research article effect of cpp/acp on initial bioadhesion...

Research ArticleEffect of CPPACP on Initial Bioadhesion toEnamel and Dentin In Situ

Susann Grychtol1 Sabine Basche1 Matthias Hannig2 and Christian Hannig1

1 Clinic of Operative Dentistry Faculty of Medicine Carl Gustav Carus TU Dresden Fetscherstraszlige 74 01307 Dresden Germany2 Clinic of Operative Dentistry Periodontology and Preventive Dentistry University Hospital Saarland UniversityBuilding 73 66421 Homburg Germany

Correspondence should be addressed to Susann Grychtol susanngrychtoluniklinikum-dresdende

Received 24 June 2014 Accepted 17 September 2014 Published 21 October 2014

Academic Editor Ana Lucia MacHado

Copyright copy 2014 Susann Grychtol et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present in situ study investigated the influence of a preparation containing CPPACP (caseinphosphopeptide-amorphouscalcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin Therefore pellicle formationwas performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects After1min of pellicle formation GC Tooth mousse was used according to manufacturerrsquos recommendations Rinses with chlorhexidineserved as positive controls Specimens carried without any rinse served as negative controls After 8 h overnight exposure of thesplints bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight livedead staining) Additionallythe colony forming units (CFU) were determined after desorption Furthermore the effects on Streptococcus mutans bacteria weretested in vitro (BacLight) There was no significant impact of CPPACP on initial bacterial colonization proved with DAPI andBacLight Determination of CFU showed statistical significance for CPPACP to reduce bacterial adherence on enamelThe in vitroinvestigation indicated no antimicrobial effects for CPPACP on Streptococcus mutans suspension Under the chosen conditionsCPPACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissuesThe tested preparationcannot be recommended for biofilm management

1 Introduction

The prevalence of caries caused by adherence of bacteria andbiofilm formation is still one of the greatest challenges indentistry Biomimetic and bioinspired oral health productscontaining nanoparticles have been developed to improvebiofilm management [1 2]

GC Tooth Mousse (Tooth Mousse MI Paste GCAmerica) is a water based cream containing CPPACPCaseinphosphopeptide- (CPP-) amorphous calcium phos-phate (ACP) molecules (Recaldent) are derived from caseina natural protein extracted from milk [3] The caseinphos-phopeptides stabilize nanoclusters of amorphous calciumphosphate (ACP) in a metastabile solution [3 4] The multi-factorial anticariogenic mechanisms of CPPACP result frommaintaining a supersaturated state of enamel minerals atthe tooth surface [3] CPPACP has already been shown toprevent demineralization and to promote remineralization

of subsurface enamel lesions in animal and human in situstudies [5ndash7]

However there are contradictory research findings aboutthe anticariogenic effects of CPPACP Morgan et al investi-gated using digital bitewing radiographs approximal cariesprogression of 2720 subjects [8] One group chewed agum containing CPPACP whereas the other group chewedidentical gum without CPPACP for 3 times 10minday over aperiod of 24 months Finally for the subjects who chewedthe CPPACP gum caries progression was 18 less thanfor the CPPACP free gum group In conclusion CPPACPsignificantly reduced progression and enhanced regression ofapproximal caries [8] A recent study by Sitthisettapong et alcame to a different result [9] The double blind placebocontrolled clinical trial determined the effect of CPPACPpaste on dental caries in primary teeth [9] High-caries-riskchildren aged between 25 and 35 years received CPPACP

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 512682 8 pageshttpdxdoiorg1011552014512682

2 The Scientific World Journal

paste (119899 = 150) in addition to regular tooth brushing withfluoridated toothpaste The placebo control (119899 = 146) onlyused fluoridated toothpaste The results were recorded withICDAS after 6 months and one year The clinical trial didnot detect any difference between both groups Enamelcaries increment was recorded in both the experimentaland the control group In conclusion as compared with thefluoridated toothpaste CPPACP had no additional cariespreventive properties [9]

There is evidence that CPPACP nanocomplexes influ-ence bacterial adhesion to the tooth surface both in vitroand in situ [10ndash12] The effect of CPPACP on Streptococcusmutans biofilm formation has been investigated in vitro [13]A decrease in the total number of adherent bacteria wasdetected on HA disks pretreated with CPP-ACP in combina-tion with acidulated phosphate fluoride [13] Former in vitrostudies by Vacca-Smith et al examined the effect of milk andcasein on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite [14] They pointed out that kappa-Casein when bound to sHA inhibited streptococcal adher-ence to sHA in vitro [14] Immunolocalization studies indicatethe incorporation of CPPACP into plaque by binding tothe surfaces of bacterial cells to components of the intercel-lular plaque matrix and to adsorbed proteins on the toothsurfaces thereby possibly influencing the process of biofilmformation [1 7 12] Furthermore CPPACP might becomeincorporated into the pellicle layer in exchange for thestreptococci-related receptors thus inhibiting or modifyingthe bacterial adherence [12]

The effect of CPP-ACP on intraoral biofilm formation hasalso been determined in an in situ study using germaniumsurfaces as substrate for the investigation of bacterial biofilmformation [11] This study showed that the presence ofCPPACP agent delayed the biofilm formation considerablyby changing the surface charges [11 15] However little isknown about the interaction of CPPACP with the in vivopellicle layer on dental hard tissues Particularly the impactof CPPACP on initial biofilm formation on dentin has notbeen considered yet Therefore the aim of this study wasto investigate the antibacterial and antiadhesive propertiesof CPPACP using elaborate methods such as fluorescencemicroscopy [16 17] It was hypothesized that the testedpreparation GC Tooth mousse containing CPPACP reducesinitial bacterial colonization on enamel and dentin in situ

2 Materials and Methods

21 Specimens and Subjects Eight subjects (age 24ndash42 years)all members of the laboratory staff participated in the studyVisual oral examination was carried out by an experienceddentist The subjects showed plaque indices and gingivitisindices close to zero and no untreated carious lesions Thesalivary flow rate was physiological The subjects gave theirinformed written consent to participate in the study Thestudy design has been approved by both the Medical EthicsCommittee of the Medical Association of Saarland Germany( 1810) and the Medical Ethics Committee of TU Dres-den ( EK 147052013) Cylindrical enamel and dentin slabs(diameter 5mm surface area 1963mm2 and height 10mm)

were obtained from the facial surfaces of bovine incisorsof BSE-negative 2-year-old cattle For preparation of dentinspecimens the enamelwas removed and the outer dentin layerwas used for the oral exposure experiments The surfacesof all samples were polished by wet grinding with abrasivepaper (400ndash4000 grit) Disinfection of the dentin sampleswas performed by ultrasonication in ethanol (70) for 1minfollowed by air drying and 1min ultrasonication in EDTA(3) to remove the smear layer [17] The slabs were washedtwice for 5min in distilled water The enamel samples weretreated by ultrasonication for 1min in 3 NaOCl [18 19]Then they have been washed twice for 5min in distilled waterand disinfected by 10min ultrasonication in ethanol (70)All specimens were stored in distilled water for 24 h beforeexposure in the oral cavity

22 Tested Preparation GC Tooth mousse (Tooth MousseMI Paste GC America topical creme with bioavailablecalcium und phosphate) was tested A chlorhexidine-basedmouth rinse (02 chlorhexidin-digluconate meridol medCHX 02 GABA Lorrach Germany) served as a reference

23 BacLight Viability Assay In Vitro Experiment TheLIVEDEAD BacLight Bacterial Viability Kit (InvitrogenMolecular probes Darmstadt Germany) adopts two stainsmdashgreen fluorescent SYTO 9 stain (component A) and red-fluorescent propidium iodide stain (component B) [20] Theassays were performed according to manufacturerrsquos instruc-tions The fluorescence intensity was measured in a 96-wellplate reader A suspension of Streptococcus mutans in salinesolution was prepared after cultivation overnight 50 of thebacteria were inactivated with heat (1 h 95∘C) Dilutions ofTooth mousse and chlorhexidine were prepared with salinesolution (09NaCl sterile)The diluted preparations (CHXTooth mousse) were mixed 1 1 with vital bacteria andincubated for 10min Subsequently these suspensions weremixed with heat-inactivated bacteria (0 100 5 95 25 7545 55 50 50) A volume of 05 120583L of BacLight-stainingsolution (component A B = 1 1) was added to 250 120583L ofthese mixtures Afterwards the staining-preparation wasincubated for 10min in a dark chamber A volume of 100 120583Lfrom each sample was pipetted twice in amicrotiter plate andthe fluorescence was measured The excitation wavelengthwas 470 nm emission was recorded at 530 nm for the vitaland at 620 nm for the avital cells Duplicates were measuredto equalize inhomogeneity of the suspension For evaluationof the recorded data the ratio of vital and deadavital cells wascalculated (ratio = emission vitalemission dead bacteria)Experiments with saline solution served as a reference

231 Oral Exposure of the Samples For in situ bioadhesionindividual upper jaw splints were vacuum formed from15mm thick methacrylate foils Cavities were prepared inthe buccal aspects of the splints at the sites of the premolarsand the 1st molar The slabs were fixed on the splints usingpolyvinyl siloxane impressionmaterial (Provil novo light reg-ular set Heraeus Kulzer Germany) Before oral exposure ofthe splints the subjects brushed their teethwithout toothpasteand rinsed with tap-water The splints were carried intraoral

The Scientific World Journal 3

for 1min to ensure pellicle formation on the surfaces There-after the subjects applied 05 grams of GC Tooth mousse onthe samples and left it undisturbed for 3min in the oral cavityThen the remainingGCToothmoussewas spread throughoutthe mouth for further 2min and finally spit out thoroughlyRinseswith chlorhexidine (8mL for 1min)were performed aspositive controls specimens carried without any rinse servedas negative controls Subsequently the splints were kept inthe oral cavity for another 8 h overnight After oral exposurethe slabs were removed immediately from the splints andthoroughly rinsed with running distilled water for 15 s toremove nonadsorbed salivary remnants Six passes of thetrial were performed per subject The enamel and dentinslabs were tested for the amount of adherent bacteria withDAPI (410158406-diamidino-2-phenylindole) livedead staining(BacLight) and the CFU method (colony forming units)each one determined twofold

232 Fluorescence Microscopic Assays The epifluorescenceassays were conducted at 1000-fold magnification as in sev-eral previous studies (Axioskop II ZEISS Oberkochen Ger-many) [20] The number of cells observed in 10 randomizedmicroscopic ocular grid fields per sample was counted Thearea of ocular grid fields (00156mm2) allowed calculating thenumber of cells per square centimeter of the evaluated surface[16 17]

24 DAPI Staining DAPI (410158406-diamidino-2-phenylindole)forms fluorescent complexes by binding to AT-rich regionsof the double-stranded DNA [17 21] The staining wasconducted as described previously [22] First the sampleswere rinsed with saline solution For staining the sampleswere covered with 1mL DAPI working-solution (MerckDarmstadt Germany) and incubated in a dark chamber for15min at room temperature The solution was then pouredoff and the specimens were covered with methanol and air-dried at room temperatureThe slabs were stuck to a slide andanalyzed by epifluorescence microscopy

241 BacLight Viability Assay In Situ Experiments TheBacLight kit was adopted for visualization of vital and avitalbacteria on enamel and dentin slabs after oral exposureSimilar amounts of component A (Syto9 167mMpropidiumiodide 167mM 300 120583L DMSO) and B (Syto 9 dye167mMpropidium iodide 183mM 300 120583L DMSO) weremixed 2 120583L was added to 1mL of saline solution Theslabs were incubated in this solution for 10min Then thesamples were rinsed with saline solution and examined byfluorescence microscopy with the fluorescein-diacetate filter(FDA) and the ethidium-bromide filter

25 Colony-Forming Units For determination of the colony-forming units (CFU) the specimens were vortexed for 2minin sterile tubes with 1mL 09 sodium chloride and for1min in an ultrasonic bath on ice This solution was seriallydiluted up to 1 104 in saline solution and plated on Columbiablood agar (CBA aerobic bacteria and facultative anaerobicbacteria) or on yeast-cysteine-blood agar respectively (HCBanaerobic bacteria) The HCB plates were incubated for

7 days in anaerobic jars (Merck) at 37∘C using BBL GasPakAnaerobic System Envelopes (Becton Dickinson NJ USA)the CBA plates under aerobic conditions with 5 CO

2for 2

days

26 Statistics Statistical evaluation was performed by meansof the Kruskal-Wallis test and additional pair-wise compari-son was carried out using theMann-Whitney119880 test 119875 valueswere evaluated according to Bonferroni correction (119875 lt0017) The Software used was SPSS 170 (IBM EhningenGermany) [23 24]

3 Results

Initial microbial colonization was visualized with DAPIstaining and BacLight (Figure 1) CPPACP had no effect onthe amount of adherent bacteria as compared with unrinsedcontrols (Figures 1 2 and 3) After 8 h of oral exposuremonolayers and chains of microorganisms were visible onthe enamel slabs covering more than 50 of the surfaceThe respective dentin slabs were colonized preferentiallyat the orifices of the dentin tubules After application ofTooth mousse no modification of bacterial adherence isvisible neither on the enamel nor on the dentin samplesBoth quantification and viability of the microorganisms werenot significantly affected by CPPACP (Figures 1 2 and 3Table 1) As compared with controls chlorhexidine hadconsiderable effects on the amount of adherent bacteria onenamel and dentin samples (Figures 1 2 and 3 Table 1)Adherent bacteria on the enamel and dentin slabs werequantifiedwithDAPI and livedead staining (Figures 2 and 3)According to DAPI-staining there was no significant impactof CPPACP on the initial bacterial colonization on enamel(108 times 106plusmn 687 times 105cm2) and dentin samples (270 times106plusmn 155 times 106cm2) as compared with unrinsed controls(enamel 357 times 106plusmn 541 times 106cm2 dentin 143 times 106plusmn 101 times106cm2) All methods showed a high interindividual andintraindividual variability A significant effect was observedfor chlorhexidine as expected (enamel 319 times 105plusmn 435 times 105bacteriacm2 dentin 611 times 105plusmn 114 times 106 bacteriacm2)

The determination of the colony forming units showedsignificant influence of CPPACP on initial bacterial colo-nization on enamel samples (Figure 4 Table 1)TheCFU datafor aerobic and anaerobic bacteria showed a high variabilityNevertheless statistical evaluation by Kruskal-Wallis testand Mann-Whitney 119880 test indicated a significant impact ofchlorhexidine rinses on the amount of culturable bacteria(Table 1)

The ratio of viable and dead bacteria amounted to 164for enamel and to 100 for dentin in the unrinsed controlgroup It was decreased by the application of chlorhexidineon enamel (067) and remained unchanged on dentin (098)CPPACP also showed a decline of the ratio on enamel (098)but an increase on dentin (152) that is there were higherproportions of viable bacteria The in vitro experiments withsuspensions of Streptococcus mutans indicated no antibacte-rial effects for CPPACP (Figure 5) As expected the goldstandard chlorhexidine showed antibacterial properties in adose-dependent manner (Figure 5)


Figure 1 DAPI staining (blue) and livedead staining (redgreen) representative images of CPPACP samples and control-specimens (CHXno rinse) after 8 h oral exposure On enamel control samples vast areas were coated by bacterial aggregates or monolayers dentin samplesshow a preferred colonization at the dentin tubules Initial rinses of chlorhexidine reduced the bacterial adhesion considerably CPPACPhad no impact on the amount of adherent bacteria as compared with unrinsed controls

0

2000000

4000000

6000000

8000000

10000000

Enamel Dentin Enamel Dentin Enamel DentinChlorhexidine

DAPI

minus2000000

minus4000000 Tooth mousseNo rinse

Bact

eria

cm

2

Figure 2 DAPI staining for detection of total amount of bacteriaafter application of GC Tooth mousse rinse with chlorhexidine orno rinse After 1min of pellicle formation GC Tooth mousse wasapplied and rinses with chlorhexidine (8mL1min)were performedrespectively The enamel and dentin samples were carried overnightfor 8 h in situ fixed on individual upper jaw splints MV plusmn SD 119899 = 8subjects two samples per subject and subgroup

4 Discussion

For the first time the present in situ study investigatedthe influence of a customary oral care product containing

1000000

3000000

5000000

7000000

Enamel Dentin Enamel Dentin Enamel Dentin

ViableDead

Chlorhexidine Tooth mousseNo rinse

minus1000000

minus3000000

Bact

eria

cm

2

Figure 3 Livedead staining (BacLight) for detection of viable anddead bacteria after application of GC Tooth mousse rinses withchlorhexidine or no rinse Exposure of enamel and dentin samplesfor 8 h overnight in situ MV plusmn SD 119899 = 8 subjects two samples persubject and subgroup

CPPACP (Tooth Mousse MI Paste GC America) on theinitial bacterial colonization of enamel and dentin Our studyrevealed no antibacterial and antiadherent effects for CPPACP The bioadhesion processes were determined in situ


Table 1 Statistical evaluation of the in situ experiments with the different preparations Kruskal-Wallis test and additional pairwisecomparison with Mann-Whitney 119880 test (119875 lt 005)

Kruskal-Wallis test119875 lt 0001

Enamel DentinTooth mousse Chlorhexidine No rinse Tooth mousse Chlorhexidine

DAPIEnamel

No rinse ns lt0001 ns ns nsTooth mousse 0001 ns lt0001 nsChlorhexidine lt0001 lt0001 ns

DentinNo rinse 0008 0001Tooth mousse lt0001

BacLight viable bacteriaEnamel

No rinse ns ns ns ns nsTooth mousse ns ns ns nsChlorhexidine lt0001 lt0001 ns

DentinNo rinse ns lt0001Tooth mousse lt0001

BacLight dead bacteriaEnamel

No rinse ns 0001 ns ns lt0001Tooth mousse 0012 ns ns lt0001Chlorhexidine ns lt0001 ns


CFU aerobicEnamel 0007 lt0001 0001 ns lt0001

No rinse lt0001 ns ns lt0001Tooth mousse ns lt0001 nsChlorhexidine

DentinNo rinse 0004 lt0001Tooth mousse lt0001

CFU anaerobicEnamel

No rinse 0011 lt0001 ns ns lt0001Tooth mousse 0008 ns lt0001 lt0001Chlorhexidine ns lt0001 ns

DentinNo rinse lt0001 lt0001Tooth mousse lt0001

considering that in vivo and in vitro conditions differ con-siderably [25] The methodical approach including bovineenamel and dentin specimens fixed to individual upper jawsplints has already been used in previous studies [20 25]Theovernight trial is an approved method to evaluate the impact

of oral therapeutics of initial biofilm formation [25] Duringthis time ingestion potation and other external influencescan be excluded It has to be considered that the applicationof the tested preparation GC Tooth mousse was performedwith paste whereas chlorhexidine rinses served as controls



AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2

Figure 4 Quantification of colony forming units (CFU) afterdesorption for determination of adherent aerobic and anaerobicbacteria Exposure of the specimens at the buccal sites of the upper1st and 2nd premolar and 1st molar for 8 h overnightMV plusmn SD 119899 = 8subjects 2 samples per subject and preparation

0

2

4

6

8

10

12

0 10 20 30 40 50Initial concentration of viable cells ()

NaCl-control

TM 100mgmLTM 10mgmLTM 1mgmL

Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50

Figure 5 Effect of CPPACP (red) chlorhexidine (CHX) (blue)and saline solution (control) (black) on the viability of S mutansin vitro A suspension of S mutans was incubated with CPPACPand chlorhexidine solutions in different concentrations After incu-bation the samples were admixed to proportions of the basicsuspension The amount of vital and dead bacteria was determinedby BacLight bacterial viability assayThemeasurements were carriedout in duplicate and the ratio was calculated emission 530 nm andemission 620 nm are representing emission vital and emission deadbacteria respectively

Accordingly a direct comparability was not ensured Yetboth application types were performed in accordance withthe manufacturerrsquos recommendations After the applicationof GC Tooth mousse or rinses with chlorhexidine the splintswere carried for 8 h overnightThe chlorhexidinemouth rinseis considered as gold standard in plaque reducing propertiesand served as positive control [26] Samples carried withoutany rinse served as negative controls This technique allowsthe investigation of initial bacterial colonization with thegreatest possible standardization under in situ conditionsDespite these precautionary measures high interindividualand intraindividual variability was observed This drawback

seems to be characteristic for initial bioadhesion in the oralcavity and corresponds to previous investigations for exam-ple recently published pilot studies of edible oils or Biorepair[17 20 22] Another limitation of the study is the smallnumber of subjects It has to be kept in mind when inter-preting the data However the aim of this in situ study wasto gain insight into the principal effect of a preparationcontaining CPPACP on initial biofilm formation DAPIand livedead staining demonstrated the lacking effect ofCPPACPon both quantification and viability of the adherentbacteria Statistical evaluation pointed out that there wasno significant influence of CPPACP on initial bacterialcolonization after 8 h in situ exposure of the samples (Table 1)The CFU method showed a pronounced variability andsignificant impact of GC Tooth mousse on initial biofilmformation on enamel samples However with the cultureplate method based on desorption and ultrasonication only50 of the bacterial strains can be cultivated whereas fluo-rescence microscopic methods (DAPI BacLight) investigatethe bacteria in the adherent state [27]

Former in vitro investigations indicated that CPPACPmay affect bacterial adhesion at the tooth surface [10]Schupbach et al visualized the incorporation of CPP intothe salivary pellicle with SEM und TEM with reductionof the adhesion of mutans streptococci [12] So far in situstudies regarding the effect of CPPACP on bacterial adher-ence are confined to investigations of plaque samples [28]In the present in situ study with enamel and dentin samplesthe investigation of bioadhesion took place in a standard-ized manner Although bovine dentin tubes have a greaterdiameter than those in human dentin and there are moretubulescm2 human and bovine dentin is alike and biofilmformation on these surfaces is similar [25 29]

The missing effect of CPPACP on bacterial adhesionto the dental hard tissues is contradictory to previous invitro and in situ studies [10ndash12] It was suggested that theadsorption of CPPACP on enamel could cause an increase inthe surface net negative charge thereby influencing the long-range interactions with microbes through the developmentof repulsive forces [15 30] Furthermore CPPACP mightmodify long-term adhesion by masking the streptococci-related receptors on salivary molecules [31] Bacterial recep-tors might be blockedcoaggregation and bacterial adhesionto the pellicle might be reduced [10 32 33] On the onehand the lacking influence of CPP-ACP on initial bacterialcolonization might be caused by the masking effect of thepellicle Otherwise a slight local stability of CPP-ACP at thetooth surface is conceivable

Rose demonstrated in vitro that CPPACP and calciumcompete for the same binding sites on S mutans It wasconfirmed that CPPACP binds with twice the affinity to bac-terial cells compared to calcium [10]The therefore enhancedavailability of calcium in the plaque has anticaries protec-tive effects by promoting remineralization and hamperingdemineralization Furthermore there is evidence that a highconcentration of free calciummay have bactericidal or bacte-riostatic effects [10]These investigations are limited to Strep-tococcus pneumonia and Streptococcus faecalis [34 35] Directantibacterial effects of CPPACP on suspensions of S mutans


were investigated in our in vitro experiment There were noantibacterial effects for CPPACP on S mutans detectableThis was to be expected antibacterial characteristics forcaseine calcium or phosphate on Streptococcus mutans havenot been observed yet

In general the efficacy of CPP-ACP in oral biofilm man-agement is doubtful As aforementioned there are contradic-tory results for the effects of CPPACP in vivo and in situBeside our investigations the potential of CPP-ACP to rem-ineralize subsurface enamel lesions was often provided withthe use of CPP-ACP chewing gums [7] However Schirrmeis-ter et al found no significant differences between chewinggums containing calcium and calcium-free chewing gumswith regard to change of mineral loss and lesion depth ofenamel samples [36] Referring to the impact of CPP-ACPon dentin hypersensitivity different findings were achieved aswell Kowalczyk et al revealed insufficient effectiveness andshort-term therapeutic effects in treating hypersensitivity ofdentine using GC Tooth mousse [37] A recent in situ studyshowed on the contrary that CPP-ACP combined with fluo-ride is slightly effective in reducing dentinal hypersensitivity[38]

5 Conclusions

Under the conditions chosen in the present in situ study thetested preparation containing CPPACP (Tooth Mousse MIPaste GC America) has no significant impact on the initialbacterial colonization on enamel and dentin Thus it cannotbe recommended for biofilm management

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] M Hannig and C Hannig ldquoNanomaterials in preventivedentistryrdquo Nature Nanotechnology vol 5 no 8 pp 565ndash5692010

[2] M Hannig and C Hannig Nanobiomaterials in PreventiveDentistry Elsevier New York NY USA 2013

[3] E C Reynolds ldquoAnticariogenic complexes of amorphous cal-ciumphosphate stabilized by casein phosphopeptides a reviewrdquoSpecial Care in Dentistry vol 18 no 1 pp 8ndash16 1998

[4] E C Reynolds ldquoAnticariogenic casein phosphopeptidesrdquo Pro-tein and Peptide Letters vol 6 no 5 pp 295ndash303 1999

[5] E C Reynolds C J Cain F L Webber et al ldquoAnticariogenicityof calcium phosphate complexes of tryptic casein phosphopep-tides in the ratrdquo Journal of Dental Research vol 74 no 6 pp1272ndash1279 1995

[6] E C Reynolds C L Black F Cai et al ldquoAdvances in enamelremineralization casein phosphopeptide-amorphous calciumphosphaterdquo Journal of Clinical Dentistry vol 10 no 2 pp 86ndash88 1999

[7] E C Reynolds F Cai P Shen and G D Walker ldquoRetention inplaque and remineralization of enamel lesions by various forms

of calcium in amouthrinse or sugar-free chewing gumrdquo Journalof Dental Research vol 82 no 3 pp 206ndash211 2003

[8] M V Morgan G G Adams D L Bailey C E Tsao S L Fis-chman and E C Reynolds ldquoThe anticariogenic effect of sugar-free gum containing CPP-ACP nanocomplexes on approximalcaries determined using digital bitewing radiographyrdquo CariesResearch vol 42 no 3 pp 171ndash184 2008

[9] T Sitthisettapong P Phantumvanit C Huebner and T Der-ouen ldquoEffect of CPP-ACP paste on dental caries in primaryteeth a randomized trialrdquo Journal of Dental Research vol 91 no9 pp 847ndash852 2012

[10] R K Rose ldquoBinding characteristics of Streptococcus mutans forcalcium and casein phosphopeptiderdquo Caries Research vol 34no 5 pp 427ndash431 2000

[11] C Rahiotis G Vougiouklakis and G Eliades ldquoCharacteriza-tion of oral films formed in the presence of a CPP-ACP agentan in situ studyrdquo Journal of Dentistry vol 36 no 4 pp 272ndash2802008

[12] P Schupbach J R Neeser M Golliard M Rouvet and BGuggenheim ldquoIncorporation of caseinoglycomacropeptide andcaseinophosphopeptide into the salivary pellicle inhibits adher-ence of mutans streptococcirdquo Journal of Dental Research vol75 no 10 pp 1779ndash1788 1996

[13] A P Erdem E Sepet T Avshalom V Gutkin andD SteinbergldquoEffect of CPP-ACP and APF on Streptococcus mutans biofilma laboratory studyrdquoAmerican Journal of Dentistry vol 24 no 2pp 119ndash123 2011

[14] A M Vacca-Smith B C van Wuyckhuyse L A Tabakand W H Bowen ldquoThe effect of milk and casein proteinson the adherence of Streptococcus mutans to saliva-coatedhydroxyapatiterdquo Archives of Oral Biology vol 39 no 12 pp1063ndash1069 1994

[15] C Rahiotis and G Vougiouklakis ldquoEffect of a CPP-ACP agenton the demineralization and remineralization of dentine invitrordquo Journal of Dentistry vol 35 no 8 pp 695ndash698 2007

[16] A Al-Ahmad M Follo A-C Selzer E Hellwig M Hannigand C Hannig ldquoBacterial colonization of enamel in situinvestigated using fluorescence in situ hybridizationrdquo Journal ofMedical Microbiology vol 58 no 10 pp 1359ndash1366 2009

[17] D J Jung A Al-Ahmad M Follo et al ldquoVisualization of initialbacterial colonization on dentine and enamel in siturdquo Journal ofMicrobiological Methods vol 81 no 2 pp 166ndash174 2010

[18] D Deimling L Breschi W Hoth-Hannig et al ldquoElectronmicroscopic detection of salivary 120572-amylase in the pellicleformed in siturdquo European Journal of Oral Sciences vol 112 no6 pp 503ndash509 2004

[19] D Deimling C Hannig W Hoth-Hannig P Schmitz JSchulte-Monting and M Hannig ldquoNon-destructive visualisa-tion of protective proteins in the in situ pelliclerdquo Clinical OralInvestigations vol 11 no 3 pp 211ndash216 2007

[20] C Hannig S Basche T Burghardt A Al-Ahmad and MHannig ldquoInfluence of a mouthwash containing hydroxyapatitemicroclusters on bacterial adherence in siturdquo Clinical OralInvestigations vol 17 no 3 pp 805ndash814 2013

[21] T Schwartz S Hoffmann and U Obst ldquoFormation of naturalbiofilms during chlorine dioxide and uv disinfection in a publicdrinking water distribution systemrdquo Journal of Applied Micro-biology vol 95 no 3 pp 591ndash601 2003

[22] C Hannig M Hannig O Rehmer G Braun E Hellwigand A Al-Ahmad ldquoFluorescencemicroscopic visualization andquantification of initial bacterial colonization on enamel insiturdquoArchives of Oral Biology vol 52 no 11 pp 1048ndash1056 2007


[23] F Faul E Erdfelder A Buchner and A G Lang ldquoStatisticalpower analyses using GlowastPower 31 tests for correlation andregression analysesrdquo Behavior Research Methods vol 41 no 4pp 1149ndash1160 2009

[24] F Faul E Erdfelder A-G Lang and A Buchner ldquoGlowastPower 3 aflexible statistical power analysis program for the social behav-ioral and biomedical sciencesrdquo Behavior ResearchMethods vol39 no 2 pp 175ndash191 2007

[25] C Hannig A Gaeding S Basche G Richter R Helbig andM Hannig ldquoEffect of conventional mouthrinses on initialbioadhesion to enamel and dentin in siturdquo Caries Research vol47 no 2 pp 150ndash161 2013

[26] L Netuschil E Reich and M Brecx ldquoDirect measurementof the bactericidal effect of chlorhexidine on human dentalplaquerdquo Journal of Clinical Periodontology vol 16 no 8 pp484ndash488 1989

[27] J A Aas B J Paster L N Stokes I Olsen and F E DewhirstldquoDefining the normal bacterial flora of the oral cavityrdquo Journalof Clinical Microbiology vol 43 no 11 pp 5721ndash5732 2005

[28] G D Walker F Cai P Shen G G Adams C Reynolds andE C Reynolds ldquoCasein phosphopeptide-amorphous calciumphosphate incorporated into sugar confections inhibits the pro-gression of enamel subsurface lesions in siturdquo Caries Researchvol 44 no 1 pp 33ndash40 2010

[29] I Nakamichi M Iwaku and T Fusayama ldquoBovine teeth aspossible substitutes in the adhesion testrdquo Journal of DentalResearch vol 62 no 10 pp 1076ndash1081 1983

[30] E C Reynolds and A Wong ldquoEffect of adsorbed protein onhydroxyapatite zeta potential and Streptococcus mutans adher-encerdquo Infection and Immunity vol 39 no 3 pp 1285ndash1290 1983

[31] B Guggenheim R Schmid J-M Aeschlimann R Berrocaland J-R Neeser ldquoPowdered milk micellar casein prevents oralcolonization by Streptococcus sobrinus and dental caries in ratsa basis for the caries-protective effect of dairy productsrdquo CariesResearch vol 33 no 6 pp 446ndash454 1999

[32] R K Rose ldquoThe role of calcium in oral streptococcal aggrega-tion and the implications for biofilm formation and retentionrdquoBiochimica et Biophysica Acta vol 1475 no 1 pp 76ndash82 2000

[33] R K Rose ldquoEffects of an anticariogenic casein phosphopeptideon calcium diffusion in streptococcal model dental plaquesrdquoArchives of Oral Biology vol 45 no 7 pp 569ndash575 2000

[34] M-C Trombe C Clave and J-MManias ldquoCalcium regulationof growth and differentiation in Streptococcus pneumoniaerdquoJournal of General Microbiology vol 138 no 1 pp 77ndash84 1992

[35] H Kobayashi J van Brunt and F M Harold ldquoATP-linked cal-cium transport in cells andmembrane vesicles of Streptococcusfaecalisrdquo Journal of Biological Chemistry vol 253 no 7 pp2085ndash2092 1978

[36] J F Schirrmeister R K SegerM J Altenburger A Lussi and EHellwig ldquoEffects of various forms of calcium added to chewinggum on initial enamel carious lesions in siturdquo Caries Researchvol 41 no 2 pp 108ndash114 2007

[37] A Kowalczyk B Botulinski M Jaworska A Kierklo MPawinska and E Dabrowska ldquoEvaluation of the product basedon Recaldent technology in the treatment of dentin hypersen-sitivityrdquo Advances in medical sciences vol 51 pp 40ndash42 2006

[38] S Madhavan M Nayak A Shenoy R Shetty and K PrasadldquoDentinal hypersensitivity a comparative clinical evaluation ofCPP-ACP F sodium fluoride propolis and placebordquo Journal ofConservative Dentistry vol 15 no 4 pp 315ndash318 2012

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paste (119899 = 150) in addition to regular tooth brushing withfluoridated toothpaste The placebo control (119899 = 146) onlyused fluoridated toothpaste The results were recorded withICDAS after 6 months and one year The clinical trial didnot detect any difference between both groups Enamelcaries increment was recorded in both the experimentaland the control group In conclusion as compared with thefluoridated toothpaste CPPACP had no additional cariespreventive properties [9]

There is evidence that CPPACP nanocomplexes influ-ence bacterial adhesion to the tooth surface both in vitroand in situ [10ndash12] The effect of CPPACP on Streptococcusmutans biofilm formation has been investigated in vitro [13]A decrease in the total number of adherent bacteria wasdetected on HA disks pretreated with CPP-ACP in combina-tion with acidulated phosphate fluoride [13] Former in vitrostudies by Vacca-Smith et al examined the effect of milk andcasein on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite [14] They pointed out that kappa-Casein when bound to sHA inhibited streptococcal adher-ence to sHA in vitro [14] Immunolocalization studies indicatethe incorporation of CPPACP into plaque by binding tothe surfaces of bacterial cells to components of the intercel-lular plaque matrix and to adsorbed proteins on the toothsurfaces thereby possibly influencing the process of biofilmformation [1 7 12] Furthermore CPPACP might becomeincorporated into the pellicle layer in exchange for thestreptococci-related receptors thus inhibiting or modifyingthe bacterial adherence [12]

The effect of CPP-ACP on intraoral biofilm formation hasalso been determined in an in situ study using germaniumsurfaces as substrate for the investigation of bacterial biofilmformation [11] This study showed that the presence ofCPPACP agent delayed the biofilm formation considerablyby changing the surface charges [11 15] However little isknown about the interaction of CPPACP with the in vivopellicle layer on dental hard tissues Particularly the impactof CPPACP on initial biofilm formation on dentin has notbeen considered yet Therefore the aim of this study wasto investigate the antibacterial and antiadhesive propertiesof CPPACP using elaborate methods such as fluorescencemicroscopy [16 17] It was hypothesized that the testedpreparation GC Tooth mousse containing CPPACP reducesinitial bacterial colonization on enamel and dentin in situ

2 Materials and Methods

21 Specimens and Subjects Eight subjects (age 24ndash42 years)all members of the laboratory staff participated in the studyVisual oral examination was carried out by an experienceddentist The subjects showed plaque indices and gingivitisindices close to zero and no untreated carious lesions Thesalivary flow rate was physiological The subjects gave theirinformed written consent to participate in the study Thestudy design has been approved by both the Medical EthicsCommittee of the Medical Association of Saarland Germany( 1810) and the Medical Ethics Committee of TU Dres-den ( EK 147052013) Cylindrical enamel and dentin slabs(diameter 5mm surface area 1963mm2 and height 10mm)

were obtained from the facial surfaces of bovine incisorsof BSE-negative 2-year-old cattle For preparation of dentinspecimens the enamelwas removed and the outer dentin layerwas used for the oral exposure experiments The surfacesof all samples were polished by wet grinding with abrasivepaper (400ndash4000 grit) Disinfection of the dentin sampleswas performed by ultrasonication in ethanol (70) for 1minfollowed by air drying and 1min ultrasonication in EDTA(3) to remove the smear layer [17] The slabs were washedtwice for 5min in distilled water The enamel samples weretreated by ultrasonication for 1min in 3 NaOCl [18 19]Then they have been washed twice for 5min in distilled waterand disinfected by 10min ultrasonication in ethanol (70)All specimens were stored in distilled water for 24 h beforeexposure in the oral cavity

22 Tested Preparation GC Tooth mousse (Tooth MousseMI Paste GC America topical creme with bioavailablecalcium und phosphate) was tested A chlorhexidine-basedmouth rinse (02 chlorhexidin-digluconate meridol medCHX 02 GABA Lorrach Germany) served as a reference

23 BacLight Viability Assay In Vitro Experiment TheLIVEDEAD BacLight Bacterial Viability Kit (InvitrogenMolecular probes Darmstadt Germany) adopts two stainsmdashgreen fluorescent SYTO 9 stain (component A) and red-fluorescent propidium iodide stain (component B) [20] Theassays were performed according to manufacturerrsquos instruc-tions The fluorescence intensity was measured in a 96-wellplate reader A suspension of Streptococcus mutans in salinesolution was prepared after cultivation overnight 50 of thebacteria were inactivated with heat (1 h 95∘C) Dilutions ofTooth mousse and chlorhexidine were prepared with salinesolution (09NaCl sterile)The diluted preparations (CHXTooth mousse) were mixed 1 1 with vital bacteria andincubated for 10min Subsequently these suspensions weremixed with heat-inactivated bacteria (0 100 5 95 25 7545 55 50 50) A volume of 05 120583L of BacLight-stainingsolution (component A B = 1 1) was added to 250 120583L ofthese mixtures Afterwards the staining-preparation wasincubated for 10min in a dark chamber A volume of 100 120583Lfrom each sample was pipetted twice in amicrotiter plate andthe fluorescence was measured The excitation wavelengthwas 470 nm emission was recorded at 530 nm for the vitaland at 620 nm for the avital cells Duplicates were measuredto equalize inhomogeneity of the suspension For evaluationof the recorded data the ratio of vital and deadavital cells wascalculated (ratio = emission vitalemission dead bacteria)Experiments with saline solution served as a reference

231 Oral Exposure of the Samples For in situ bioadhesionindividual upper jaw splints were vacuum formed from15mm thick methacrylate foils Cavities were prepared inthe buccal aspects of the splints at the sites of the premolarsand the 1st molar The slabs were fixed on the splints usingpolyvinyl siloxane impressionmaterial (Provil novo light reg-ular set Heraeus Kulzer Germany) Before oral exposure ofthe splints the subjects brushed their teethwithout toothpasteand rinsed with tap-water The splints were carried intraoral








2for 2

days


3 Results






0

2000000

4000000

6000000

8000000

10000000


DAPI

minus2000000


Bact

eria

cm

2


4 Discussion


1000000

3000000

5000000

7000000


ViableDead


minus1000000

minus3000000

Bact

eria

cm

2







DAPIEnamel












CFU anaerobicEnamel







AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2


0

2

4

6

8

10

12


NaCl-control


Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50










5 Conclusions




References
















































Biomaterials












Journal of


























2for 2

days


3 Results






0

2000000

4000000

6000000

8000000

10000000


DAPI

minus2000000


Bact

eria

cm

2


4 Discussion


1000000

3000000

5000000

7000000


ViableDead


minus1000000

minus3000000

Bact

eria

cm

2







DAPIEnamel












CFU anaerobicEnamel







AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2


0

2

4

6

8

10

12


NaCl-control


Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50










5 Conclusions




References
















































Biomaterials












Journal of





















0

2000000

4000000

6000000

8000000

10000000


DAPI

minus2000000


Bact

eria

cm

2


4 Discussion


1000000

3000000

5000000

7000000


ViableDead


minus1000000

minus3000000

Bact

eria

cm

2







DAPIEnamel












CFU anaerobicEnamel







AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2


0

2

4

6

8

10

12


NaCl-control


Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50










5 Conclusions




References
















































Biomaterials












Journal of























DAPIEnamel












CFU anaerobicEnamel







AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2


0

2

4

6

8

10

12


NaCl-control


Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50










5 Conclusions




References
















































Biomaterials












Journal of





















AerobAnaerob


100E + 09

100E + 06

100E + 03

100E + 00

Bact

eria

cm

2


0

2

4

6

8

10

12


NaCl-control


Ratio

via

ble

dead

bac

teria

CHX 1 10CHX 1 20CHX 1 50










5 Conclusions




References
















































Biomaterials












Journal of






















5 Conclusions




References
















































Biomaterials












Journal of











































Biomaterials












Journal of



















research article effect of cpp/acp on initial bioadhesion...

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