FACULTEIT GENEESKUNDE EN

GEZONDHEIDSWETENSCHAPPEN

Academiejaar 2012 - 2013

A comparison of the remineralisation effect of

CPP-ACP, fluoride and a combination on initial

caries lesion

Charlotte VAN ELST

Promotor: Prof. dr. Guy De Pauw

Copromotor: Prof. dr. Ronald Verbeeck

Masterproef voorgedragen in de Master na Master Opleiding Orthodontie

FACULTEIT GENEESKUNDE EN

GEZONDHEIDSWETENSCHAPPEN

Academiejaar 2012 - 2013

A comparison of the remineralisation effect of

CPP-ACP, CPP-ACPF and fluoride

on initial caries lesion

Charlotte VAN ELST

Promotor: Prof. dr. Guy De Pauw

Copromotor: Prof. dr. Ronald Verbeeck

Masterproef voorgedragen in de Master na Master Opleiding Orthodontie

De auteur(s) en de promotor geven de toelating deze Masterproef voor consultatie

beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander

gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking

tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit

deze Masterproef.

Datum 09/08/2013

Charlotte Van Elst Prof. dr. Guy De Pauw

Voorwoord

Deze Master na Masterproef kwam tot stand met behulp van verschillende mensen en deze wens ik hier graag te bedanken.

Speciale dank gaat uit naar Petra Maes, die niet alleen de zware taak had om mij te helpen met het analyseren van de coupes maar wiens steun en begrip eindeloos was.

Mijn oprechte dank gaat ook uit naar Lynn Hauspy voor de talloze uren van hulp en gezelschap.

Chris Vercruysse dank ik graag voor zijn hulp bij het opstellen van de onderzoeksmethode en wiens geduld groot was om mij de nodige technieken in het labo aan te leren.

Mijn copromotor, Prof. dr. R. Verbeeck, wil ik van harte danken voor het verstrekken van onontbeerlijke informatie en het constant kritisch evalueren.

Mijn promotor Prof. dr. G. De Pauw bedank ik graag voor de kritische blik en het coachen van mijn masterproef.

Tenslotte wil ik ook mijn collega’s, mijn familie en Bernard bedanken voor hun niet aflatende morele steun gedurende de voorbije jaren.

“Learn from yesterday, live for today, hope for tomorrow.”

Albert Einstein

Table of contents

Samenvatting .............................................................................................................................. 1

Abstract ...................................................................................................................................... 3

Inleiding ..................................................................................................................................... 5

Study of the literature ................................................................................................................. 6

1. Caries process and white spot lesions .......................................................................... 8

2. Anticariogenetic properties of CPP-ACP (GC Tooth MousseTM) ............................. 19

3. Mechanism of action of fluoride (Elmex Medical Gel®) ......................................... 20

4. Interaction of CPP-ACP and fluoride (MI Paste PlusTM) .......................................... 23

5. Quantifying enamel demineralisation ........................................................................ 24

Aim of this study ...................................................................................................................... 29

Methodology ............................................................................................................................ 30

Results ...................................................................................................................................... 36

Discussion ................................................................................................................................ 44

Critical evaluation .................................................................................................................... 49

Conclusions .............................................................................................................................. 50

References….. .......................................................................................................................... 51

Annexe…………...…..…………………………………………………………….……...….....…63

1

Samenvatting

Introductie: Ondanks preventie, blijft het ontstaan van “white spots” tijdens de

orthodontische behandeling met vaste apparatuur nog steeds een belangrijk klinisch probleem

(prevalentie white spot laesies -WSL- van 2% tot 97%). De verhoogde prevalentie van deze

glazuurontkalking tijdens de behandeling is deels te wijten aan het feit dat brackets voor een

onregelmatig oppervlak zorgen en dat er een beperking is van het zelfreinigend mechanisme

van speeksel en de beweging van de orale spiermassa. Na het verwijderen van de apparatuur

blijven “white spots” zichtbaar door hun opaak, kalkachtig uitzicht op het glazuur en kunnen

ze een esthetisch probleem zijn voor de patiënt. Een goede mondhygiëne tijdens een

orthodontische behandeling is daarom absoluut noodzakelijk. Verschillende producten (zoals

fluoride en meer caseïne fosfopeptide-amorf calciumfosfaat) worden gepromoot voor het

voorkomen en remineralizeren van WSL.

Doel: Het doel van deze studie was het in vitro effect van fluoride, CPP-ACP (caseïne

fosfopeptide-amorf calciumfosfaat) en een combinatie van beide (CPP-ACPF) te vergelijken

met een controlegroep op de remineralisatie van WSL na 6 en 12 weken.

Materiaal en Methoden: 96 premolaren werden in twee gesplitst (buccaal en palataal). WSL

werden gecreëerd met een demineraliserende oplossing gedurende 96 uur, gevolgd door een

pH-cycling regime van 3 dagen. De premolaren werden in vier groepen verdeeld: een fluoride

groep (Elmex Medical Gel®), een CPP-ACP groep (Tooth Mousse TM), CPP-ACPF groep

(Mi Paste Plus TM) en een controle groep. Elke groep werd behandeld volgens de instructies

van de fabrikant (GABA, Lörrach, Duitsland en GAC Europe, Nieuwegein, Nederland). Alle

tanden werden dagelijks gepoetst met een tandpasta (1450 ppm fluoride) behalve de controle

groep. Op drie verschillende plaatsen van de WSL werd met microradiografie na 6 en 12

weken de volgende acht parameters gemeten: diepte van de totale laesie (DTL), de

‘body’laesie (DBL), de diepte van de oppervlaktelaag (DSL), de maximum “mineral content

van de surface layer” (MSL), de minimum “mineral content van de body of the lesion”

(MBL), het demineralisatie volume (VD), de basis minerale hoeveelheid (BMC) en het aantal

2

“body laesies”(NBL). Verschillende statistische tests werden uitgevoerd om de groepen te

vergelijken na 6 en 12 weken. Het significantieniveau werd ingesteld op p <0.05.

Resultaten: De Student’s t-test toonde geen statistisch significant verschil tussen de palatale

en buccale zijden voor de verschillende variabelen. De Wilks' Lambda exact test vertoonde

een sterk significant verschil (p<0.001) voor de testgroepen tussen 6 en 12 weken en geen

verschil voor de controlegroep (p=0.402). Na 6 weken was het punt BMC-DTL significant

verschillend in mineraalgehalte en laesiediepte voor de fluoridegroep versus de controlegroep

(p=0.045) en voor de CPP-ACP groep versus de CPP-ACPF groep (p=0.041). Na 12 weken,

waren de testgroepen zeer sterk verschillend (p<0.001) t.o.v. de controle groep voor de

verschillende parameters. BMC-DTL was statistisch significant lager voor CPP-ACPF versus

de fluoride groep (p=0.025) en versus de CPP-ACP groep (p=0.03). De Friedman test toonde

geen significant verschil aan voor het volume van demineralisatie (VD) van de drie

testgroepen tussen 6 en 12 weken. Er was meer remineralisatie (lagere VD) bij de testgroepen

dan bij de controlegroep (p=0.007). De Pearson Chi-Square test en de Fisher ’s exact test

toonden geen verschil in het aantal NBL na 6 weken, wel na 12 weken (p< 0.001).

Conclusie: De resultaten van deze studie toonden geen significante verschillen tussen de drie

experimentele tandpasta’s voor de regressie van de WSL. Pas na 12 weken was er een

statistisch significante verbetering van de laesiedieptes van de drie experimentele tandpasta in

vergelijking met de controlegroep. De laesiediepte, maar ook het mineraalgehalte, van de

WSL is aanzienlijk lager na 12 weken voor de experimentele tandpasta.

3

Abstract

Introduction: Despite prevention, the development of white spot lesions (WSL) during

orthodontic treatment with fixed appliance still is a significant clinical problem (the overall

prevalence of WSL varies from 2% to 97%). The increased prevalence of enamel

decalcification during fixed appliances therapy is partly due to the irregular surface of

brackets, bands and other attachments and a limitation of the self-cleansing mechanism such

as saliva and the movement of the oral musculature. At the end of orthodontic treatment white

spot lesions might persist as visible damage and can be an aesthetic problem to the patient

because of the chalky, rough opaque appearance of the enamel. Good oral hygiene during

orthodontic treatment is therefore necessary. Different products (such as fluoride and more

recently casein phosphopeptide-amorphous calcium phosphate) are promoted for preventing

and remineralizing WSL.

AIM: The aim of this study was to evaluate the in vitro effects of fluoride, casein

phosphopeptide-amorphous calcium (CPP-ACP) and a combination of both (CPP-ACPF) and

to compare it with a control group on remineralisation of WSL after 6 and 12 weeks.

Materials and Method: 96 premolars were sectioned in two parts (buccal and palatal). WSL

were created with a demineralisation solution during 96h, following a pH-cycling regime

during 3 days. The premolar parts were divided into four groups: a fluoride-group (Elmex

medical gel®), a CPP-ACPF group (Tooth MousseTM), a CPP-ACPF group (Mi Paste PlusTM)

and a control group. Each group of teeth was treated according to the instructions of the

manufacturer (GABA, Lörrach, Germany and GAC Europe, Nieuwegein, the Netherlands).

All teeth were daily brushed with toothpaste (1450 ppm fluoride) except the control group.

Eight different parameters were measured with transverse microradiography after 6 and 12

weeks: the depth of the total lesion (DTL) with the basic mineral content (BMC), the depth of

the body of the lesion (DBL) with the minimum mineral content of the body of the lesion

(MBL) and the depth of the surface layer (DSL) with the maximum mineral content of the

4

surface layer (MSL). The volume of demineralisation (VD) and the amount of body lesions

(NBL) were also measured. Different statistical tests were performed to compare the four

groups after 6 and 12 weeks. The significant level is p<0.05.

Results: The Student’s t-test showed no statistically significant difference between the palatal

and buccal sides for the different measured parameters. The multivariate test, Wilks' Lambda

exact test, showed a strong significant difference (p<0.001) for the experimental groups

between 6 and 12 weeks and no significant difference for the control group (p=0.402). At 6

weeks, only BMC-DTL differed significantly for the mineral content and the lesion depth for

the fluoride group versus the control group (p=0.045) and for CPP-ACP-group versus CPP-

ACPF-group (p=0.041). At 12 weeks, the difference was highly significant (p<0.001) for the

comparison between the three experimental groups and the control group for the different

parameters. BMC-DTL was also statistically significant lower for CPP-ACPF versus the

fluoride group (p=0.025) and the CPP-ACP group (p=0.030). The Friedman test showed no

statistically significant difference for the volume of demineralisation (VD) in the 3

experimental groups between 6 and 12 weeks. The results demonstrated significantly more

remineralisation (lower VD) in the experimental groups than in the control group (p=0.007).

The Pearson Chi-Square test and the Fisher’s exact test showed no significant difference in

NBL after 6 weeks and a highly significant difference after 12 weeks (p< 0.001).

Conclusion: The results of this study revealed no significant differences between the three

different experimental toothpastes in regression of the WSL. Only after 12 weeks, there was a

statistically significant improvement of lesion depths for the three experimental toothpastes

compared with the control group. The lesion depth but also the mineral content of the WSL

decreased significantly after 12 weeks for the experimental toothpastes.

5

Inleiding

Aan de afdeling orthodontie te Gent worden onderzoeken uitgevoerd met betrekking tot de

werking van Casein Phosphopeptide in vergelijking met conventionele tandpasta’s. In een

eerder onderzoek aan onze afdeling onderzocht Veronique Noens het therapeutisch effect van

Casein PhosphoPeptide-Amorphous Calcium Phosphate (CPP-ACP) op ‘white spots’ na een

orthodontische behandeling. Silvia Dauwe spitste zich in een tweede deel toe op het

preventieve aspect van ‘white spots’ tijdens de orthodontische behandeling. In een derde luik

van deze onderzoekslijn wordt het effect van deze tandpasta’s (CPP-ACP en Fluoride) en de

combinatie CPP-ACP/fluoride op ‘white spots laesies’(WSL) in een in-vitro studie verder

onderzocht. In functie van de duurtijd zal de demineralisatie en remineralisatie vergeleken

worden.

6

Study of the literature

The following search terms were used:

- White Spot Lesions (WSL) => “Dental caries” [MeSH], “White Spot Lesions”,

- Casein PhosphoPeptide – Amorphous Calcium Phosphate (CPP-ACP) =>

“caseins”[MeSH], “CPP-ACP”

- Fluoride =>”Fluoride”[MeSH], “Fluor*”,

- Remineralisation => “ Remineralisation”[MeSH], “Reminerali*”

- Demineralisation =>“ Demineralisation”[MeSH], “Deminerali*”

- Microradiography => “Microradiography"[Mesh],”Transverse microradiography”

- Orthodontics=> “Orthodontics” [MeSH], “orthodon*”

The cursive words stand for the combination of the different search terms per term. Different

electronic databases (Medline, Web of science, Cochrane) were used. The search was

restricted to English and Dutch literature only, full texts must been available. If the amount of

selected articles was high, only the articles published in the last 10 years were selected. For

WSL and fluoride only reviews were selected.

Database Search Terms Results Selected

PubMed WSL 49 10

WSL AND Cpp-Acp 98 12

WSL AND Fluoride 871 17

WSL AND Cpp-Acp AND Fluoride 33 /

WSL AND Remineralisation 346 16

WSL AND Demineralisation 420 23

WSL AND Remineralisation AND Demineralisation 147 0

WSL AND Microradiography 86 /

WSL and Orthodontics 415 /

7

CPP-ACP AND Fluoride 221 17

CPP-ACP AND Remineralisation 148 4

CPP-ACP AND Demineralisation 103 1

CPP-ACP AND Remineralisation AND Demineralisation 74 0

CPP-ACP AND Microradiography 10 /

CPP-ACP AND Orthodontics 24 /

Fluoride AND Remineralisation 254 /

Fluoride AND Demineralisation 249 23

Fluoride AND Remineralisation AND Demineralisation 135 /

Fluoride AND Microradiography 183 12

Fluoride AND Orthodontics 310 19

Remineralisation AND Demineralisation 336 /

Remineralisation AND Microradiography 68 1

Remineralisation AND Orthodontics 33 1

Demineralisation AND Microradiography 89 5

Demineralisation AND Orthodontics 136 /

WSL AND Fluoride AND Remineralisation AND Demineralisation 142 12

Web White Spots Lesions 77 7

of CPP-ACP 90 9

Science Fluoride 1148 7

Enamel remineralisation 296 12

Enamel demineralisation 448 10

Transverse microradiography 71 2

WSL AND CPP-ACP 10 2

WSL AND Fluoride 48 8

WSL AND Remineralisation 35 5

WSL AND Demineralisation 61 6

Cochrane White Spots Lesions 3 /

Fluoride 15 3

Remineralisation 4 /

Demineralisation 4 /

8

1. Caries process and white spot lesions

Dental caries is the most prevalent human disease of bacterial origin. It is a major public

health problem for both children and adults. The involvement of microorganisms has been

recognized since the late 1800s. Nowadays, modern management of dental caries is based on

appropriate detection of the disease, diagnosis of pathological changes (including lesion

formation in its earliest stages), prevention, control and treatment (3).

The caries process is initiated by the demineralisation of tooth hard tissue by organic acids.

Dental plaque bacteria like Mutans Streptococci (MS) and Lactobacillus can fermentate

dietary sugar and produce organic acids (4). Granulicatella, Veilloellaceae, Bifidobacteriacea

and Wiggsiae were also associated with the presence of WSL in adolescents undergoing

orthodontic treatment (5). The acids decrease the pH in the biofilm and causing the

dissolution of the calcium phosphate mineral in the tooth enamel or dentin. This leads to

dissolution of the hard tissue (2). At different stages in the caries process, different

combinations of bacteria may interact to produce the overall effect of a plaque with lowered

pH (6). This demineralisation, a dynamic process, is reversible in early stages. The calcium,

phosphate and fluoride in the saliva restore the crystalline structure of the tooth. This process,

known as remineralisation, neutralizes the acids and reverses the diffusion gradient for

calcium and phosphate (2, 7). In a remineralisation process, the pH increases. An imbalance

between demineralisation and remineralisation of dental hard tissues can result in dental

caries. The caries balance is shown in figure 1. A diminution of the salivary flow decreases

the resistance to white spot formation (8).

The use of fixed appliances provides a rough surface that makes tooth cleaning more difficult.

This accelerates the rate of plaque accumulation and plaque maturation. The presence of the

carbohydrates in the plaque in combination with the plaque associated bacter

and the demineralisation starts.

correlation between plaque accumulation and development of carious lesions. The

the local environment by placing an orthodontic

acid uric bacteria. Mutans streptococci

treatment. After the removal of fixed appliances,

significantly to levels comparable with age

phase (5). The reduction in bacterial counts was more pronounced for lactobacilli, which may

indicate that MS levels need mo

these plaque bacteria is shifted during orthodontic treatment

amount of subgingival P. gingivalis remained high for 6 months after appliance removal. This

finding might imply a potential risk to periodontal health in certain patients

Enamel crystal dissolution starts

enamel rods. The early clinical sign of an e

termed white spot lesions because of the resultant

causing scattering of light and giving the enamel a chalky, rough opaque appearance

14).

Figure 1: The caries balance: a diagram of the balance between pathological and protective factors in the caries process, a balance that alternates either way several times a day in most people (2).

of fixed appliances provides a rough surface that makes tooth cleaning more difficult.

This accelerates the rate of plaque accumulation and plaque maturation. The presence of the

in the plaque in combination with the plaque associated bacter

ation starts. According to Zachrisson & Zachrisson (

correlation between plaque accumulation and development of carious lesions. The

by placing an orthodontic appliance appear to favour

streptococci can increase up to fivefold during orthodontic

After the removal of fixed appliances, microbial levels were found to decrease

significantly to levels comparable with age-matched controls, 6 to 15 weeks into retention

he reduction in bacterial counts was more pronounced for lactobacilli, which may

indicate that MS levels need more time to return to normal, or that natural balance between

these plaque bacteria is shifted during orthodontic treatment (10). According to Liu, the

amount of subgingival P. gingivalis remained high for 6 months after appliance removal. This

finding might imply a potential risk to periodontal health in certain patients

starts with subsurface demineralisation, creating pores between th

The early clinical sign of an enamel mineral loss is a white spot lesion. I

because of the resultant change in the refractive index of the enamel,

causing scattering of light and giving the enamel a chalky, rough opaque appearance

The caries balance: a diagram of the balance between pathological and protective factors in the caries process, a balance that alternates either way several times a day in most

9

of fixed appliances provides a rough surface that makes tooth cleaning more difficult.

This accelerates the rate of plaque accumulation and plaque maturation. The presence of the

in the plaque in combination with the plaque associated bacteria lowers the pH

(9) there is a linear

correlation between plaque accumulation and development of carious lesions. The changes in

favour colonization of

can increase up to fivefold during orthodontic

microbial levels were found to decrease

o 15 weeks into retention

he reduction in bacterial counts was more pronounced for lactobacilli, which may

re time to return to normal, or that natural balance between

According to Liu, the

amount of subgingival P. gingivalis remained high for 6 months after appliance removal. This

finding might imply a potential risk to periodontal health in certain patients (11, 12).

ation, creating pores between the

a white spot lesion. It is

in the refractive index of the enamel,

causing scattering of light and giving the enamel a chalky, rough opaque appearance (8, 13,

The caries balance: a diagram of the balance between pathological and protective factors in the caries process, a balance that alternates either way several times a day in most

10

This change in the refractive index of the enamel surface and subsurface are due to the

changes in physical and chemical properties, the spatial configuration of the crystals and the

presence of water (12, 15). When the demineralisation process progress, the full thickness of

the enamel and some of the dentine demineralize before the relatively hypermineralized

surface layer is actually lost (8, 14). WSL, a visual damage, may detract from the beneficial

effect of orthodontic treatment.

There seems to be a difference in progression between traditional caries formation and white

spots lesions. The latter has a rather superficial (75µm) and ‘speedy’ character and can

become apparent within 4 weeks after placement of fixed appliance, which is the average time

between two orthodontic visits (14, 16, 17). The formation of a normal caries lesion is usually

a slower process, which takes at least 6 months (18). The shape of the white-spot lesion is

determined by the distribution of the biofilm and the direction of the enamel prisms. This

emphasizes that the lesion is driven by, and reflects, the specific environmental conditions in

the overlying biofilm (19).

a) Prevalence of white spots

The prevalence of dental caries has drastically decreased the last decades but is far from being

eliminated (20-22). The age group (11-14 years) in which orthodontic treatment is usually

carried out is considered to be at greatest risk of developing caries (23). In 2011, Al Maaitah

et al reported the overall incidence of WSL reported from 2% to 97% (24). According to

Behnan et al, the incidence of WSL during orthodontic treatment with fixed appliances is 73%

to 95% (25). This large variation of prevalence-data could be attributed to the variety of

clinical investigations, registration and the difficulty in standardizing clinical examinations.

11

The increased prevalence of WSL in orthodontic patients can be attributed to the following

factors:

- Fixed orthodontic appliances form a niche for bacterial plaque to inhabit, complicating

a proper oral hygiene practice. Patients with inadequate oral hygiene tend to develop

more WSL affecting more teeth, during orthodontic treatment.

- The newly erupted teeth, seen in typical orthodontic patients, might also be more

susceptible to acid attack (13).

- The fixed appliance makes conventional oral hygiene for plaque removal more

difficult, and increases gingival inflammation. The clearance of plaque by saliva or the

movement of oral musculatures adjacent to the brackets is also reduced.

- Younger patients tend to develop WSL during orthodontic treatment more often than

adult patients.

- Patients with at least 1 decayed, missing due to caries or filled first permanent molar

before orthodontic treatment might develop more WSL after orthodontic treatment

than patients who have 4 healthy first permanent molars.

No significant association between severity of WSL and patient’s age, socio-economic status,

compliance with attendance, treatment duration or treatment type was found. Except that once

WSL have developed in male patients, the lesions have a greater degree of demineralisation

after orthodontic treatment than in female patients (24).

On average, in males 40% and in females 22% of surfaces showed white spots (10).

Zachrisson and Zachrisson (26) reported that higher caries index, recorded for male

orthodontic patients, was a result of a lower standard of oral hygiene (with higher scores for

plaque and gingival index) in male patients as compared to female patients.

12

After orthodontics, the enamel lesions have been reported to develop most frequently on the

cervical and middle third of the buccal surfaces. In the literature, different distribution of

affected teeth is mentioned. Most frequently, lateral maxillary incisors, mandibular canines

and first premolars are affected with enamel lesions, followed by maxillary central incisors

and first molars in the upper and/or the lower arch or both arches. (23, 27, 28). A significant

difference in the number of lesions was found between incisors and canines on the one hand

and molars and premolars on the other hand, indicating more lesions in the molar-premolar

region (10). When the appearance of white spots was evaluated in overall manner, there was

significantly more upper than lower decalcification (29).

The lingual surfaces are less prone to caries in comparison to the buccal surfaces (30). This

may be explained by differences in surface morphology, plaque retention, salivary flow and

mechanical cleaning of surfaces by the tongue. WSL development on buccal surfaces is 4.8

times higher than the number of new WSL developing or progressing on bracketed lingual

surfaces. The caries extent was 10.6 higher for buccal surfaces (31).

b) Prevention of white spot lesions

Many studies have been performed to find the solution to prevent caries lesions during

orthodontic treatment with fixed appliances. Oral hygiene is the most important factor. The

patient’s cooperation can be improved by repeatedly verbally praising and re-education (32).

The orthodontist prevents WSL also by starting fixed appliance treatment only in plaque-free

patients, providing a strict oral hygiene protocol during treatment, placement of the

orthodontic appliance on a less plaque-retentive way and discontinuing orthodontic treatment

as soon as incipient WSL become visible.

13

Patients, parents, orthodontists and general dentists indicate that patients are the most

responsible for the prevention of WSL. Most patients and parents are aware that WSL were

caused by inadequate or improper brushing and flossing during orthodontic treatment (33).

To obtain a good oral hygiene and to prevent WSL, there are several products like fluoride,

CPP-ACP and chlorhexidine to use. Underneath, a short explanation of the different products.

In the following chapters, the mechanism of actions of fluoride and CPP-ACP is explained.

Fluoride

The standard actions used in an orthodontic practice to prevent demineralisation are brushing

with fluoride (1000ppm) toothpaste twice daily (32, 34) combined with 0.05% sodium

fluoride mouth rinse and nutritional counselling (35-37). 0.2 % Sodium fluoride mouth rinse

helps to reduce the severity of enamel decay adjacent to fixed braces, and also inhibits lesion

development by about 60%. Unfortunately the trustworthiness of any home medication

treatment can’t be controlled. Only 42% of patients rinsed with a sodium fluoride mouth rinse

at least every other day (38).

Fluoride administration is possible in various ways and there is a great variability of products

on the market. Many fluoridated materials release large amounts of fluoride initially, but the

level drops rapidly and might not be sufficient to prevent decay over the whole course of

orthodontic treatment. Long duration and low-dose fluoride release appears to increase the

caries-resistant fluorapatite concentration in enamel and to reduce demineralisation during

orthodontic treatment (39). Evidence suggests that the use of extra fluorides during fixed

appliance treatment reduces the occurrence and severity of white spot lesions (16, 40, 41).

14

There are 2 different ways to apply fluoride: a topical way and an adhesive way.

Topical way (toothpaste, mouth rinse, gel, varnish):

- Fluoride varnish during orthodontic treatment resulted in 50% less enamel

demineralisation according to Todd et al., 48% according to Øgaard et al and 44, 3%

less demineralisation according to Vivaldi-Rodrigues et al. (11, 42, 43). Following Du

et al., varnish application should be advocated as a routine caries prevention measure

after orthodontic treatment (6).

- Topical fluoride pastes provide a protective coat which decreases enamel solubility

and an increase of fluoride uptake in enamel (32). After debonding, application of

topical fluoride on WSL remineralizes these lesions (44-46). On the contrary, topical

application of acidulated phosphate fluoride gel immediately after debonding appears

to be of little benefit in reducing the incidence of white spots (36).

- Titanium tetrafluoride (TiF4) is more effective in preventing WSL than other fluoride

agents (47). The mode of action of TiF4 is due to the formation of an acid-resistant

surface layer. This layer provides mechanical protection to the surface, and increases

the fluoride uptake, which might chemically reduce demineralisation of dental hard

tissues. Clinical trials analysing the effect of TiF4 are lacking so the potential of TiF4

to prevent acid demineralisation requires further research to confirm the promising in

vitro results obtained (48).

15

Adhesive way: fluoride releasing cements, elastomeric modules, glass ionomer, etc...

- Elastomeric or modules are used in orthodontics to hold the arch wire in the bracket

slots. Instead of conventional elastomers, there are also fluoride-releasing elastomers.

The overall reduction in score per tooth produced by the fluoride-releasing

elastomerics was 49%. A significant difference was seen in all but the occlusal enamel

zones. Fluoride-releasing elastomers have been shown to reduce significantly the

salivary levels of Mutans Streptococcus MS. However clinically, these elastomers

showed a more rapid deterioration with swelling and apparent loss of elastic properties

2-3 weeks after insertion. Apparently, these modules are less elastic, rendering figure-

of-eight ligating impossible and not available in different colours. The magnitude of

fluoride released by these modules was considerably higher than that reported for

fluoride-releasing chain (49).

- Fluoride-releasing orthodontic bonding adhesives have been available for several

years but long-term clinical trials have shown them to be ineffective in preventing

enamel damage (28, 29). The bond strength is also too weak so it is clinically not

successful for routinely bonding of brackets (29). These composites generally release

fluoride over a short time span, which is insufficient to provide protection throughout

a 2-year treatment period.

- Glass ionomer cements have been suggested as alternative bracket bonding materials.

The cements provides greater fluoride release, but their bond strengths are considered

inadequate for clinical use (49). According to Gjorgievska et al., conventional glass

ionomer has the greatest effect of caries inhibiting and shows the highest level of ion

migration (50).

- Enamel sealants/Resin sealers: An in vitro study found that Pro-seal TM® can resist

acid challenges and toothbrush abrasion in a laboratory environment. In a second in

vitro study, it was found that Pro-Seal TM® (a fluoride containing sealant) provided

significantly more protection than either fluoride varnish or an unfilled resin sealer

with 92% reduction in lesion depth compared with the controls (25).

16

Casein phosphopeptide – Amorphous calcium phosphate

Casein phosphopeptide – Amorphous calcium phosphate (CPP-ACP) would slow down the

demineralisation process and enhance the remineralisation. CPP-ACP can be incorporated

into various products (methacrylate composites, sugar-free gum, mints, topical gels, pastes,

sport drinks, glass ionomer cements) in order to exert a topical effect (51-53).

The daily topical exposure to CPP-ACP in CPP-ACP containing sugar-free chewing gum and

mint in-situ increase enamel remineralisation of artificially demineralized subsurface enamel

lesions, with an increase in enamel mineralisation of 78 to 176% (51-53). In addition, the

consumption of chewing gum and mints results in an increased production of stimulated

saliva (32). Sudjalim et al. recommended the combination of CPP-ACP with fluoride varnish

to enhance the treatment effect (54). The use of CPP-ACP in orthodontic practice has been

proposed for white spots prevention and caries prophylaxis even before bracket placing

procedures (9).

Chlorhexidine

Chlorhexidine applications, such as the chlorhexidine-thymol varnish (Cervitec, Vivadent)

used in conjunction with a fluoride varnish might help to lower the level of Mutans

Streptococci (55). Chlorhexidine inhibits acid production in plaque and thus reduces the pH

decrease during sucrose challenges (56). But the use of antimicrobials, such as chlorhexidine

against the temporary elevations in salivary MS levels may not be warranted in all orthodontic

patients (32). An adverse effect includes a bitter taste and discoloration of the teeth and

tongue (57).

17

Others

Argon lasers are also suggested in this context but there are very few data about this subject

(2, 32).

Excessive surplus orthodontic etching of the complete labial enamel surface, instead of the

bracket bases only, must be avoided to prevent iatrogenic white spot lesions. Etching times

not exceeding 15 seconds are favourable (58).

c) Treatment of white spot lesions

Despite preventive measurements, some patients have unsightly WSL after debonding

presenting a challenge for restorative treatment. While de-bonding eliminates a major

etiological factor, remineralisation of white-spot lesions is not guaranteed. During the first 6

months after de-bonding, the greatest change of the white spot can occur (59). A significant

clinical improvement of about 80% was seen of the sites after 6 months, only 40% became totally

invisible, 22% remained as an aesthetic problem (60-62).

The remineralisation of WSL can be attributed to a reduction of the cariogenic challenge. By

removal of the accumulated plaque and cariogenic microorganisms around the appliances, the

cariogenic challenge is substantially reduced. Other factors of importance in this respect could

be reduced clearance time for cariogenic food items and also more efficient tooth brushing

(63). According to Artun and Thylstrup, the result of surface abrasion with some

predisposition of minerals is to be held responsible for the loss of porous tissue and the

gradual regression of the WSL after debonding (27).

Highly concentrated fluoride solutions hypermineralize the surface of the lesion, leaving them

more resistant to acid attacks and decrease body remineralisation. Unfortunately, since these

18

solutions do not penetrate inside the lesion, they do not eliminate its opaque whitish aspect

which is less aesthetic (64). Low doses of fluoride in mouth rinses do not improve the lesions

either (61).

Allowing remineralisation by saliva and, if necessary, the use of acid microabrasion 10 weeks

after debonding is suggested by Welbury and Carter (62). The hydrochloric acid-pumice

micro-abrasion technique offers a method for improving the appearance of these lesions.

The potential of CPP-ACP to promote lesion remineralisation has been demonstrated (52, 53,

66, 67). According to Bailey et al., there is 31% more regression at 12 weeks for WSL code 2

and 3 (68). In the presence of fluoride, CPP-ACP has been shown to promote the formation of

fluorapatite deep in the subsurface lesion (69). Hence, this ability to promote remineralisation

of enamel subsurface lesions in situ explains the significant regression of WSL by CPP-ACP

in this post-orthodontic population using fluoride toothpaste and receiving supervised fluoride

mouthrinses.

The aesthetic result of bleaching spot- or stripe-shaped local opacities is rather limited

according to Glockner (70). In contrast to Knosel’s study where patients were satisfied with

the outcome (71). The susceptibility to the formation of caries-like lesions after bleaching

increases. Therefore perfect oral health and hygiene is needed. Wiegand et al. suggested

fluoridation after the bleaching therapy to enhance remineralisation of bleached teeth (72).

Featherstone demonstrated that specific pulsed carbon dioxide, or CO2 laser treatment of

dental enamel can inhibit subsequent caries like progression in a severe demineralisation-

remineralisation model in the laboratory by up to 85 per cent (2). CO2 and diode laser

irradiation of the enamel surface can both increase fluoride uptake; however, laser energy

parameters must be carefully controlled in order to limit increases in pulpal temperature and

alterations to the enamel surface (73, 74).

19

2. Anticariogenetic properties of CPP-ACP (GC Tooth MousseTM)

Solutions containing calcium and phosphate ions have limited effect on remineralisation, due

to the low solubility of calcium and phosphate salts. The multiple phosphoferyl residues of

the CPP bind to nanoclusters of ACP in supersaturated solutions, thereby preventing growth

to the critical size required for phase transformations (75). The CPP molecules contain a

cluster of phosphoseryl residues which markedly increase the apparent solubility of calcium

phosphate by stabilizing amorphous calcium phosphate (ACP) under neutral and alkaline

conditions. CPP-ACP can promote the formation of fluorapatite deep in the subsurface lesion

(69).

The proposed anticariogenic mechanism of CPP-ACP involves the incorporation of the nano-

complexes into dental plaque and onto the tooth surface, thereby acting as a calcium and

phosphate reservoir (68). This mechanism is ideal for the prevention of enamel

demineralisation as there appears to be an inverse association between plaque calcium and

phosphate levels and measured caries experience. The localized CPP-ACP nanocomplexes act

to buffer free calcium and phosphate ions in the plaque fluid, in order to maintain a state of

supersaturation of ACP with respect to enamel mineral, thereby limiting enamel

demineralisation and enhancing remineralisation (53, 76).

CPP-ACP can be incorporated into supragingival dental plaque by binding to the surfaces of

bacterial cells, to components of the intercellular plaque matrix and to adsorbed

macromolecules on the tooth surface. All these interactions may then lead to the formation of

a less cariogenic plaque (75). Enamel lesions which have been remineralized with topical

exposure to CPP-ACP have been shown to be more resistant to subsequent acid challenge

compared with normal remineralized enamel as CPP-ACP is able to promote the

remineralisation of enamel subsurface lesions with hydroxyapatite.

20

In addition, the relatively low carbonate environment of the CPP-ACP treated subsurface

lesion may also exhibit both improved crystallinity and lower microstrain than might be found

in normal tooth enamel (32, 77)

CPP stabilize calcium, phosphate and hydroxide (fluoride) ions preventing spontaneous

precipitation of hydroxyapatite (fluorapatite). The CPP stabilized calcium, phosphate and

hydroxide (fluoride) ions substantially promote remineralisation of enamel subsurface lesions

and therefore have the potential as a superior form of fluoride for the prevention and

remineralisation of incipient carious lesions and for the remineralisation of hypomineralized

enamel lesions generally.

3. Mechanism of action of fluoride (Elmex Medical Gel®)

Why caries prevalence reduced during the last 20 years is difficult to say. Strong evidence

exists, however, that the near universal use of fluoride containing products such as toothpaste,

mouthrinses and topical gels (used at home or applied in the dental office by the dentist) have

been major contributors. Earlier reductions of caries has resulted from the fluoridation of

public water supplies in many communities (2). Fluoride has three principal topical

mechanisms of action:

Inhibiting bacterial metabolism.

The bacteria in the plaque produce acids and decrease the pH. A portion of the fluoride

present in the plaque fluid combines with hydrogen ions to form hydrogen fluoride (HF). The

HF molecules diffuse rapidly into the cell. The HF molecules dissociates in the cell which

makes the inner cell more acid and release fluoride ions, that interfere with the enzymatic

activity of the bacteria (2).

21

Inhibiting demineralisation

Different forms of apatite are present. The carbonated hydroxyapatite, or CAP, of our teeth is

more soluble in acid than hydroxyapatite, or HAP (HAP = Ca10 (PO4)6(OH)2. Fluorapatite, or

FAP (unit cell stoichiometry of Ca10 (PO4)6F2) in which the OH- ion in pure hydroxyapatite is

completely replaced by an F– ion, is even more resistant to dissolution in an acid environment,

so fluoride inhibit demineralisation. If fluoride is present in the aqueous solution surrounding

the crystals, it absorbs strongly to the surface of the CAP and acts as a potent protection

mechanism against dissolution of the CAP of the enamel of the tooth. When the bacteria

generate acids, the fluoride present in the plaque fluid, travel with the acid into the subsurface

of the tooth and adsorb to the crystal surface and protect it. When the fluoride is concentrated

into a new crystal by remineralisation, it modifies the crystal resistance to dissolution.

Fluoride incorporated into the tooth during tooth development is insufficient to play an

important role in the caries protection. So it is important to have a high level of fluoride

surrounding CAP crystals to inhibit the demineralisation by an acid attack of bacteria (2).

Fluoride concentrations as found in dental plaque have biological activity on critical virulence

factors of Mutans Streptococci in vitro, such as acid production and glucan synthesis (78).

Enhancing remineralisation

When fluoride is adsorbed to the crystal surface, it attracts calcium ions, followed by

phosphate ions leading to new mineral formation. The new formed crystal structure is

composed of HAP and FAP. CAP is no longer present in the crystal. Since the solubility of

HAP and FAP is much lower than that of CAP, the newly formed crystal is less soluble in an

acid environment. The critical pH for hydroxyapatite (the pH at which it starts to dissolve and

hence it lost) is 5.5, however between pH 4.5 and 5.5 saliva and plaque fluids are still

supersaturated with regard to fluorapatite and will tend to precipitate and preventing the loss

22

of mineral ions. pH levels lower than 4.5 have been measured in the plaque around the

brackets and the bands. Several methods have been suggested to improve the cariostatic effect

of fluoride at low pH. Acid-resistant coatings of calcium fluoride or titanium fluoride on the

enamel surface and the use of fluoride in combination with anti-microbial have been

suggested (43).

A problem with fluoride ions is that, in the presence of salivary calcium and phosphates ions,

a rapid deposition of these ions occurs in the surface layer of the enamel lesion. This

deposition in the surface layer then blocks further ion penetration into the body of the carious

lesion thus limiting deeper remineralisation (7). Two mol fluoride ions are required for every

10 mol of calcium and 6 mol of phosphate ions to remineralize enamel subsurface lesions

with fluorapatite.

Figure 2: Schematic representation of demineralisation followed by remineralisation in the caries process. If remineralisation is successful, the final result is a crystal with a surface layer of "fluorapatitelike" mineral of low solubility. (Reproduced from Featherstone (1) with permission of the publisher. Copyright ©1999, Munksgaard.)

23

Hence for many applications of topical fluorides the level of calcium and phosphate ions is

the limiting factor for remineralisation (69, 79).

Elmex medical gel® contains the amine fluorides: olaflur and dectaflur. Amine fluorides

(NaF) act like surfactants, reducing the surface tension of saliva, and forming a homogeneous

film on all oral surfaces. This is due to their molecular structure: the fluoride ion is bound to

an organic fatty acid amine fragment. Amine fluorides cover the tooth surfaces with a

homogenous molecular layer. This continuous film prevents rapid rinsing off by the saliva

and thus active for a longer period (80).

4. Interaction of CPP-ACP and fluoride (MI Paste PlusTM)

Recently, fluoride has been incorporated into the CPP-ACP complex (CPP-ACPF) and was

shown to promote remineralisation; the deposited mineral is with fluorapatite. The level of

remineralisation (up to 57%) was able to return translucency of the treated enamel to that of

sound enamel. The use of CPP-ACP with fluoride and its synergistic effect on enamel

remineralisation have been attributed to the formation of CPP-stabilized amorphous calcium

fluoride phosphate resulting in the increased concentrations of bioavailable calcium and

phosphate ions. (69, 79)

24

5. Quantifying enamel demineralisation

White spot lesions are not detectable visually until they progressed 200-300µm into the

enamel (81). A reliable and reproducible detection of dental caries by clinical examination has

been recognized as a problem for decades. Variable approaches has been taken to recognize

WSL (82).

There are invasive and non-invasive diagnostic methods. In this in-vitro study we choose the

micro-radiography, which is considered as the ‘‘gold standard’’.

Non-invasive diagnostic methods:

A non-invasive diagnostic method, such as visual detection and observation of fluorescence,

preservers the tooth in its entirety. It can be used in vivo.

Visual detection

The most evident method is based on a visual detection of the labial surfaces of the teeth, but

it cannot help detecting caries in very early stage. There are different indices for scoring the

amount of decalcification like for example the white spot lesion index from Gorelick (table 1),

from Andersson (table 2) or the white spot lesion index from Banks and Richmond (table 3)

(8, 49, 60). According to Torlakovic et al., WSL’s colour intensity might predict the depth of

enamel demineralisation as well as traditional WSL scoring (7).

An indirect way of visual inspection is by computerized image analysis of digital photographs.

This is a more reliable and reproductive way than direct visual detection (83).

25

Score Definition

1 No white spots

2 Thin white border: With a guideline of <33% of the vestibular surface

3 Thick white border. With a guideline between 33 and 66% of the vestibular surface

4 White spot and cavitation or covering more than 66% of the vestibular surface

TABLE 1 The white spot lesion index from Gorelick (8).

Score Definition

0 No visible colour change

1 Slight white colour change, only visible after air drying

2 Slight colour change with certain marked white areas

3 White consistent colour change

4 Distinct white colour change

TABLE 2 The white spot lesion index from Andersson (60).

Score Definition

0 No decalcification

1 Decalcification covering <50% of the gingival/ mesial/ distal/ occlusal zone

2 Decalcification covering >50% of the gingival/ mesial/ distal/ occlusal zone

3 Decalcification covering 100% of the gingival/ mesial/ distal/ occlusal zone or severe

decalcification with cavitations

TABLE 3 The white spot lesion index from Banks and Richmond (49).

26

Observation of fluorescence

Fluorescence makes it possible to detect caries in an initial stage. The QLF (Quantitative

Light induced Fluorescence) is an optical, visible light-based system that can be used to detect

and quantify early demineralisation of enamel. The clinical use of QLF is limited by several

confounding factors in caries-risk adolescents but seems to be a sensitive method that is

suitable for the detection of visually undetected initial caries lesions (10, 84).

Organic (bacterial by-products) and inorganic materials present on the tooth surface absorb

the red laser light (wave length of 655 nm) and emit this light as near-infrared fluorescent

light. Changes in the tooth substance associated with the amount of bacterial by-products are

reflected as an increase in the re-emitted fluorescent light and thus the difference between

sound and decayed areas is recognized by the PLM (Polarized Light Microscope, Kavo

DIAGNOdent). The amount of fluorescence relates more closely to bacterial presence than to

the mineral content of the tooth (63, 85).

Other detection methods with fluorescence is possible with a Vistaproof, Dye-Enhanced laser

fluorescence or DELF, optical coherence tomography or OCT, fiberoptic transillumination,

Electronic caries monitor or ECM, a colorimeter, etc (3, 21, 86, 87).

Invasive diagnostic methods:

To quantify a demineralisation by the invasive way, the teeth need to be extracted. For in-

vitro and microscopic investigation, slices are needed.

27

Microscopes

Transverse microradiography (TMR) is a standardized method in studies on

de/remineralisation of teeth for decades and considered as the ‘‘gold standard’’ for the

determination of mineral loss or gain in experimentally induced incipient carious lesions. This

method has been used for the comparison and validation of other newly developed caries

detection techniques. The TMR provides a very precise quantitative measurement of the

mineral content (88). A representative microradiography image of sectioned enamel is

represented on figure 3. The body of the lesion with the remineralisation is clearly visible.

The scanning electron microscopy SEM can be used to observe the surface morphology (89).

The transmission electron microscopy or TEM, Atomic Force Microscopy, Confocal laser-

scanning microscope and X-ray scanning analytical microscope are alternative microscopes

(90).

Figure 3: Representative microradiography images of sectioned enamel specimens. The body of the lesion with the remineralisation is clearly visible.

28

Others

Micro-CT, a 3-D tool, is also able to detect artificial caries lesions after remineralisation and

may be used to substitute TMR and PLM in in-vitro studies of caries (88). However, micro-

CT is less precise than microradiography.

Cross sectional hardness or CSH: is not very accurate for estimating the mineral content, but

it gives some information regarding the mechanical (physical strength) properties of the

lesions, which are not provided by TMR; therefore, it should be advised to combine different

methods to analyse enamel demineralisation, in order to get more information about the

properties of the lesions (91).

The nano indentation (92), the electron probe micro-analyser (93) and superficial

microhardness (SMH) can also be used for quantifying WSL.

29

Aim of this study

The aim of this study was to compare the in vitro effects of fluoride, casein phosphopeptide-

amorphous calcium phosphate (CPP-ACP) and CPP-ACPF products on remineralisation of

white spots after 6 and 12 weeks, and to compare them with a control group.

The null hypothesis to be tested was that there is no statistically significant difference in

remineralisation between the fluoride, the CPP-ACP and the CPP-ACPF products on white

spot lesions after 6 and 12 weeks.

30

Methodology

Preparation of enamel blocks

96 premolars (24 patients with 4 premolars) were collected from various dental practices

(informed consent was obtained nr B 670 2010 10019). The teeth were washed in tap-water

and stored in 0.1% thymol solution at 4° C. Premolars with a healthy enamel surface were

selected and included in the study. Teeth with dental caries, fluorosis and other

hypermineralized lesions, pitting or cracks, hypoplastic areas, large irregularities of enamel

structure were excluded. An enamel surface layer was removed to avoid a potentially fluoride

rich layer and the individual enamel differences as shown in figure 4 (13). The premolars

were sectioned in two parts: a buccal side and a palatal side. In order to divide the tooth, a

diamond saw, water-cooled, cutting machine was used. To avoid interference of general tissue

demineralisation of the crown, the entire part was covered by acid-resistant nail varnish

except the area of interest.

Figure 4: An enamel surface layer was removed to avoid a potentially fluoride rich layer and the individual enamel difference.

31

Artificial white spot lesions

White spot lesions were created in this area with the demineralisation solution of Ten Cate

and Duijsters (2.2 mM CaCl2.2H2O + 2.2 mM NaH2PO4 + 50 mM CH3COOH (94). pH was

adjusted to pH 4.4 using 1M KOH) during 96 h to have a lesion depth of 150-200 µm deep on

every half premolar (95). This method produces a subsurface enamel demineralisation without

surface erosion.

pH-cycling regime

Ten Cate and Duijsters introduced a dynamic model concept with alternating periods of de-

and remineralisation (94). The pH-cycling protocol simulates in vivo high caries risk

condition and simultaneously measures the net result of the inhibition of demineralisation and

the enhancement of remineralisation. This solution approximates the mineral ion composition

and supersaturation of saliva as originally reported by ten Cate and Duijsters (78, 94). The re-

and demineralisation solutions in the pH-cycling regime were renewed every day.

- Demineralisation stage (20ml/sample, during 6h/day at 37°C) used an acid buffer

containing demineralized H2O with 2mM Ca(Ca(NO3)2), 2 mM PO4 (KH2PO4) and 75 mM

acetate. pH was adjusted to 4.3 using 1M KOH.

- Remineralisation solution (20ml/sample, during 17h/day at 37°C) contained calcium

and phosphate at a known degree of saturation (1.5mM Ca and 0.9mM PO4) to mimic the

remineralizing properties of saliva. This solution contained demineralized H2O with 130-150

mM KCL (to provide background ionic strength) 100 mM TRIS, 1.5 mM Ca(NO3)2. 4H2O,

0.2 mM KH2PO4, and 140mM KCL. pH was adjusted to 7 using 1M HCL.

32

All 192 parts underwent 3 days the pH-cycling regime without the fluoride dentifrices

treatment to allow baseline values of calcium uptake and loss to be determined. After these 3

days brushing of the teeth was started.

Study design

The premolar parts were divided in four groups: a fluoride group (Elmex medical gel®,

GABA Benelux), a CPP-ACP group (Tooth MousseTM, GC Europe), a CPP-ACPF (Mi Paste

PlusTM, GC Europe) and a control group. The three active groups were brushed with toothpaste

(1450 ppm fluoride); the control group just underwent the pH-cycling regime. Table 4

represents the different test products, their application and their content.

Table 4: Ingredients and concentration range of tested agents, according to manufacturers’ specification.

Test products Application Content

Elmex Medical Gel® GABA, Lörrach, Germany

Gel 1x / week 3 minutes

Water, Amine fluoride (Olaflur and Dectaflur) Sodium fluoride Propylene glycol, Hydroxyethyl cellulose Fluoride content: 12,500 ppm

Gc Tooth MousseTM GAC Europe, Nieuwegein, the Netherlands

Gel Daily 3 minutes

Water CPP-ACP glycerol, D-sorbitol, CMC-Na, propylene glycol, silicon dioxide, titanium dioxide, xylitol, phosphoric acid, flavoring, zinc oxide, sodium saccharin, ethyl p-hydroxybenzoate, magnesium oxide, guar gum, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate

Mi Paste PlusTM GAC Europe, Nieuwegein, the Netherlands

Gel Daily 3 minutes

CPP-ACP Sodium Fluoride (900 ppm) water, glycerol, d-sorbitol, CMC-Na, propylene glycol, silicon dioxide, titanium dioxide, xylitol, phosphoric acid, flavoring, sodium saccharin, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate

33

Fluoride group (Elmex medical gel®): The premolar parts were brushed with conventional

toothpaste (1450 ppm fluoride) every day. Once a week, after brushing with conventional

toothpaste, Elmex medical gel® was applied during 180 seconds and rinsed before putting it

back in the pH-cycling regime. These were the instructions of the manufacturer.

CPP-ACP group (GC Tooth MousseTM): Every day after brushing with conventional

toothpaste (1450 ppm fluoride) the GC Tooth MousseTM was applied during 3 minutes.

Afterwards the teeth were placed in the remineralisation solution.

CPP-ACPF group (Mi Paste PlusTM): Every day after brushing with conventional toothpaste

(1450 ppm fluoride) the Mi Paste PlusTM was applied during 3 minutes. Afterwards the teeth

were placed in the remineralisation solution.

Control group: Control group without fluoride or CPP-ACP, underwent the pH-cycling

regime.

Microradiography

Changes in the mineral content and the lesion depths of the experimental caries lesions were

measured using transverse microradiography (TMR). At least three different enamel sections

were cut across the lesion window. These sections were x-rayed with an aluminium step-

wedge at 20kV and 15mA for 6 minutes (PW1830, Philips, Eindhoven, the Netherlands).

WSL exhibiting an intact surface zone were included. A second examiner evaluated the

photos. The average of three measurements per WSL was obtained for the different

parameters.

34

The lesion characteristics as defined by Theuns were the different parameters, evaluated in

this study (1). The mineral content in the lesion (volume fraction of the mineral) was plotted

against the depth.

- DTL = total depth of the lesion, defined as the depth at which no further increase in

mineral content was observed (µm)

- DBL = depth of the MBL point = depth of the body of the lesion (µm)

- DSL = depth of the MSL point = depth of the surface layer (µm)

- MSL= maximum mineral content of the surface layer (%)

- MBL= minimum mineral content of the body of the lesion (%)

- BMC= the basic mineral content (%)

- VD = volume of demineralisation (as shown in figure 5B) (µm %)

- NBL= the number of body lesions

The mineral contents were calculated with the standard use of 85% apatite.

Figure 5: Schematic drawing of the microradiography procedure. A Lesion characteristics as defined by Theuns: the maximum mineral content of the surface layer (MSL) and the point at which this maximum is reached (DSL), the minimum mineral content of the lesion body (MBL) and the point at which this minimum is reached (DBL) and the depth of the lesion (DTL). B Schematic drawing of the area representing the dissolved mineral (shaded part) (1).

35

Statistics

Descriptive statistics to calculate the range, mean, median and standard deviation.

A Student’s t-test was used to compare the palatal and the buccal side for the different

parameters (DBL, DSL, DBL, MSL, MBL, BMC and VD).

The Mann–Whitney U test, a non-parametric test, was used to compare the volume of

demineralisation between 6 and 12 weeks in the 4 groups.

The MANOVA, multivariate analysis of variance, compared the multivariate means (DSL-

MSL, DBL-MBL, and DTL-BMC) between the four groups at the different time periods and

between the 6 and 12 weeks.

The Friedman test, a non-parametric statistical test, was used to compare the volume of

demineralisation for the three experimental groups mutually.

The Wilcoxon signed-rank test, a non-parametric statistical hypothesis test, was used to

compare the volume of demineralisation between two related samples.

A Pearson Chi-Square test and a Fisher’s exact test were used to compare the amount of body

lesions between the groups mutually and between 6 and 12 weeks. .

The Intraclass Correlation Coefficient, a descriptive statistic, was used for assessing the

reliability between two measurements.

A p-value less than 0.05 was considered as statistically significant.

36

Results

The Student’s t-tests for different parameters indicated that there were no statically significant

differences between the palatal and buccal sides of the teeth. Most of the p-values were larger

than 0.344 showing no difference between buccal and palatal side (table 5). Only the p-value

for the DBL measurements after 6 weeks was smaller (p=0.096) but was still not significant.

Table 5: P-values after different Student’s t-tests comparing the buccal and palatal side at 6 weeks and

12 weeks. P>0.05 is not significant.

Time

Parameters 6w 12w DTL(µm) 0,870 0,922 DBL(µm) 0,096 0,770 DSL(µm) 0,408 0,804 MSL (%) 0,855 0,664 MBL (%) 0,495 0,854 BMC (%) 0,838 0,344

VD (%.µm) 0,783 0,655

37

In this in vitro study, due to the fact that there was no statically significant difference between

buccal and palatal side, these sides are considered as the same side. For the different

parameters, the average of the buccal and palatal values was taken. Descriptive statistics of

the different parameters at 6 and 12 weeks are shown in table 6.

Table 6: Descriptive statistics, mean and standard deviation, of the different parameters after 6 and 12 weeks.

Time Parameters Elmex® Gc Tooth MousseTM Mi Paste PlusTM Control

Mean (SD) Mean (SD) Mean (SD) Mean (SD)

6w DTL(µm) 155,69 (38,62) 188,18 (50,94) 153,12 (64,93) 203,26 (55,06)

DBL(µm) 68,98 (26,88) 80,98 (46,67) 57,76 (34,94) 91,08 (33,21)

DSL(µm) 41,13 (16,76) 47,46 (26,71) 32,59 (17,38) 49,91 (17,84)

MSL (%) 44,74 (15,29) 46,69 (18,70) 44,89 (39,74) 45,50 (12,82)

MBL (%) 33,35 (11,80) 32,74 (11,89) 26,15 (10,01) 27,08 (11,04)

BMC (%) 68,70 (1,91) 69,88 (1,91) 59,73 (11,24) 65,39 (5,83)

VD(%.µm) 3607,43 (1010,54) 3532,84 (1545,52) 3689,70 (1988,62) 4702,64 (2677,47)

12w DTL(µm) 72,38 (44,49) 77,12 (35,61) 83,59 (21,10) 231,73 (74,24)

DBL(µm) 10,20 (16,03) 10,62 (13,50) 16,82 (13,91) 105,67 (55,31)

DSL(µm) 7,51 (14,02) 5,71 (7,32) 9,59 (8,06) 52,83 (22,38)

MSL (%) 5,82 (10,92) 9,09 (12,27) 10,98 (9,20 38,79 (12,34)

MBL (%) 4,72 (9,16) 6,89 (8,72) 8,96 (7,80) 20,22 (5,11)

BMC (%) 63,98 (3,75) 64,08 (3,99) 67,28 (1,77) 67,45 (3,41)

VD(%.µm) 1461,93 (673,36) 1727,80 (814,16) 2339,18 (799,61) 6801,59 (3111,57)

38

The multivariate test (Wilks' Lambda) of the three different points, DSL-MSL, DBL-MBL

and DTL-BMC, revealed a strong significant difference (p<0.001) for the different

experimental groups between 6 and 12 weeks. No significant difference was found for the

control group (p=0.402). A graphical demonstration or is shown in figure 6.

A B

C D

Figure 6: Mean changes of mineral loss and lesion depth for the different groups after 6 and 12 weeks. A = Elmex Medical Gel®, B = GC Tooth Mousse™, C= Mi Paste Plus™ D = Control group. Each curve shows the three different points, DSL-MSL, DBL-MBL and DTL-BMC.

39

A Mann-Whitney U test showed a significant decrease (p<0.05) for the volume of

demineralisation for the different experimental groups but again not significant difference for

the control group.

The multivariate test (Wilks' lambda exact test) compared the groups two by two at 6 weeks

and at 12 weeks.

At 6 weeks, only BMC-DTL differed significantly for the mineral content and the lesion

depth for the fluoride group versus the control group (p=0.045) and for CPP-ACPgroup

versus CPP-ACPFgroup (p=0.041) (figure 7).

At 12 weeks, the difference was highly significant (p<0.001) for the comparison between the

three experimental groups and the control group for the different parameters. BMC-DTL was

also statistically significant lower for CPP-ACPF versus the fluoride group (p=0.025) and the

CPP-ACP group (p=0.030) (table 7).

The Friedman test showed no statistically significant difference for the volume of

demineralisation (VD), for the 3 experimental groups between 6 and 12 weeks. When

comparing pairwise the different experimental groups with the control group using the

Wilcoxon matched-pairs signed rank test, the VD was statistically significant lower for each

experimental group versus the control group (p=0.007).

40

Figure 7: Schematic representations of the experimental groups and the control group at 6 weeks (A) and at 12 weeks (B).

Table 7: P-values were calculated using the Wilks' lambda exact test, for 6 weeks and 12 weeks. (* Significant p-values).

Significance level

Time Parameter Elmex® vs.

GC Tooth MousseTM

Elmex® vs.

Mi Paste PlusTM

Elmex® vs.

control group

GC Tooth MousseTM

vs. Mi Paste PlusTM

GC Tooth MousseTM

vs. control group

Mi Paste PlusTM

vs. control group

6w MSL-DSL 0,790 0,547 0,492 0,357 0,894 0,129

MBL-DBL 0,543 0,378 0,098 0,406 0,269 0,132

BMC-DTL 0,249 0,067 0,045* 0,041* 0,096 0,142

12w MSL-DSL 0,446 0,388 < 0.001* 0,108 < 0.001* < 0.001*

MBL-DBL 0,681 0,429 < 0.001* 0,318 < 0.001* < 0.001*

BMC-DTL 0,953 0,025* < 0.001* 0,030* < 0.001* < 0.001*

A B

41

The number of body lesions resembles the mineral uptake and loss patterns of mineral

distribution in the subsurface layer. A variety in number of body lesions (NBL) is presented in

this study (figure 8), NBL=0, lack of body lesions; NBL=1, one body lesion; NBL=2, two or

more body lesions.

Figure 8: Histograms of the number of body lesions at 6 weeks (time 1) and 12 weeks (time 2) in intervention

and control groups.

42

The Pearson Chi-Square test and the Fisher’s Exact test showed no significant difference

between the four groups after 6 weeks, and a highly significant difference after 12 weeks

(p<0.001) (Table 8).

Time Chi-square tests Exact Sig. (2-sided) 1 Pearson Chi-Square 0,70 Fisher ‘s Exact Test 0,55

2 Pearson Chi-Square < 0.001* Fisher ‘s Exact Test < 0.001*

A strong statistically significant difference (p<0.001) was demonstrated by the same tests (the

Pearson Chi-Square and the Fisher‘s Exact Test) for the experimental groups between 6 and

12 weeks. Despite the fact that the p-value for the control group equals 0.026 the results are

still significant (table 9).

Table 9: P-values are calculated using the Pearson Chi-Square test and Fisher‘s Exact Test for the four experimental groups between 6 and 12 weeks.

Paste Chi-square tests Exact Sig. (2-sided) Elmex® Pearson Chi-Square < 0.001 Fisher ‘s Exact Test < 0.001 Gc Tooth MousseTM Pearson Chi-Square < 0.001

Fisher ‘s Exact Test < 0.001 Mi Paste Plus TM Pearson Chi-Square < 0.001 Fisher ‘s Exact Test < 0.001

Control Pearson Chi-Square 0,022

Fisher ‘s Exact Test 0,024

Table 8: P-values are calculated using the Pearson Chi-Square test and Fisher‘s Exact Test between the four

groups, for 6 weeks (time 1) and 12 weeks (time 2) (* Significant p-values).

43

The Intraclass Correlation Coefficient, a descriptive statistic, was used for assessing the

reliability between two measurements on the same white spots but on a different time by the

same examiner. The Intraclass Correlation Coefficient (ICC) varying from 0.671 (substantial

agreement) to 0.918 (almost perfect) showed a strong correlation.

Due to the fact that one examiner did all the measurements exclusively, only the ICC for

single measures is demonstrated in table 10.

Table 10: The Intraclass Correlation Coefficients for the different parameters.

*Fleiss Kappa Coefficient Interpretation

< 0 Poor agreement

0.01 – 0.20 Slight agreement

0.21 – 0.40 Fair agreement

0.41 – 0.60 Moderate agreement

0.61 – 0.80 Substantial agreement

0.81 – 1.00 Almost perfect agreement

Parameters ICC 95% Confidence Interval Fleiss interpretation*

Lower Bound Upper Bound DTL (µm) .761 0,294 0,935 Substantial agreement DBL (µm) .918 0,706 0,979 Almost perfect DSL (µm) .846 0,496 0,959 Almost perfect MSL (%) .665 0,105 0,905 Substantial agreement MBL (%) .657 0,091 0,902 Substantial agreement BMC (%) .833 0,464 0,956 Almost perfect VD (%.µm) .671 0,116 0,907 Substantial agreement NBL .816 0,421 0,951 Almost perfect

44

Discussion

A pH-cycling model mimics the dynamics of mineral loss and gain. The high level of

scientific control, should result in lower intrinsic variability of the in vitro models and so

smaller sample size are required (78). However, in vitro experiments cannot fully simulate the

physiologic conditions of the mouth and statistically significant findings do not always equate

to clinically significant findings (96).

Despite many investigations, the efficacy of various remineralisation methods is still difficult

to define. Comparison of the results of these studies is not always possible due to the lack of

homogeneity in the research protocols. In the literature, different methodologies were

described to create white spot lesions. Some authors used polyacrylic acid (Carbopol), others

used 37% phosphoric acid for 30 seconds or a demineralisation solution during 14 days (15,

25, 96, 97). In our study, we used a demineralisation solution by ten Cate and Duijsters for

96h to produce artificial carious lesions (depth of 150-200 µm) (94). A similar method is

used by several other authors (78, 98-100).

Furthermore, there are several ways to quantify remineralisation of WSL. Bailey et al. used a

visual inspection to rate WSL, while others used more quantitative evaluation. Jayarajan et al.

observed the surface morphology (SEM) in combination with a polarized light microscope

(DIAGNOdent®) (101). Pai et al. chose the QLF computer imaging and SEM to make their

measurements (89). The QLF measuring method is used in many studies (31, 102, 103). This

technique has the ability to quantify enamel lesions in an objective way, but it has been stated

that QLF is not suitable for inter-patient comparison (104). In our study, transverse

microradiography (TMR) was used to quantify the mineral content. TMR is considered as the

‘‘gold standard’’ for the determination of mineral loss or gain in experimentally induced

initial carious lesions. The TMR provides a very precise quantitative measurement of the

mineral content (88, 105).

45

In the literature, different experimental periods for the intervention were found. The total

experimental period of this study was based on the instructions of the manufacturer of

toothpastes: a clinical advice to the patient to use the pastes for at least 8 to 12 weeks before

expecting a visually clinical result. A similar protocol was used in other studies (103, 106,

107). In this study we evaluated a first group after 6 weeks to see the results on a short time

usage of the experimental toothpastes and a second group after 12 weeks, which is the

recommended therapy duration. The lesions depth and the mineral content decreased

significantly between 6 and 12 weeks. This could be due to the distribution of the mineral.

The TMR could not show the organic matrix, so further study is needed to explain this

phenomena.

Care has to be taken to standardize the participant’s background. In many studies, variables in

the model (such as hard tissue substrate, diet, biofilm formation, and intra-oral site) are

controlled and standardized to improve the power of the study. In our study we wanted to

exclude this factor by distributing four premolars from the same patient into the four study

groups. An enamel surface layer was removed from each premolar (buccal and palatal) to

avoid a potentially fluoride rich layer and to reduce the individual enamel differences. Despite

these measures, a large diversity of results in each study group was observed.

Clinically lingual surfaces are less prone to caries in comparison to the buccal surfaces (30).

This may be explained by differences in surface morphology, plaque retention, salivary flow

and mechanical cleaning of surfaces by the tongue but also by the shape of the brackets. WSL

development on buccal surfaces is 4.8 times higher than the number of new WSL developing

or progressing on bracketed lingual surfaces. Van der Veen et al. concluded in an in vivo

study that lingual brackets are in favour of buccal brackets, when considering caries outcome

on the smooth surfaces (31). In this study, the buccal of the palatal/lingual side were

separated. However, Student’s t-tests for different parameters showed no statistically

significant differences between both sides. Due to these results no distinction was made

between the palatal and buccal sides.

46

In this in vitro study, the lesion depth but also the mineral content of the WSL decreased

significantly after 12 weeks for the experimental toothpastes (the fluoride, the CPP-ACP and

the CPP-ACPF group) but there were no significant differences between the three different

experimental groups in regression of the WSL.

Fluoride therapy is the most frequently used for caries prevention and remineralisation

methods (38). The repeated application of highly concentrated fluoride gels, like Elmex

Medical gel 12.500ppm, prevent enamel loss by demineralisation (108). However, from the

literature we know that high doses of fluoride are less appropriate for remineralisation or

treatment of white spots. A high concentration of fluoride indeed blocks the surface layer

pores due to crystal growth and prevents further remineralisation of the lesion once the

surface layer is full remineralized (109). Therefore, Øgaard et al. warned against treating

WSL with concentrated fluoride agents (16, 101).

Professor Reynolds (University of Melbourne, Australia) could isolate calcium and phosphate

transporter (CPP) out of cow milk which can capture calcium and phosphate in a soluble

estate. This complex was called CPP-ACP (“casein phosphopeptide” and “amorphous calcium

phosphate”) (76). CPP-ACP was claimed to have the ability to reduce demineralisation and

promote remineralisation by releasing calcium and phosphate ions.

Demineralisation occurs when the pH in the oral environment falls below 5.5, allowing

calcium and phosphate ions to diffuse from the enamel surface. The localized CPP-ACP

nanocomplexes act to buffer free calcium and phosphate ions in the plaque fluid, thereby

limiting enamel demineralisation and enhancing remineralisation (53, 76). Calcium and

phosphate needs to penetrate the surface layer in order to deposit minerals within the body of

the lesion (101).

47

Contradictory results have been reported in the literature about the remineralisation effect of

CPP-ACP on WSL. Recent clinical investigations showed less promising results. Ogata et al.

reported an insufficient effect of remineralisation on enamel subsurface lesions with the use of

CPP-ACP alone. There was no statistically significant difference between the CPP-ACP and

the control with respect to mineral loss; the addition of low levels of fluoride was needed to

observe lesion regression. In contrast, Wu et al. concluded that ACP with or without fluoride

reduced WSL size. After 3 months, Wu’s study demonstrated that fluoride toothpaste alone

was equally effective as CPP-ACP treatment at lesion remineralisation. In a clinical trial by

Bailey et al., the use of CPP-ACP cream enhanced the regression of WSL compared with

placebo (68, 106). The clinical benefits of CPP-ACP paste for the treatment of WSL are not

yet substantiated by credible scientific evidence. In theory, CPP-ACP would seem like an

ideal remineralizing agent for early caries, as discussed in a review by Zero in 2009 (110).

Since 2006 there exists MI Paste Plus which combines CPP-ACP and fluoride. CPP-ACPF,

containing 900 ppm of NaF, increases the salivary concentrations of calcium, inorganic

phosphate and fluoride. CPP prevents spontaneous precipitation and allowed penetration of

the ions deep into the subsurface lesion. This resulted in an increased remineralisation

throughout the body of the lesion whereas the fluoride-alone products tended to remineralize

predominantly in the surface layer (111).

Jayarajan et al. studied the remineralisation effect of CPP-ACP and CPP-ACPF compared to

the remineralisation potential of saliva. Even though saliva has some remineralisation

potential it cannot increase the levels of calcium and phosphate release. CPP-ACPF, an

excellent local slow-delivery system, showed a much higher amount of remineralisation of

WSL. CPP-ACPF is an. The three different groups suggest remineralisation in the same order,

with the CPP-ACPF showing the greatest amount of mineral deposits. CPP-ACPF can

enhance the level of remineralisation of enamel subsurface lesions when the product is used in

situ. The fluoride ions are adsorbed onto the surface of enamel crystals, inhibiting dissolution

and increasing remineralisation. The softened surface lesions remineralize faster and more

48

completely than subsurface lesions. Jayarajan et al. advised to include CPP-ACPF in the

routine hygiene and maintenance instructions for reversing or arresting white spot lesions in

orthodontic patients (101). The Beerens’ group compared, during 3 months CPP-ACPF, with

a control paste in a clinical study. They found no advantage for use of the fluoridated CPP-

ACP paste over regular oral hygiene in WSL regression as measured by QLF (103). Similarly,

Bröchner et al. in a prospective clinical trial using non-fluoridated CPP-ACP paste, found

WSL regression to be comparable with traditional toothpaste after a 4- week treatment period

(102, 106). The study of Shen et al. showed additional benefit in remineralisation of enamel

subsurface lesions with the CPP-ACPF (111). A significant enhancement of fluoride ion’s

ability to remineralize enamel subsurface lesions in situ by CPP–ACP confirmed previous

results showing the same effect as Reynolds' studies where the CPP–ACP was used

immediately after a fluoride-containing toothpaste (69). The conflicting results of these

clinical trials may be related to the method of assessment of WSL regression but also to the

different time periods of application.

49

Critical evaluation

In our study we used conventional toothpaste (1450ppm) in combination with three

experimental products (fluoride, CPP-ACP and CPP-ACPF). The control group had no

contact with toothpaste or experimental products. To investigate the difference in

remineralisation capacity of the tooth paste of experimental products, further research is in

progress.

The formula for 2 means from Pocock was used to calculate the sample size.

n= ����� x = ���

�µ�µ���

n = sample size

�����= is a function of power and significance level

�= the standard deviation

µ1 = the anticipated mean response for the standard or control treatment.

µ2 = the anticipated mean response for the alternative treatment and hence the minimum

clinically important difference (µ2 – µ1) between treatment arms that we would like to detect.

With 27 subjects per group, the trial has 80% power to detect a significant difference of 32,7%

in lesion depth between the 6 and 12 weeks at the 5% significance level. In this study, we had

only 24 subjects in each group.

50

Conclusions

Based on the results of this study, there were no significant differences between the three

different experimental groups in regression of the WSL.

Only after 12 weeks, there was a statistically significant improvement of lesion depths for the

three experimental toothpastes compared with the control group. The lesion depth but also the

mineral content of the WSL decreased significantly after 12 weeks for the experimental

toothpastes. Tough, there is a strong significant difference between the experimental groups

and the control group.

The application of a CPP-ACP or CPP-ACPF paste provides compared with a highly

concentrated amine fluoride, no additional benefit in the regression of WSL.

51

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Annexe

Prof. Dr. G. De Pauw, Afdelingshoofd Dienst Orthodontie De Pintelaan 185 9000 GENT, BELGIË

Informatie-toestemmingsformulier (voor

Geachte heer, mevrouw Beste patiënt, Eerder heeft uw tandarts/kaakchirurg u voorgesteld bij u meerdere tanden om bepaalde redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. Invan mijn master-na-master opleiding orthodontie ben ik een experiment gestart, waarbij ik tanden verzamel, met de bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u willen vragen of u bereid bent uw getrokken tanden af te staan in hetwetenschappelijk onderzoek. Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuring dient niet beschouwd te worden als aansporing om deel te nemen aan deze studie. Doel en verloop van het onderzoekGraag zou ik 3 verschillende tandpasta’s vergelijken (Elmex Medical GelMousseTM, MI Paste PlusTM) met een controlegroep (gewone tandpasta).Het wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de tand) ga evalueren na ze zelf te hebben gecreëerd. White spots komen in veel gevallen voor na een orthodontische behandeling. Voor mijn studie heb ik 4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden er een 40-tal patiënten deelnemen (160 tanden). Praktische gegevens:

- Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. - Er zullen voor u geen extra kosten zijn. Alle onkosten i.v.m. de studie worden door

het ziekenhuis gedragen.- Het onderzoek is volledig anoniem. De gegevens worden strikt v

zonder vermelding van uw naam bewaard. De privacy zal dus volledig worden gerespecteerd.

- Als u toch liever heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uwstaan zal zeker geen invloed hebben op een eventuele verdere behandeling.

Mag ik u vragen om op de ommezijde te tekenen voor akkoord, indien u bereid bent uw tanden af te staan in het kader van dit experiment? Vriendelijk dank. Charlotte Van Elst Specialist orthodontie in opleidingTel: 093326893

toestemmingsformulier (voor patiënten tussen de 12 en 18 jaar en ouders)

Eerder heeft uw tandarts/kaakchirurg u voorgesteld bij u meerdere tanden om bepaalde redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. In

master opleiding orthodontie ben ik een experiment gestart, waarbij ik tanden verzamel, met de bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u willen vragen of u bereid bent uw getrokken tanden af te staan in het

Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuring dient niet beschouwd te worden als aansporing om deel te nemen aan deze studie.

Doel en verloop van het onderzoek verschillende tandpasta’s vergelijken (Elmex Medical Gel®, Gc Tooth

) met een controlegroep (gewone tandpasta). Het wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de

f te hebben gecreëerd. White spots komen in veel gevallen voor na een orthodontische behandeling. Voor mijn studie heb ik 4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden

tal patiënten deelnemen (160 tanden).

Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. Er zullen voor u geen extra kosten zijn. Alle onkosten i.v.m. de studie worden door het ziekenhuis gedragen. Het onderzoek is volledig anoniem. De gegevens worden strikt vzonder vermelding van uw naam bewaard. De privacy zal dus volledig worden

Als u toch liever heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uwstaan zal zeker geen invloed hebben op een eventuele verdere behandeling.

Mag ik u vragen om op de ommezijde te tekenen voor akkoord, indien u bereid bent uw tanden af te staan in het kader van dit experiment?

Specialist orthodontie in opleiding

63

patiënten tussen de 12 en 18 jaar en ouders)

Eerder heeft uw tandarts/kaakchirurg u voorgesteld bij u meerdere tanden om bepaalde redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. In functie

master opleiding orthodontie ben ik een experiment gestart, waarbij ik tanden verzamel, met de bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u willen vragen of u bereid bent uw getrokken tanden af te staan in het kader van dit

Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuring dient niet beschouwd te worden als aansporing om deel te nemen aan deze studie.

®, Gc Tooth

Het wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de f te hebben gecreëerd. White spots komen in veel gevallen voor

Voor mijn studie heb ik 4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden

Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. Er zullen voor u geen extra kosten zijn. Alle onkosten i.v.m. de studie worden door

Het onderzoek is volledig anoniem. De gegevens worden strikt vertrouwelijk en zonder vermelding van uw naam bewaard. De privacy zal dus volledig worden

Als u toch liever heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uw tanden af te staan zal zeker geen invloed hebben op een eventuele verdere behandeling.

Mag ik u vragen om op de ommezijde te tekenen voor akkoord, indien u bereid bent uw

Wij, ondergetekende, ………………………………………………………………, ouders van …………………………………………………………geven hierbij de toestemming om zijn/haar tanden, die om tandheelkundige/orthodontische redenen werden verwijderd, af te staan in het kader van een onderzoek. Voor de vrijwilliger: Datum Handtekening Voor de ouders: Datum

Wij, ondergetekende, ………………………………………………………………, ouders van ………………………………………………………… geven hierbij de toestemming om zijn/haar tanden, die om tandheelkundige/orthodontische

rd, af te staan in het kader van een onderzoek.

Datum Handtekening

Handtekening

64

Wij, ondergetekende, ………………………………………………………………, ouders van

geven hierbij de toestemming om zijn/haar tanden, die om tandheelkundige/orthodontische

Datum Handtekening

Handtekening

Prof. Dr. G. De Pauw Afdelingshoofd Dienst Orthodontie De Pintelaan 185 9000 GENT BELGIË Informatie-toestemmingsformulier (voor patiënten ouder dan 18 jaar ) Geachte heer, mevrouw Beste patiënt, Eerder heeft uw tandarts/kaakchirurg u redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. In functie van mijn master-na-master opleiding orthodontie ben ik een experiment gestart, waarbij ik tanden verzamel, met de bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u willen vragen of u bereid bent uw getrokken tanden af te staan in het kader van dit wetenschappelijk onderzoek. Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuriniet beschouwd te worden als aansporing om deel te nemen aan deze studie. Doel en verloop van het onderzoekGraag zou ik 3 verschillende tandpasta’s vergelijken (Elmex Medical GelMousseTM, MI Paste PlusTM) met een controlegroep (gewoHet wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de tand) ga evalueren na ze zelf te hebben gecreëerd. White spots komen in veel gevallen voor na een orthodontische behandeling. Voor mijn studie heb ik 4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden er een 40-tal patiënten deelnemen (160 tanden). Praktische gegevens:

- Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. - Er zullen voor u geen extra kosten zijn.

het ziekenhuis gedragen.- Het onderzoek is volledig anoniem. De gegevens worden strikt vertrouwelijk en

zonder vermelding van uw naam bewaard. De privacy zal dus volledig worden gerespecteerd.

- Als u toch liever heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uw tanden af te staan zal zeker geen invloed hebben op een eventuele verdere behandeling.

Mag ik u vragen om op de ommezijde tanden af te staan in het kader van dit experiment? Vriendelijk dank. Charlotte Van Elst Specialist orthodontie in opleidingTel: 093326893 Faculteit Geneeskunde en Gezondheidswetenschappen

De Pintelaan 185 P8, B-9000 Gent

toestemmingsformulier (voor patiënten ouder dan 18 jaar )

Eerder heeft uw tandarts/kaakchirurg u voorgesteld bij u meerdere tanden om bepaalde redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. In functie

master opleiding orthodontie ben ik een experiment gestart, waarbij ik bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u

willen vragen of u bereid bent uw getrokken tanden af te staan in het kader van dit

Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuriniet beschouwd te worden als aansporing om deel te nemen aan deze studie.

Doel en verloop van het onderzoek Graag zou ik 3 verschillende tandpasta’s vergelijken (Elmex Medical Gel®

) met een controlegroep (gewone tandpasta). Het wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de tand) ga evalueren na ze zelf te hebben gecreëerd. White spots komen in veel gevallen voor na een orthodontische behandeling.

4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden tal patiënten deelnemen (160 tanden).

Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. Er zullen voor u geen extra kosten zijn. Alle onkosten i.v.m. de studie worden door het ziekenhuis gedragen. Het onderzoek is volledig anoniem. De gegevens worden strikt vertrouwelijk en zonder vermelding van uw naam bewaard. De privacy zal dus volledig worden

heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uw tanden af te staan zal zeker geen invloed hebben op een eventuele verdere behandeling.

Mag ik u vragen om op de ommezijde te tekenen voor akkoord, indien u bereid bent uw tanden af te staan in het kader van dit experiment?

Specialist orthodontie in opleiding

Faculteit Geneeskunde en Gezondheidswetenschappen – Vakgroep Tandheelkunde 9000 Gent

65

voorgesteld bij u meerdere tanden om bepaalde redenen (bijvoorbeeld omwille van te weinig plaats in de kaak) te laten verwijderen. In functie

master opleiding orthodontie ben ik een experiment gestart, waarbij ik bedoeling verschillende tandpasta’s te vergelijken. Nu zou ik u

willen vragen of u bereid bent uw getrokken tanden af te staan in het kader van dit

Dit onderzoek is goedgekeurd door het ethisch comité UZGent. Maar deze goedkeuring dient niet beschouwd te worden als aansporing om deel te nemen aan deze studie.

®, Gc Tooth

Het wordt een studie waarin ik de grootte en diepte van white spots (= ontkalkingen op de tand) ga evalueren na ze zelf te hebben gecreëerd. White spots komen in veel gevallen voor

4 intacte voorkiezen van dezelfde patiënt nodig. En in totaal zouden

Deelname aan deze studie brengt geen therapeutisch voordeel met zich mee. Alle onkosten i.v.m. de studie worden door

Het onderzoek is volledig anoniem. De gegevens worden strikt vertrouwelijk en zonder vermelding van uw naam bewaard. De privacy zal dus volledig worden

heeft dat uw tanden niet gebruikt worden voor dit experiment, dan kan u zonder enig probleem weigeren. Een eventuele weigering om uw tanden af te staan zal zeker geen invloed hebben op een eventuele verdere behandeling.

te tekenen voor akkoord, indien u bereid bent uw

Ik, ondergetekende, ………………………………………………………………, geefhierbij de toestemming om mijn werden verwijderd, af te staan in het kader van een onderzoek. Voor de vrijwilliger: Datum Handtekening

Ik, ondergetekende, ………………………………………………………………, geefmijn tanden, die om tandheelkundige/orthodontische redenen

werden verwijderd, af te staan in het kader van een onderzoek.

Datum Handtekening

66

Ik, ondergetekende, ………………………………………………………………, geef tanden, die om tandheelkundige/orthodontische redenen

Datum Handtekening