can be reduced by altering pH Periodontol2008,35(8Suppl):305–315.
before mechanical instrumentation, this could be a future 10. MombelliA,LangNP:Thediagnosisandtreatmentofperi-implantitis.
Periodontol20001998,17:63–76.
way to establish implant surfaces that are free from bac-
11. BowdenGH,LiYH:Nutritionalinfluencesonbiofilmdevelopment.Adv
teriaandnewwaysoftreatinginfectionsaroundimplants. DentRes1997,11(1):81–99.
12. KonttinenYT,TakagiM,MandelinJ,LassusJ,SaloJ,AinolaM,LiTF,Virtanen
I,LiljestromM,SakaiH,etal:AcidattackandcathepsinKinbone
Abbreviations resorptionaroundtotalhipreplacementprosthesis.JBoneMinerRes
BHI:Brainheartinfusion;CFU:Colonyformingunits;CLSM:Confocallaser 2001,16(10):1780–1786.
scanningmicroscopy;LPD:Lagphaseduration;Sa:Surfaceroughness; 13. NyakoEA,WatsonCJ,PrestonAJ:DeterminationofthepHofperi-implant
UV:Ultraviolet. crevicularfluidinsuccessfulandfailingdentalimplantsites:apilot
study.ArchOralBiol2005,50(12):1055–1059.
14. Roos-JansakerAM:Longtimefollowupofimplanttherapyand
Competinginterests treatmentofperi-implantitis.SwedDentJSuppl2007,188:7–66.
JSCandJFareemployedbyNobelBiocare.
15. LoescheWJ,GusbertiF,MettrauxG,HigginsT,SyedS:Relationship
betweenoxygentensionandsubgingivalbacterialflorainuntreated
Authors’contributions humanperiodontalpockets.InfectImmun1983,42(2):659–667.
JSCparticipatedinplanningthestudyandtheexperimentaldesign, 16. BowdenGH,HamiltonIR:EnvironmentalpHasafactorinthe
performedthelaboratorywork,theCLSManalysisandthestatisticalanalysis competitionbetweenstrainsoftheoralstreptococciStreptococcus
andparticipatedindraftingthemanuscript.MLV,JFandMLparticipatedin mutans,S.sanguis,and"S.mitior"growingincontinuousculture.CanJ
thestudyplanning,experimentaldesign,dataanalysisanddraftingofthe Microbiol1987,33(9):824–827.
manuscript.Allauthorshavereadandapprovedthefinalmanuscript. 17. MatsuiR,CvitkovitchD:Acidtolerancemechanismsutilizedby
Streptococcusmutans.FutureMicrobiol2010,5:403–417.
18. LiYH,LauPC,TangN,SvensaterG,EllenRP,CvitkovitchDG:Noveltwo-
Acknowledgements componentregulatorysysteminvolvedinbiofilmformationandacid
WeacknowledgethefinancialsupportofTheKnowledgeFoundation, resistanceinStreptococcusmutans.JBacteriol2002,184(22):6333–6342.
throughTheResearchSchoolforSurfaceChemistry(YPK)(JSC).Theauthors 19. LiYH,BowdenGH:TheeffectofenvironmentalpHandfluoridefromthe
wouldalsoliketothankSiwKidmanforprofessionalhelpwithCLSM substratumonthedevelopmentofbiofilmsofselectedoralbacteria.
imagingandthelaboratorypersonnelatTheInstituteforFoodand JDentRes1994,73(10):1615–1626.
Biotechnology:LisbethMärs,MarieBlomqvistandIngelaKarlssonfor 20. GomesBP,SouzaSF,FerrazCC,TeixeiraFB,ZaiaAA,ValdrighiL,Souza-Filho
valuablehelpinthelaboratory. FJ:Effectivenessof2%chlorhexidinegelandcalciumhydroxideagainst
SethCaousetal.BMCOralHealth2013,13:4 Page10of10
http://www.biomedcentral.com/1472-6831/13/4
Enterococcusfaecalisinbovinerootdentineinvitro.IntEndodJ2003, 44. BurgersR,GerlachT,HahnelS,SchwarzF,HandelG,GosauM:Invivoand
36(4):267–275. invitrobiofilmformationontwodifferenttitaniumimplantsurfaces.Clin
21. BystromA,SundqvistG:Theantibacterialactionofsodiumhypochloriteand OralImplantsRes2010,21(2):156–164.
EDTAin60casesofendodontictherapy.IntEndodJ1985,18(1):35–40. 45. SubramaniK,JungRE,MolenbergA,HammerleCH:Biofilmondental
22. DelCurtoB,BrunellaMF,GiordanoC,PedeferriMP,ValtulinaV,VisaiL, implants:areviewoftheliterature.IntJOralMaxillofacImplants2009,
CigadaA:Decreasedbacterialadhesiontosurface-treatedtitanium.IntJ 24(4):616–626.
ArtificOrg2005,28(7):718–730. 46. ElterC,HeuerW,DemlingA,HannigM,HeidenblutT,BachFW,Stiesch-
23. ScaranoA,PiattelliA,PolimeniA,DiIorioD,CarinciF:Bacterialadhesion ScholzM:Supra-andsubgingivalbiofilmformationonimplant
oncommerciallypuretitaniumandanatase-coatedtitaniumhealing abutmentswithdifferentsurfacecharacteristics.IntJOralMaxillofac
screws:aninvivohumanstudy.JPeriodontol2010,81:1466–1471. Implants2008,23(2):327–334.
24. HallJ,LausmaaJ:Propertiesofanewporousoxidesurfaceontitanium 47. QuirynenM,BollenCM:Theinfluenceofsurfaceroughnessandsurface-
implants.ApplOsseointRes2000,1(1):5–8. freeenergyonsupra-andsubgingivalplaqueformationinman.A
25. AlbrektssonT,WennerbergA:Oralimplantsurfaces:Part2–review reviewoftheliterature.JClinPeriodontol1995,22(1):1–14.
focusingonclinicalknowledgeofdifferentsurfaces.IntJProsthodont 48. FröjdV,dePazLEC,AnderssonM,WennbergA,DaviesJR,SvensäterG:In
2004,17(5):544–564. situanalysisofmultispeciesbiofilmformationoncustomizedtitanium
26. WennerbergA,OhlssonR,RosenBG,AnderssonB:Characterizingthree- surfaces.MolOralMicrob2011,26:241–252.
dimensionaltopographyofengineeringandbiomaterialsurfacesby
confocallaserscanningandstylustechniques.MedEngPhys1996, doi:10.1186/1472-6831-13-4
18(7):548–556. Citethisarticleas:SethCaousetal.:AdhesionofStreptococcusmitisand
27. BaranyiJ,RobertsTA:Adynamicapproachtopredictingbacterialgrowth Actinomycesorisinco-culturetomachinedandanodizedtitanium
infood.IntJFoodMicrobiol1994,23(3–4):277–294. surfacesasaffectedbyatmosphereandpH.BMCOralHealth201313:4.
28. MarshP,MartinMV,LewisMAO,WilliamsDW:OralMicrobiology.UK:
ChurchillLivingstoneElsevier;2009.
29. GagnonGA,SlawsonRM:Anefficientbiofilmremovalmethodfor
bacterialcellsexposedtodrinkingwater.JMicrobiolMethods1999,
34:203–214.
30. KnoxKW,HardyLN,MarkevicsLJ,EvansJD,WickenAJ:Comparative
studiesontheeffectofgrowthconditionsonadhesion,hydrophobicity,
andextracellularproteinprofileofStreptococcussanguisG9B.Infect
Immun1985,50(2):545–554.
31. RosanB,AppelbaumB,CampbellLK,KnoxKW,WickenAJ:Chemostat
studiesoftheeffectofenvironmentalcontrolonStreptococcussanguis
adherencetohydroxyapatite.InfectImmun1982,35(1):64–70.
32. HoriuchiM,WashioJ,MayanagiH,TakahashiN:Transientacid-impairment
ofgrowthabilityoforalStreptococcus,Actinomyces,andLactobacillus:
apossibleecologicaldeterminantindentalplaque.OralMicrobiol
Immunol2009,24(4):319–324.
33. SvensäterG,LarssonU-B,GreifECG,CvitkovitchDG,HamiltonIR:Acid
toleranceresponceandsurvivalbyoralbacteria.OralMicrobiolImmunol
1997,12:266–273.
34. JeffersonKK,PierDB,GoldmannDA,PierGB:Theteicoplanin-associated
locusregulator(TcaR)andtheintercellularadhesinlocusregulator(IcaR)
aretranscriptionalinhibitorsoftheicalocusinStaphylococcusaureus.
JBacteriol2004,186(8):2449–2456.
35. FuxCA,CostertonJW,StewartPS,StoodleyP:Survivalstrategiesof
infectiousbiofilms.TrendsinMicrob2005,13(1):34–40.
36. SzomolayB,KlapperI,DockeryJ,StewartPS:Adaptiveresponsesto
antimicrobialagentsinbiofilms.EnvironMicrob2005,7(8):1186–1191.
37. KnoblochJK,BartschtK,SabottkeA,RohdeH,FeuchtHH,MackD:Biofilm
formationbyStaphylococcusepidermidisdependsonfunctionalRsbU,
anactivatorofthesigBoperon:differentialactivationmechanismsdue
toethanolandsaltstress.JBacteriol2001,183(8):2624–2633.
38. RachidS,ChoS,OhlsenK,HackerJ,ZiebuhrW:Inductionof
Staphylococcusepidermidisbiofilmformationbyenvironmentalfactors:
thepossibleinvolvementofthealternativetranscriptionfactorsigB.Adv
ExpMedBiol2000,485:159–166.
39. RachidS,OhlsenK,WallnerU,HackerJ,HeckerM,ZiebuhrW:Alternative
transcriptionfactorsigma(B)isinvolvedinregulationofbiofilm Submit your next manuscript to BioMed Central
expressioninaStaphylococcusaureusmucosalisolate.JBacteriol2000, and take full advantage of:
182(23):6824–6826.
40. dePazLEC,BergenholtzG,DahlénG,SvensäterG:Responsetoalkaline
• Convenient online submission
stressbyrootcanalbacteriainbiofilms.IntEndodJ2007,40(5):11.
41. ZhaoL,ChuPK,ZhangY,WuZ:Antibacterialcoatingsontitanium • Thorough peer review
implants,Review.JBiomedMaterResBApplBiomater2009,91B:470–480. • No space constraints or color figure charges
42. GopolJ,MuraleedharanP,GeorgeP,KhatakHS:Investigationsofthe
• Immediate publication on acceptance
antibacterialpropertiesofananodizedtitaniumalloy.TrendsBiomater
ArtifOrgans2003,17(1):13–18. • Inclusion in PubMed, CAS, Scopus and Google Scholar
43. YamauchiM,YamamotoK,WakabayashiM,KawanoJ:Invitroadherence • Research which is freely available for redistribution
ofmicroorganismstodenturebaseresinwithdifferentsurfacetexture.
DentMaterJ1990,9(1):19–24.
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