University of Groningen

Bacterial adhesion forces and biofilm prevention on orthodontic materialsMei, Li

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Chapter 1

General introductionand aim of this thesis

Biofilms in orthodontics: formation,consequences, treatment and prevention

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Introduction

Orthodontic treatment is becoming increasingly popular. Whereas two decades

ago, it was exclusively for juveniles, adult orthodontic treatment is now very

common. Together there are more than four million juvenile and one million adult

patients in North America alone reported by the American Association of

Orthodontists. However, the downside of orthodontic treatment has not been much

addressed. The region of the tooth surface around the brackets is prone to

adhesion of oral bacteria and subsequent biofilm formation. Oral biofilm, or “dental

plaque”, is difficult to remove and regular brushing is often insufficient to remove

plaque from retention sites, such as the vulnerable bracket-adhesive-enamel

junction and the sensitive region between brackets and the gingival (Fig. 1).

Figure 1. Orthodontic biofilm, visualized by staining with GUM red-cote, before (lowerdentition) and after (upper dentition) removal of brackets. Stained areas can be clearly seenon the tooth surfaces around the area where the brackets have been bonded or around thebrackets still present.

Biofilms on dental hard and soft tissues, as well as on different biomaterials

employed for restoration of function in the oral cavity, are the main cause of dental

disease1,2. In orthodontics, the great variety of biomaterials used provides

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numerous additional surfaces to which microorganisms can adhere and form a

bioflm (Fig. 2). Moreover, orthodontic appliances severely hamper the efficacy of

toothbrushing3, reduce the self-clearance by saliva4, change the composition of the

oral flora5, increase the amount of oral biofilm formed6 and the colonization of oral

surfaces by cariogenic7 and periodontopathogenic bacteria8. These factors strongly

complicate orthodontic treatment, and illustrate that the need for oral biofilm control

is even greater during orthodontic treatment than usual4. Despite current

preventive measures to control biofilm formation during orthodontic treatment, the

prevalence of biofilm-related problems has remained high.

Figure 2. Components of orthodontic appliances influencing biofilm formation.(A): adhesive; (B): bracket; (C): ligating with elastomeric ring; (D): ligating with steel ligature;(E): self-ligating bracket with the clip open; (F): self-ligating bracket with a closed clip; (G):arch wire; and (H): bonded retainer.

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Biofilm formation on orthodontic materials

Composition of orthodontic biofilms

Oral biofilms, including orthodontic biofilms (oral biofilms formed on orthodontic

biomaterials during active orthodontic treatment or retention phase), are diverse

communities of microorganisms on dental hard and soft tissues and dental

biomaterials. These biofilms are embedded in an extracellular matrix of polymers of

host and microbial origin, possessing complex spatial, heterogeneous and dynamic

structures9. Oral biofilms in general comprise about 80% water and 20% of solid-

phase components including proteins, carbohydrates, fat, and inorganic

components. The bacterial diversity in the oral cavity is estimated to include at

least 800 different species, consisting of a wide variety of Gram-positive and Gram-

negative bacteria, such as facultative anaerobic and obligatory anaerobic species.

The number of detectable bacterial species is expected to rise into the thousands

with advances in modern detection techniques1. The extracellular matrix of biofilms

is a self-produced, gel-like structure mainly made of polysaccharides, proteins,

nucleic acids and lipids10.

The composition of orthodontic biofilms varies during the course of treatment.

Placement of an orthodontic appliance increases not only the amount of biofilm,

but also the prevalence of cariogenic bacteria such as mutans streptococci and

lactobacilli7 and periodontopathogenic bacteria such as Porphyromonas gingivalis,

Prevotella intermedia, Prevotella nigrescens, Tannerella forsythia, and

Fusobacterium species8. The elevated prevalence of Streptococcus mutans during

active treatment, decreases quickly to normal levels within a few months after

removal of the orthodontic appliances11. In regions with dental crowding, more

biofilm accumulates with higher levels of periodontopathogens, such as

Fusobacterium species, Capnocytophaga species, Campylobacter rectus, and

Peptostreptococcus micros12.

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Mechanism of biofilm formation

Oral biofilms accumulate through sequential and ordered colonization of oral

surfaces by the different strains and species present in the oral cavity1,2,9. Distinct

phases can be discriminated in the formation of oral biofilms (Fig. 3):

Figure 3. Steps of biofilm formation: 1. Conditioning film formation; 2. Reversible adhesion;3. Transition to irreversible adhesion; 4. Co-adhesion of microorganisms to alreadyadhering; 5. Growth into a mature biofilm. (Illustration adapted from Stoodley et al13).

1. Conditioning film formation: a salivary conditioning film, known in dentistry as

the “acquired pellicle”, forms immediately after cleaning or introducing new

surfaces into the oral cavity.

2. Reversible adhesion: an interplay of attractive Lifshitz-Van der Waals forces

and electrostatic repulsion between bacteria and substratum surfaces yields an

initially reversible adhesion. Since conditioning films form more rapidly than

bacteria can be transported to the surface, bacteria mostly adhere to a salivary

conditioning film and seldom to a bare substratum surface9.

3. Transition to irreversible adhesion: when close contact between an adhering

bacterium and a substratum surface is established, adhesion becomes irreversible

through the involvement of acid-base interactions, forming the basis for the strong,

stereo-chemical interactions between adhesins on the bacterial surface and

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receptors in the salivary conditioning film (M proteins, antigen I/II, Ig-binding

proteins, and Fn-binding proteins)10.

4. Co-adhesion of microorganisms to already adhering, early colonizers, which

is also mediated by long-range, attractive Lifshitz-Van der Waals forces and highly

specific stereo-chemical interactions at close approach2.

5. Growth into a biofilm: adhering bacteria grow into a biofilm and produce

extracellular polymeric substances (EPS) by metabolism of e.g. sucrose, yielding

the complex biofilm matrix. EPS matrices are three-dimensional, spatially and

functionally organized structures, protecting the microorganisms against outside

attacks, such as from antimicrobials1.

Physico-chemical properties, such as hydrophobicity, surface charge and

surface free energy are generally believed to play an important role, particularly in

the initial stages of bacterial adhesion to surfaces, although no physico-chemical

explanation of microbial adhesion to surfaces has been forwarded that goes

beyond validity for selected collections of strains and surfaces14. Once biofilm

formation has passed the stage of initial adhesion, environmental factors like pH

and nutrient availability often play a more decisive role in the final amount of biofilm

formed than differences in initial adhesion that can be achieved by altering the

biomaterials surface properties, which yields limited reductions in microbial

adhesion numbers of utmost a factor of ten.

Under clinical conditions, roughness overrides all beneficial effects of

biomaterials coatings, especially in supra-gingival regions15. Roughness is of less

importance in relatively stagnant regions, such as in sub-gingival pockets16. Also

the removal of biofilm from rougher surfaces is more difficult than from smooth

surfaces. Consequently, in vivo the natural oral cleansing forces are less effective

in removing oral biofilm. Moreover, rough surfaces offer protection against oral

shear forces, but also provide a protective shelter against environmental attacks

such as by antibacterial oral health care components14.

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Factors influencing orthodontic biofilm formation

Orthodontics adhesives, fixed appliances including brackets, ligating devices and

arch wires, or removable acrylic plates are important factors influencing orthodontic

biofilm formation (see also Fig. 2).

Adhesives. Composite resins for orthodontic bonding are in direct contact with

the vulnerable enamel surface and their properties with respect to bacterial

adhesion may be more important than of other orthodontic materials. In general,

excessive composite resin at the bracket-enamel-adhesive junction is prone to

bacterial adhesion, especially since polymerization shrinkage may yield a gap with

a width of up to 10 m at the adhesive-enamel interface where bacteria find

themselves protected against oral cleansing forces and antibacterial components

of toothpastes and mouthrinses17. Moreover, bacterial adhesion forces to

composite resin, which often have a rougher surface than enamel or brackets,

were stronger than to brackets or saliva-coated enamel, and depended on the

bacterial strain involved18. Different filler-volume fractions of silicon dioxide in the

composite did not have an effect on the adhesion of S. mutans19.

Brackets. Some in vitro studies have demonstrated significant differences in

bacterial adhesion and biofilm formation between brackets made from different

materials. Initial adhesion of S. mutans was higher on plastic brackets than on

stainless steel and ceramic brackets, on which adhesion was lowest20. In contrast,

another in vitro study showed no significant differences of S. mutans adhesion to

stainless steel, ceramic or plastic brackets21. However, the difference between

metallic and ceramic brackets in the adhesion of S. mutans could not be confirmed

in vivo, neither was there a difference in adhesion of Lactobacillus species22. In

vivo, maxillary brackets harvested more S. mutans and S. sobrinus than

mandibular brackets23, while labial brackets harvested more biofilm than lingual

brackets24. Also a split-mouth study indicated more anaerobic and aerobic

organisms in self-ligating than in conventional bracket sites25. Combined with the

observation that the occurrence of white spot enamel lesions and gingival

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inflammation was similar in patients with self-ligating and conventional brackets26,

this may indicate that biofilm formation on the brackets themselves is less harmful

than when formed at the bracket-enamel-adhesive junction.

Ligating Devices. Steel ligature wires and elastomeric rings are the most

frequently used devices for ligating orthodontic wires into brackets before the

introduction of self-ligating brackets. Between elastomeric rings and steel ligature

wires, no difference was found regarding biofilm weight or biofilm-related clinical

indices. However, elastomeric rings are related to a higher possibility of enamel

demineralization, and are not recommended in patients with poor oral hygiene27.

Self-ligating devices, made of stainless steel or titanium, accumulate less biofilm

than elastomeric rings28.

Arch Wires. Complicated appliance designs with loops and auxiliary arch wires

create areas that are difficult to clean and may therefore enhance biofilm

formation4. The inter-bracket part of arch wires is relatively distant from the enamel

surface and gingival tissues, and biofilms formed here may also be considered

relatively harmless to the enamel and gingival tissues. Moreover, biofilms on these

parts are easier to remove by brushing, compared with those formed on brackets,

adhesives, and ligating devices. Biofilms on the arch wires ligated in the bracket

slot may however, compromise the efficiency of the sliding mechanics29.

Retainers. Removable orthodontic retainers may attract oral biofilm and

present new retention sites, similar to removable acrylic plates, favoring bacterial

adhesion and growth30. Biofilms have been observed on acrylic base-plates after

one week of wear31, harvesting different strains of streptococci and candida30.

Fixed retainers are in direct contact with the enamel surface and cannot be

removed for extensive cleaning like removable ones. Therefore they are generally

considered to yield increased biofilm formation with negative consequences with

respect to gingival inflammation32. No differences were found in the clinical indices

between fixed retainers made of multistrand wire or single-strand wire, but more

biofilm was isolated from the multistrand wire33. Retainers made of multistrands

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have niches where biofilms can be easily formed to provide shelter to adhering

bacteria against environmental attacks, in contrast to retainers made of a single

strand (Fig. 4).

Figure 4. Scanning electron micrograph of a multistrand wire used for fixed retainers.Biofilm formation in the niches between the wires is clearly visible.

Other Factors. Banding induced more orthodontic biofilm formation, gingival

inflammation and white spot lesions than bonding34. Most biofilm was located at the

gingival margin, with more band surface being covered by biofilm at the supra-

gingival area than at the sub-gingival one35. Coil springs had the lowest affinity for

S. sobrinus, followed by intra-oral elastics and elastic modules36. External factors

such as smoking, have been shown to increase bacterial adhesion and biofilm

formation by S. mutans and Candida albicans on orthodontic materials37.

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Consequences of orthodontic biofilms

Enamel demineralization

Enamel demineralization surrounding brackets is the most common side-effect of

orthodontic treatment, affecting around 50% of all patients4. The severity of

demineralization can range from white spot lesions to cavitations upon bracket

removal, which can occur on both vestibular and lingual surfaces, with the most

affected sites being the bracket-adhesive-enamel junction and the most affected

teeth being the first molars, upper lateral incisors, and lower canines4,17,18. White

spot lesions (Fig. 5) can develop rapidly in susceptible individuals within the first

month of treatment, and can remain visible many years after debonding, or in

severe cases appear as a permanent enamel scar4.

Figure 5. White spot lesions, cavities and gingival inflammation caused by orthodonticbiofilms, after removal of fixed orthodontic appliances.

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Soft tissue inflammation

Almost all orthodontic patients experience some degree of soft tissue inflammation

(see also Fig. 5). Gingivitis during orthodontic treatment is often temporary and

rarely progresses to periodontitis, however, biofilms on the retention sites increase

the possibility of the development of periodontitis.

Biofilm formation on temporary anchorage devices (Fig. 6), such as mini-

screws, micro-implants, or mini-plates, especially on the trans-gingival parts of the

devices, can cause inflammation of the surrounding soft tissues similar to peri-

implantitis. These inflammations are associated with a 30% increase in the failure

rate of temporary anchorage devices38. In addition, biofilms on the head of a

temporary anchorage device may infect the adjacent contacting buccal mucosa

resulting in aphthous ulceration which is not a direct risk factor for the stability of

temporary anchorage devices, but might forewarn of a greater soft tissue

inflammation39.

Figure 6. Biofilms on and around temporary anchorage devices causing soft tissueinflammation. (A): Gingival inflammation around a temporary anchorage device (see arrow).(B) and (C): Scanning electron micrographs of biofilm formed on a temporary anchoragedevice at low (B) and high magnification (C).

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Other consequences of orthodontic biofilms

Bacteremia related to orthodontics, caused by trauma during the placement or

removal of fixed appliances, is usually transient and occurs with an incidence of up

to 10% during fixed orthodontic treatment40 and about 30% at removal of bonded

maxillary expansion appliances41.

The orthodontic biofilms may also affect the appliance itself and cause pitting

and crevice corrosion of metallic biomaterials42, or affect the mechanical properties,

surface roughness or topographies of composite adhesives43. The increase in

roughness of the appliance materials due to biofilm is especially troublesome,

since a rougher surfaces promote biofilm formation14, providing a protective niche

against environmental challenges. Hence a vicious cycle develops in which biofilm

formation amplifies itself.

Treatment and prevention of orthodontic biofilms

Treatment of enamel demineralization

White Spot Lesions. Enamel remineralization of white spot lesions can be achieved

passively by human saliva or actively by fluoride or calcium-phosphate-based

remineralization delivery systems44. Whether complete remineralization occurs or

not is related to the type or severity of the white spot lesion4. Fast developing

lesions (soft lesions), most often enamel surface defects, may almost completely

remineralize within a few weeks after the removal of cariogenic challenges. Lesions

that developed gradually during orthodontic treatment usually remineralize

extremely slow. It is therefore recommended that orthodontists wait for at least 2

months to allow a lesion to remineralize spontaneously by saliva before taking

other measures. When spontaneous remineralization by saliva is insufficient,

topical fluoride agents as e.g. in toothpastes, mouthrinses or gels, or calcium-

phosphate-based remineralization delivery systems, e.g. casein phosphopeptide-

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stabilized amorphous calcium phosphate complexes or casein phosphopeptide-

stabilized amorphous calcium fluoride phosphate complexes44, may be applied to

accelerate remineralization. Even after active remineralization procedures, severe

lesions may persist.

Micro-abrasion. An effective treatment approach for the cosmetic improvement

of persistent or stabilized lesions is micro-abrasion45. Similar as remineralization,

minor micro-abrasion may also take place spontaneously leading to a gradual

regression of the white spot lesion. Although micro-abrasion may remove a very

thin layer of surface enamel, it is still appropriate to employ this technique prior to

initiating more aggressive procedures.

Cavitation. More extensive lesions that can not be restored by remineralization

or micro-abrasion may be referred to cosmetic dentistry for resin bonding, Class V

restorations, labial veneers, or crowns.

Treatment of soft tissue inflammation

The symptomatic treatments for gingivitis or peri-implantitis in orthodontics include

local cleaning, application of antimicrobial containing products, such as

chlorhexidine, cetylpyridinium chloride or triclosan preferably followed by brushing

with a fluoridated toothpaste39. Dental professionals remove biofilms and calculus

using root debridement, sometimes coupled with the administration of adjunctive

antimicrobial drug therapy46. If gingivitis develops into periodontitis, which happens

in rare occasions, the treatment should be done by a periodontist.

Prevention of orthodontic biofilms

Mechanical Removal. Effective manual or powered brushing and the use of

interdental brushes is still by far the most important measure for oral hygiene

control in orthodontic patients3,47. Manual toothbrushes with a special head design

for orthodontics, such as staged, v-shaped, or triple-headed, are more efficient

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than brushes with a conventional planar bristle field48. Powered toothbrushes for

removing orthodontic biofilms are difficult to compare because of the diversity of

frequencies or types of vibration, areas or types of bristle, and criteria or methods

for assessment3,49. The auxiliary interdental brush is helpful in removing biofilm

formation behind the wire during orthodontic treatment47. Despite the fact that new

designs of general toothbrushes came on the market, longer brushing time and

proper brushing techniques are still necessary for good oral hygiene in orthodontic

patients.

Chemical Biofilm Control. A variety of chemical biofilm control measures

including incorporation of antimicrobials in toothpastes, mouthrinses, varnishes and

adhesives are currently used by the dental profession, including orthodontists (see

Table 1). Chlorhexidine however, still remains the most effective antimicrobial in

reducing biofilm-induced iatrogenic side effects in orthodontic patients50,51 and S.

mutans levels52. Unfortunately, long-term use of chlorhexidine is known to stain

teeth and tongue and affect taste sensation. The benefits of fluoride containing

toothpastes and mouthrinses in preventing caries have been well established53;

and besides aiding enamel remineralization, fluoride acts as a buffer to neutralize

acids produced by bacteria and suppresses their growth14. Stannous fluoride

provides dual benefits with respect to caries and biofilm prevention by stannous

ions54. The combination of an aminefluoride/stannous fluoride containing

toothpaste or mouthrinse showed greater inhibition of biofilms, less white spot

lesions and gingivitis during orthodontic treatment than sodium fluoride containing

products55. Laser irradiation in addition to fluoride treatment has been suggested to

prevent the formation of white spot lesions both in vitro and in vivo56,57.

Modification of Orthodontic Materials. Modification of orthodontic materials is

either aimed at reducing the consequences of orthodontic biofilms or at preventing

biofilm formation (see Table 1), and includes incorporation of chemicals in the

adhesive or coating of bracket and wire materials.

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Table 1. Antimicrobial products and their active ingredients, as used by profession inorthodontics and antimicrobial products suggested by current research papers but not yetused by the profession.

Products Active ingredient Used by profession Reference

Fluoride Yes 53,55,58-60

Stannous fluoride Yes 55Toothpaste

Triclosan Yes 61,62

Fluoride Yes 55

Chlorhexidine Yes 50,52

Chlorhexidine digluconate Yes 63

Cetylpyridinium chloride Yes 64

Octenidine dihydrochloride Yes 65

Listerine Yes 66

Mouthrinse

Amine-fluoride-triclosan No 63

Fluoride Yes 67

VarnishChlorhexidine-thymol Yes 68

Fluoride Yes 69-74

Chlorhexidine Yes 62,75

Quaternary ammonium compound No 62,76

ZnO No 77

Cetylpyridinium chloride No 78

12-methacryloyloxydodecylpyridinium bromide

No 79,80

Casein phosphopeptide-amorphouscalcium phosphate

No 81

Adhesive

Silica nanofillers and silvernanoparticles

No 82

Titanium tetrafluoride No 83

Polytetrafluoroethylene No 84Bracket

Fluoride No 85

Photocatalytic TiO2 No 86

WireCaF2 No 87

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Fluoride has been incorporated into various orthodontic adhesives69,73 to yield

a slow release system with direct, beneficial clinical effects on enamel de- and

remineralization. Other fluoride applications, which have not yet found their way to

extensive clinical use, include coating of brackets and wires e.g. titanium

tetrafluoride83 or calicium fluoride87, demonstrating sustained release of fluoride

and associated reductions in lesion depths and total mineral loss around the

bracket-adhesive-enamel junction. Fluoride-containing elastomeric rings have also

been demonstrated to release significant amounts of fluoride with a concurrent

clinical reduction in the degree of decalcification around the brackets88, although

the number of S. mutans or anaerobic bacterial growth in saliva or biofilms

surrounding the brackets remained the same89,90.

Incorporation of antimicrobial agents in adhesives is more directly aimed at

biofilm prevention. Antimicrobial release kinetics depends on the solubility of the

antimicrobial in water, while the build-up of sufficiently high concentrations

preventing microbial growth in saliva may be impossible due wash-out in vivo. The

solubility of chlorhexidine and triclosan in water for instance, is low and their

release from adhesives may be less than required to reach a minimal inhibitory

concentration preventing microbial growth62. However, the validity of this statement

greatly depends on the volume considered into which the antimicrobials are

released and it can be argued that when released into biofilm formed at the

enamel-adhesive junction it may yield locally high concentrations effective in

preventing growth or killing colonizing bacteria91. The release of cetylpyridinium

chloride in water from adhesives showed a burst release during the first two weeks,

followed by a much lower tail-release78, and in vitro caused an inhibition zone on

agar inoculated with bacteria. Other antimicrobials as e.g. benzalkonium chloride62

are only effective for two weeks after an initial burst release. Silver nanoparticles

and quaternary ammonium polyethylenimine nanoparticles mixed into adhesives

with an antibacterial activity upon contact are preferred since they are long-

lasting76,82, but the safety of nanoparticles for human use is still a matter of

17

controversy92. Considering the duration of orthodontic treatment, more permanent

non-adhesive or antimicrobial coatings are preferable. However, low surface free

energy polytetrafluoroethylene coatings on brackets84 or photocatalytic TiO2 on

wires86 have not yet been developed into a stage of clinical application. A strategy

that has not yet found its way into orthodontic materials but that could make them

almost fully non-adhesive might be based on polymer brush-coatings. Polymer

brush coatings have been shown to yield several log-units of reduction in microbial

adhesion numbers, and therewith may leave an influence also after growth93, unlike

common biomaterial coatings only showing reductions by a factor of utmost 3-4.

Especially under dynamic shear conditions, as in the oral cavity, weakly adhering

biofilms on polymer brush coatings can be expected to detach easily, leaving the

impression of a totally non-adhesive coating.

Summary and conclusions

Orthodontic appliances severely hamper biofilm control in the oral cavity, but once

treatment has successfully ended, it creates a better condition for oral cleaning and

strongly aids oral health by a properly aligned dentition. The negative side-effects

of orthodontic treatment as related to biofilms, such as white spot lesions and

gingivitis, compromise the facial esthetics aimed for by the treatment after an often

lengthy and costly course of orthodontic treatment. In severe cases, orthodontic

appliances have to be removed before the treatment goal has been reached. Most

orthodontists are aware of these problems, but yet effective preventive programs

are lacking. To date, less than 3% of all papers in the orthodontic field could be

found in the Pubmed database dealing with topics as: biofilm, plaque,

decalcification, demineralization, white spot lesion, gingivitis, or gingival

inflammation. Considering the prevalence and seriousness of the consequences of

orthodontic biofilms, more attention from orthodontic societies is needed, both in

terms of research as well as patient education about the potential side-effects of

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orthodontic treatment. A combination of proper oral hygiene instruction, preventive

measures, early diagnosis, and timely treatment would contribute greatly to the

management of orthodontic biofilms. Moreover, new orthodontic materials should

be developed attracting less biofilm and with appropriate anti-bacterial properties.

Aim of this thesis

The aim of this thesis is to investigate the adhesion forces of different oral bacterial

strains to orthodontic materials with and without a salivary conditioning film. After

having identified the adhesive as the site of strongest adhesion forces, adjacent in

addition to the vulnerable enamel surface, an antimicrobially modified adhesive

based on incorporation of a quaternary ammonium compound into the adhesive is

designed and evaluated in vitro.

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