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Oral health benefits

of

chewing gum

Stefan W. Wessel

2016

Oral health benefits of chewing gum

University Medical Center Groningen, University of Groningen

Groningen, The Netherlands

Copyright © 2016 by S.W. Wessel

Cover photography by E.U.D. Kaiser

Lay-out by S.W. Wessel

Printed by Off-Page, Amsterdam, The Netherlands

ISBN: 978-94-6182-656-5

The printing of this thesis was financially supported by the Graduate School of

Medical Sciences and William Wrigley, Jr. company

Oral health benefits of chewing gum

Proefschrift

ter verkrijging van de graad van doctor aan de

Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 9 maart 2016 om 11:00 uur

door

Stefan Wouter Wessel geboren op 3 mei 1987

te Emmen

Promotores: Prof. dr. ir. H.J. Busscher

Prof. dr. H. C. van der Mei

Beoordelingscommissie:

Prof. dr. J.M. ten Cate

Prof. dr. ir. W. Norde

Prof. dr. Y. Ren

Aan mijn ouders

Paranimfen: T.H. Kraaij

S.T. Mulder

Table of contents

Chapter 1 General introduction and aim of this thesis

1

Chapter 2 Chewing gum: From candy to nutraceutical - Advantages in oral

biofilm control Stefan W. Wessel, Henny C. van der Mei, Amarnath Maitra, Michael W.J.

Dodds and Henk J. Busscher (Submitted to Clinical Oral Investigations)

5

Chapter 3 Effects of chewing gum with and without active ingredients on oral

biofilm viability and composition (To be submitted)

31

Chapter 4 Effects of chewing gum on tooth surface wettability and mouthfeel

perception (To be submitted)

43

Chapter 5 Adhesion forces and composition of planktonic and adhering

microbiomes Stefan W. Wessel, Yun Chen, Amarnath Maitra, Edwin R. van den Heuvel,

Anje M. Slomp, Henk J. Busscher and Henny C. van der Mei

(J. Dent. Res., 2014; 93 (1): 84-88)

Appendices

57

71

Chapter 6 Quantification and qualification of bacteria trapped in chewed gum Stefan W. Wessel, Henny C. van der Mei, David Morando, Anje M Slomp,

Betsy van de Belt-Gritter, Amarnath Maitra and Henk J. Busscher

(PLoS One., 2015; 10(1): e0117191)

Appendix: Societal versus Scientific impact of “Quantification and

qualification of bacteria trapped in chewed gum”

77

95

Chapter 7

Magnolia bark extract increases adhesion of oral Gram-negative

bacteria to a hydrophobic ligand Stefan W. Wessel, Henny C. van der Mei, Amarnath Maitra, Michael W.J.

Dodds and Henk J. Busscher

(Submitted to Journal of Agricultural and Food Chemistry)

103

Chapter 8 General discussion

Summary

Dutch summary / Samenvatting

Acknowledgements / Dankwoord

Curriculum Vitae

115

123

129

135

139

General introduction and

aim of this thesis

Chapter 1

CHAPTER 1

2

Throughout history, humans have sought various aids to maintain oral health. In ancient

times, by rinsing their mouth with vinegar, later by chewing on the end of a twig of the

Salvadora persica tree, which eventually has led to the modern day toothbrush. Today’s

society can choose a vast array of toothbrushes, dentifrices, mouthrinses, toothpicks and

dental floss, which are continuously developing to serve more specific needs (1). Besides

the traditional oral health care products, also chewing gum has developed into an oral care

product.

Undoubtedly, oral health has advanced tremendously over the last decades,

reflected in a decrease in the number of total denture wearers in The Netherlands from

32% in 1981 to 12% in 2009 (2). Nevertheless, prevalence of oral diseases is still one of

the most important diseases present in daily life, as dental caries is one of the most non-

communicable diseases among children worldwide and affecting the vast majority of adults

in industrialized countries (3,4). Only 16% of all young adults in the Netherlands have a

perfect dentition without any restorations (5) and approximately 12% of the Dutch

population receives a dental filling every year (6). Numbers on the epidemiology of

gingivitis vary greatly but estimates are that more than 50% of all adults experience

gingivitis from which roughly 10% advances to severe periodontitis (7–9). These figures

indicate the difficulty of maintaining oral health and are illustrative for the necessity of

continuous improvement of oral health care products.

Causative to most oral diseases, including caries, gingivitis and periodontitis, is

the formation of oral biofilm. Planktonic bacteria in saliva adhere to the salivary

conditioning film on the tooth surface to form a biofilm; a complex structure of multiple

bacterial species, protected by a matrix of extracellular polymeric substances from

environmental forces and antimicrobials. If the oral biofilm is not mechanically removed,

the composition of the biofilm changes which is the onset of diseases (10–12). In the case

of caries, specific bacteria in the biofilm ferment environmental sugars into acids, causing

softening of the enamel. Normally this is counteracted by minerals in saliva which recover

hardness of the enamel, however when this balance is lost the tooth surface is prone to

cavities (13). As gingivitis is concerned, specific pathogenic bacteria in biofilm in the

gingival margin and in between teeth excrete products that cause a signaling cascade in

the gingival tissue of the host, resulting in an inflammatory response. If left untreated, the

inflammation may advance to periodontitis, affecting the bone around the teeth which can

eventually lead to tooth loss(14).

Since the 1970s, chewing gum developed from a candy into an oral care product.

Replacement of conventional sugars by artificial sweeteners, which cannot be fermented

into acids by oral bacteria, together with the stimulation of saliva during chewing made

1

GENERAL INTRODUCTION AND AIM

3

chewing gum an established oral care product (15). Currently, dictated by the urge for

continuous product development, various active ingredients are incorporated in chewing

gum to chemically influence the oral biofilm, for instance by reducing the number of

bacteria in saliva or preventing bacterial adhesion to the tooth surface, all aiming to

enhance the current oral health benefits (16–18).

Aims of this thesis

The general aim of this thesis is to explore new possibilities to further develop the oral

health benefits of chewing gum. To this end, we first evaluated the current oral health

benefits of chewing gum, emphasizing the effects of active ingredients on biofilm formation.

The effects of two active ingredients in chewing gum on oral biofilm after 4 weeks of use

and the effects on the mouthfeel perception in relation to tooth surface properties was

investigated in an in vivo study. Next, we looked into the importance of adhesion forces of

bacteria in the oral cavity and its role in the eventual bacterial composition of the biofilm.

Subsequently, the possibilities of using a piece of chewing gum to trap oral bacteria and to

remove them from the oral cavity was investigated both in vitro and in vivo. Finally, we

explore the first step towards the development of an oral care agent that targets specific

bacteria within the oral cavity.

1

CHAPTER 1

4

References

1. Fischman SL. The history of oral hygiene products: how far have we come in 6000 years? Periodontol 2000. 1997; 15:7–14.

2. Centraal Bureau voor de Statistiek. Gebruik medische voorziening; t/m 2009. Statline. 2010. Available from: http://statline.cbs.nl/StatWeb/publication/?VW=T&DM=SLnl&PA=7042MC&LA=nl

3. Preet R. Health professionals for global health: include dental personnel upfront! Glob Health Action. 2013;6:21398.

4. World Health Organization. Oral Health. 2012. Available from: http://www.who.int/mediacentre/factsheets/fs318/en/

5. Schuller AA, Kampen I van, Poorterman J, Verrips G. Kies voor tanden. Een onderzoek naar mondgezondheid en preventief tandheelkundig gedrag van jeugdigen. Hoofdmeting 2011, een vervolg op de reeks TJZ-onderzoeken. Rapportnummer: TNO/LS 2013 R10056. 2013.

6. Centraal Bureau voor de Statistiek. Medische contacten, ziekenhuisopname, medicijnen; pers. kenmerken, 2010-2013. StatLine. 2015. Available from: http://statline.cbs.nl/Statweb/publication/?DM=SLNL&PA=81027ned&D1=34-35,38&D2=0-13,32-37&D3=0&D4=a&HDR=T&STB=G1,G2,G3&VW=T

7. Van der Velden U. Epidemiologie van gingivitis en parodontitis. In: Beertsen W, Quirynen M, van Steenberghe D, van der Velden U, editors. Parodontologie. 1st ed. Houten: Bohn Stafleu van Loghum; 2009. p. 33–9.

8. Burt B. Position paper - Epidemiology of periodontal diseases. J Periodontol. 2005; 76(8):1406–19.

9. Teeuw WJ. Parodontitis en levenskwaliteit. Ned Tijdschr Tandheelkd. 2011; 118(4):199–201.

10. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000. 2011; 55(1):16–35.

11. Hojo K, Nagaoka S, Ohshima T, Maeda N. Bacterial interactions in dental biofilm development. J Dent Res. 2009; 88(11):982–90.

12. Kolenbrander PE. Multispecies communities: interspecies interactions influence growth on saliva as sole nutritional source. Int J Oral Sci . 2011; 3(2):49–54.

13. Edgar M, Dawes C. Saliva and oral health. 3rd ed. London: BDJ Books; 2004. 146 p.

14. Scannapieco FA. Periodontal inflammation: from gingivitis to systemic disease? Compend Contin Educ Dent. 2004; 25:16–25.

15. Imfeld T. Chewing gum - facts and fiction: A review of gum-chewing and oral health. Crit Rev Oral Biol Med. 1999; 10(3):405–19.

16. Dodds M. The oral health benefits of chewing gum. J Ir Dent Assoc . 2012; 58(5):253–61.

17. Marwaha M, Bhat M. Antimicrobial effectiveness of chlorhexidine chewing gums on Streptococcus mutans counts – An in vivo microbiological study. J Clin Pediatr Dent. 2010; 35(1):31–5.

18. Hayashi Y, Ohara N, Ganno T, Ishizaki H, Yanagiguchi K. Chitosan-containing gum chewing accelerates antibacterial effect with an increase in salivary secretion. J Dent. 2007; 35(11):871–4.

1

Chewing gum:

From candy to nutraceutical

-

Advantages in oral biofilm control

Stefan W. Wessel, Henny C. van der Mei, Amarnath Maitra,

Michael W.J. Dodds and Henk J. Busscher

Submitted to Clinical Oral Investigations

Chapter 2

CHAPTER 2

6

2

Abstract

Objectives

Over the years, chewing gum has developed from a candy towards an oral-health

promoting nutraceutical. This review summarizes evidence for oral-health benefits of

chewing gum, with emphasis on identification of active ingredients in gum that facilitate

prevention and removal of oral biofilm.

Results

Chewing of sugar-free gum yields oral-health benefits that include clearance of interdental

debris, reduction in oral dryness and amount of occlusal oral biofilm. These basic effects of

the chewing of gum are attributed to increased mastication and salivation. Active

ingredients incorporated in chewing gums aim to expand these effects to inhibition of

extrinsic tooth stain and calculus formation, stimulation of enamel remineralization,

reduction of the numbers of bacteria in saliva and amount of oral biofilm, neutralization of

biofilm pH, and reduction of volatile sulfur compounds. However, clinical benefits of

incorporating active ingredients are often hard to prove, since they are frequently

overshadowed by the effects of increased mastication and salivation and require daily

chewing of gum for prolonged periods of time.

Conclusion

Evidence for oral-health benefits of chewing gum additives is hard to obtain viz a viz

additives in advanced toothpaste formulations or mouthrinses due to their relatively low

concentrations and rapid wash-out. Clinical effects of gum additives are overshadowed by

effects of increased mastication and salivation due to the chewing of gum.

Clinical relevance

Chewing of sugar-free gum can contribute to oral health provided used on a daily basis,

but clinical benefits of incorporating active ingredients into chewing gum are hard to

demonstrate over the beneficial effects of increased mastication and salivation.

CHEWING GUM: FROM CANDY TO NUTRACEUTICAL

7

2

Introduction

Many oral diseases, most notably caries, gingivitis and periodontitis are caused by oral

biofilms. The development of a pathogenic biofilm depends to a major part on the amount

and composition of the biofilm. The formation of oral biofilm constitutes the transition of

bacteria from their freely suspended or planktonic state in saliva to an adhering or sessile

state on oral hard and soft tissues. In the oral cavity, due to the abundant presence of

salivary proteins, bacteria never adhere to bare surfaces but always to an adsorbed

salivary conditioning film (1). Small differences in the forces by which bacteria are attracted

to these salivary conditioning films play a determining role in the composition of oral biofilm

on intra-oral surfaces (2). Upon further growth of the biofilm, more strains and species

become incorporated in a biofilm through co-adhesion with other colonizers, governed by

an interplay between specific ligand-receptor binding and non-specific bacterial interactions

(3,4). Oral diseases develop when cariogenic strains in a biofilm produce an excess of

acids through the fermentation of environmental sugars causing enamel demineralization

or when periodontopathogens residing mostly in gingival pockets, cause gingivitis or in

more advanced state, periodontitis (5).

Although much has been achieved with respect to the prevention of oral diseases

like caries, gingivitis and periodontitis (6,7), maintenance of effective oral hygiene by

toothbrushing, using advanced toothpaste formulations and mouthrinses remains beyond

reach for many people. Therefore a variety of other mechanical aids such as toothpicks,

floss wire and chewing gum has been promoted for the removal of oral biofilm (8). In this

review we evaluate possible oral health benefits of chewing gum, with special emphasis on

the identification of active ingredients incorporated in gum that facilitate prevention and

removal of oral biofilm.

History and development of chewing gum

Throughout history, various materials have been used by people to chew upon in order to

refresh their breath or relieve oral dryness. Early types of chewing gum are based on tree

resins. It was not until 1870 that Thomas Adams was able to successfully market chewing

gum on a mass scale (9,10). Since its first introduction, chewing gum has developed into a

multi-billion dollar industry (9,11) aided by the invention of rubbers in the 1930s and 1940s

(12). In the 1970s, chewing gum developed more and more from a candy towards a

functional food aiming for niche markets, like non-stick gum for denture wearers, gums with

tooth-whitening properties or preventing halitosis (13,14). The current portfolio of chewing

CHAPTER 2

8

2

gums meets the specific demands of various types of consumers and follows the need to

differentiate in a competitive world (15). Following compliance with different consumer

requirements, chewing gum has become recognized for its role in maintenance and

improvement of oral health (9).

Current chewing gums consist of a few basic ingredients. The gum base provides

elasticity to a gum and should not dissolve during chewing. Moreover, it should allow a

gum to be chewed for relatively long periods of time without major changes in structure.

Generally, gum base consists of a mixture of elastomers, like polyvinylacetate or

polyisobutylene, that are complemented with softeners, texturizers and other ingredients as

emulsifiers and plasticizers. Hydrophilicity of the base system is an important determinant

for the ability of gums to take up water or saliva, which influences chewing gum texture.

Molecular weight of the polymer ingredients, together with the interaction with the other

ingredients, determine gum viscosity. Tendency to absorb saliva is mainly determined by

emulsifiers, which create a stable mixture of normally immiscible ingredients. Formulation

of the latter ingredients is adjusted based on desired functionality.

Approximately 70 % of all gums marketed do not contain conventional

sweeteners, like sucrose, but have sugar substitutes like xylitol, sorbitol, mannitol and/or

maltitol and are considered to be sugar-free gums (16). Aspartame, acesulfame-K and

glycerine add an extra degree of sweetness and also provide longer lasting flavor duration

(15,17,18). Polyols are widely used instead of conventional sugars and the main

advantage is that they are not or hardly fermented by bacteria, classifying them as non-

acidogenic (19) and cariostatic (20). The replacement of conventional sugars to create

sugar-free gums, was the most important development advancing chewing gum from a

candy to a nutraceutical with specific oral health benefits.

Benefits of chewing gum on oral health

Chewing of gum stimulates the salivary glands, causing approximately a tenfold increase in

salivary flow over unstimulated salivation during the first five minutes of chewing (21).

Increased salivation together with the mechanical action of mastication provides the basis

for many effects of chewing gum on oral health (See Fig. 1 and Table 1 for an overview).

Furthermore, chewing gum is an excellent vehicle for administering active ingredients to

the oral cavity. Possible oral health benefits of the chewing of gum are summarized in the

different circle segments in Fig. 1, together with the active ingredients assumed

responsible for these benefits.


9

2

A gradual release profile of active ingredients from chewing gum can readily be

achieved, potentially making their prolonged presence and substantive action in the oral

cavity possible (18). However, at the same time, increased salivation stimulates rapid

wash-out of active ingredients from the oral cavity making their clinical efficacy hard to

demonstrate over the basic effects of increased salivation and mastication.

Figure 1 Basic effects of the chewing of regular sugar-free gum on oral health are displayed in the inner ring,

and are predominantly due to increased mastication and salivation. Potential effects of active

ingredients used in chewing gum on oral health are displayed in the outer ring.

CHAPTER 2

10

2

Reduction of oral dryness Individuals suffering from xerostomia or the subjective feeling of dry mouth, can relief their

symptoms by the use of regular sugar-free chewing gum (Fig. 1), which is generally

preferred by dry mouth patients over the use of artificial saliva (22). Symptom relief is

related to mastication and increased salivation and not to any specific additive incorporated

in chewing gums (23). Importantly, the claim that the chewing of gum reduces dry mouth

perception (23,24), is supported by the European Food Safety Authority (EFSA).

Clearance of interdental debris The chewing of gum can stimulate removal of interdental debris left after food

consumption, as illustrated in Fig. 2. Removal is partly due to direct attachment of debris to

the gum but also due to increased mastication and salivation which aids to wash away

debris (25,26). Since debris left after food consumption often contains fermentable sugars,

its removal prevents oral bacteria from producing acids that desorb calcium (Ca2+) and

phosphates (PO43-) from the enamel (21,27–30), which constitutes a clear oral health

benefit (Fig. 1).

Inhibition of calculus formation

Calculus formation involves the formation of calcium phosphate mineral salts, that calcify

and harden oral biofilm. Among many other factors, biofilm pH and salivary calcium

phosphate saturation play an important role in the rate of calculus formation (31,32).

Chewing of regular sugar-free gum did not have a pronounced effect on inhibiting

calculus formation and it has even been suggested that calculus formation is promoted by

chewing sugar-free gum, due to higher biofilm pH and salivary calcium phosphate

saturation (33–35). Therefore, active ingredients have been incorporated in chewing gums

aiming to maintain calcium phosphate deposits in an amorphous state, preventing

hardening and facilitating removal. When chewing vitamin C supplemented chewing gum

at least five times per day for three months, a reduction in supra-gingival calculus formation

was found compared to not chewing gum (35). Similar results were obtained for pyro/tri-

phosphates supplemented chewing gum after six weeks use (36). Nevertheless, since

reductions in calculus formation were only demonstrated for supra-gingival surfaces, and

not for gingival margins and interproximal spaces that matter most in oral health, a

negative verdict was released by the EFSA on oral health claims regarding a reduction in

calculus formation by pyro/triphosphates in sugar-free gums (37) (Table 1).


11

2

Figure 2 Removal of food debris (Cookie) from occlusal surfaces after 2 min chewing of regular sugar-free gum

(top panel) or without the chewing of gum (bottom panel). No specific instructions were given to the

volunteer.

Inhibition of extrinsic tooth stain

Aesthetics, including the appearance of white teeth, is more and more considered as an

important component of oral health. Extrinsic tooth stain is caused by chromogens from

food, drinks or smoking that absorb in superficial enamel layers (or calculus). Extrinsic

tooth stain is more susceptible to whitening regimens than intrinsic tooth stain, but still

usually requires professional removal, depending on the causative chromogen. Chewing of

regular sugar-free gum multiple times per day for four weeks or longer has been shown to

prevent and remove extrinsic tooth stain caused by chromogens (13,38,39), likely again as

a result of increased salivation (Fig. 1).

To enhance extrinsic stain prevention and removal (13), sugar-free chewing gum

has been supplemented with polyphosphates (40,41). Sodium hexametaphosphate (Table

1) in a sugar-free gum, reduced stain formation better than a control gum in short-term, two

day studies during which volunteers chewed eight times two tablets of gum throughout the

day (41–43). When chewing three times two tablets per day for six weeks or longer, tooth

stain prevention has been shown for hexametaphoshate, pyrophosphate and

tripolyphosphate supplemented sugar-free gums (44,45). Stain prevention by

polyphosphates has been attributed to adsorption of these highly negatively charged and

hydrophilic molecules to salivary conditioning films which makes incorporation of

Before

Removal of food debris without chewing of gum

Removal of food debris after chewing of gum

CHAPTER 2

12

2

chromogens in superficial enamel layers more difficult. Administration of sodium

hexametaphosphate through the chewing of gum produced a more hydrophilic tooth

surface in vivo than a control gum (46), while sodium hexametaphosphate caused

desorption of proteins from adsorbed salivary conditioning films and created a more open

film structure in vitro (47,48).

Reduction of volatile sulfur compounds

Oral malodor, or halitosis, results from the production of volatile sulfur compounds (VSCs),

such as hydrogen sulfide and methyl-mercaptan by anaerobic Gram-negative bacteria

adhering to the tongue or associated with periodontitis (49). Regular sugar-free chewing

gum has been shown to successfully reduce VSCs and thereby freshen breath (14) (Fig.

1). Besides the reduction of VSCs by the regular chewing of sugar-free gum, active

ingredients incorporated in a gum have aimed to further reduce halitosis either by directly

interacting with VSCs or by targeting bacteria responsible for oral malodor (Table 1). Zinc

has high affinity for sulfur compounds (50) and, especially in combination with allyl

isothiocyanate (51), results in reduced VSC levels directly after chewing compared to a

control gum (52), although this could not be confirmed in another study (14). Furthermore

magnolia bark extract and eucalyptus both target the viability of VSC producing bacteria

and were shown to be effective against oral malodor in a chewing gum (53,54), especially

when magnolia bark extract was combined with zinc (55) (Table 1).

Neutralization of biofilm pH The pH buffering ability of saliva counteracts acids produced in oral biofilm and is therefore of importance to maintain the intra-oral balance between enamel re- and demineralization. Bicarbonate (HCO3

-) provides the main buffering system of saliva and neutralizes oral

biofilm pH (56,57). Neutralization of biofilm pH is also achieved via a different mechanism

involving carbamide ((NH2)2CO) or urea. Oral bacteria that produce urease hydrolyze and

convert carbamide into ammonia and create a more alkaline environment. Chewing of

regular sugar-free gum has been shown to increase biofilm pH, as also recognized by the

EFSA (58). Furthermore it increases the resting pH of oral biofilm and resistance of the

enamel surface to acid challenges (59) as a result of increased salivation. This effect was

enhanced by the addition of xylitol to chewing gum (Table 1) (60).

Addition of other actives such as bicarbonate to chewing gum also caused an

increase in the buffering capacity of saliva (61). Accordingly, interproximal biofilm pH, after

a sucrose challenge, was elevated more rapidly and maintained at a higher level compared

to a gum without bicarbonate (62). Furthermore chewing of carbamide supplemented gum


13

2

yielded a concentration dependent rise in biofilm pH (63,64). The EFSA has concluded that

the claim that chewing gum containing carbamide stimulates biofilm pH neutralization

directly after chewing is justified when the gum contained at least 20 mg of carbamide and

was chewed for 20 min after food intake (65). However, when effects of the chewing of

carbamide supplemented gum were evaluated for four weeks or longer, no change in acid

production by oral biofilm was observed (64), neither were caries preventive effects

observed after three years of use in terms of a reduced number of decayed, missing or

filled surfaces (66). This shows that short term results cannot be readily extrapolated to

long term effects, most likely because short term studies do not include enamel

demineralization due to food and drink consumption, effects that are apparently only

influential in long-term studies.

Enamel remineralization

Saliva is rich in calcium and phosphates, facilitating enamel remineralization and

preventing demineralization (9). Chewing of regular sugar-free gum can enhance calcium

and phosphate levels in the oral cavity through increased salivation. Long-term clinical

studies showed that chewing of regular sugar-free gum multiple times per day, especially

after a meal in addition to normal oral hygiene, can results in lower caries incidence

(66,67). The latter was acknowledged by the EFSA (24,68) (Fig. 1).

In order to increase the effects of the chewing of gum on remineralization, calcium

has been added to chewing gums either in the form of ionic calcium or casein-calcium

conjugates (CPP-ACP) (Table 1). In situ studies with demineralized enamel slabs placed in

the oral cavity using specific intra oral appliances and removed after the chewing of gum

supplemented with calcium phosphates, demonstrated increased remineralization

compared to chewing of regular sugar-free gum (69,70). Unfortunately, in these studies the

intra oral appliances were worn only for approximately forty minutes after the chewing of

gum or were removed during food intake. Therewith demineralization is largely left out of

consideration (71). A review on calcium phosphate supplemented chewing gum concluded

that these additives to chewing gum did not yield increased caries prevention (72). In

accordance with the latter study, the EFSA does not support a health claim on increased

remineralization of chewing gum containing calcium phosphates as compared to regular,

sugar-free gums (73).

CPP-ACP has been suggested to deposit a calcium and phosphate reservoir on the tooth

surface and the surface of oral biofilm, inhibiting enamel demineralization and promoting

remineralization (74). Similar to calcium phosphates, significant, dose-dependent, enamel

remineralization and increased resistance against demineralization were reported in situ

CHAPTER 2

14

2

after use of CPP-ACP containing gum compared to a control gum (75–77). However,

contrary to calcium phosphates, the caries preventive effect of CPP-ACP could be

demonstrated in a long-term, two year study involving 2720 volunteers, showing that when

CPP-ACP gum was chewed three times per day, there was 18% less chance of a tooth

surface progressing to caries compared to a control gum (78). Nonetheless there is no

unanimous positive judgment on the remineralization potential of CPP-ACP in chewing

gum and while most studies on CPP-ACP were done by the same research group, studies

by others did not confirm beneficial effects of chewing CPP-ACP supplemented gums on

remineralization (79,80).

Fluoride (Table 1) hardens the enamel as it is incorporated in the hydroxyapatite

lattice network of the crystallites, creating less soluble fluorohydroxyapatite (81). Its

incorporation in a chewing gum was shown in four week in situ studies to enhance

remineralization of enamel compared to a control gum (82), likely to be more effective on

the side of the dentition which is used mostly for chewing (83). The EFSA considers that

the general health claims with respect to the use of fluoride also apply to fluoridated

chewing gum (84). However, the chewing of fluoridated gum did not yield additional

benefits when used in combinations with a regular oral hygiene with fluoridated products

(85).

Reduction of oral biofilm formation and impact on biofilm composition

Oral bacteria adhere to salivary conditioning films in order to avoid being washed away by

salivary flow, and adhesion is governed by the forces by which specific planktonic bacteria

are attracted to the salivary conditioning film (2). Only the so-called initial colonizers adhere

directly to the conditioning film and later colonizers co-adhere with initial colonizers to yield

a multispecies biofilm (4). Disease usually develops when the composition of oral biofilm

shifts towards a predominance of specific pathogens. Active ingredients in chewing gums

(Table 1) can affect oral biofilm formation at various stages either by reducing the number

of specific planktonic bacteria, preventing their adhesion or reducing growth of adhering

bacteria to yield less biofilm or biofilm with a different microbial composition.

Effects on planktonic bacteria in saliva

Planktonic bacteria suspended in saliva constitute the source of bacteria for initial adhesion

and biofilm formation on oral surfaces. Therefore the effects of active ingredients in

chewing gum on the amount of salivary pathogens such as cariogenic Streptococcus

mutans or Streptococcus sobrinus, commonly referred to as mutans streptococci, are often

used as an indicator of potential oral health benefits. Chewing of regular, sugar-free gum


15

2

was shown to be able to non-specifically trap oral bacteria within a piece of gum and

thereby remove bacteria from the oral cavity, although it could not be firmly established

whether the bacteria trapped in chewed gum originated from saliva or the biofilm formed on

the dentition (86). Chewing of gum had no specific effect on salivary mutans streptococcal

concentrations (87,88). However, some artificial polyol sweeteners, particularly xylitol when

used exclusively, were reported to reduce salivary mutans streptococcal numbers (89). A

minimum of 6 g of xylitol per day during five weeks was necessary to reach a significant

reduction in mutans streptococcal numbers (90–93). Xylitol should be preferred over other

sweeteners such as sorbitol which are reported to be low cariogenic, which again should

highly be preferred over conventional sugars (94,95). Other ingredients incorporated in

chewing gum such as chlorhexidine, chitosan, magnolia bark extract and mastic were also

shown to lower the number of planktonic bacteria in saliva compared to a control gum

(88,96–98).

Effects on bacterial adhesion to oral surfaces

Adhesion of planktonic bacteria to oral surfaces is the first step in the formation of oral

biofilm and is mediated by attractive forces between oral surfaces and adhering bacteria.

Accordingly, the properties of the oral surfaces play a major role in the development of

these adhesion forces and changing the forces may impact the amount and composition of

oral biofilm formed (2,99). Chewing a gum containing polyphosphates made adsorbed

salivary conditioning films more hydrophilic and more negatively charged as compared with

other gums. Since most oral bacterial strains are negatively charged (46,48), this implies

weaker adhesion of oral bacteria and polyphosphates may even promote detachment of

bacteria from salivary conditioning films on enamel surfaces (100).

Effects on biofilm formation, composition and removal

Chewing of regular sugar-free gum dislodges loosely bound bacteria from the oral mucosa

(101) and inhibits regrowth and maturation of oral biofilm on occlusal surfaces (102) (Fig.

1). Nonetheless, biofilm regrowth was not inhibited on smooth lingual and buccal surfaces

and a relation between complete biofilm removal directly after a single gum chew has not

been established (103,104), not even when abrasive agents were included in the gum

(105). Therefore the EFSA concluded that the claim that the chewing of regular sugar-free

gum “reduces plaque formation” is unsubstantiated (103) so direct and clinically relevant

biofilm reduction is not a supportable claim for chewing gum without active ingredients,

although it is possible that gum chewing could modify the biofilm composition to a less

cariogenic state.

CHAPTER 2

16

2

Chlorhexidine is the most effective antimicrobial for the chemical control of oral

biofilm (106). Its antimicrobial properties are based on disturbing the bacterial cell-

membrane and its binding to intra-oral surfaces ensures substantive action (106).

Chlorhexidine tastes bitter, alters long term taste perception (106) and causes (reversible)

tooth stain (107). Antimicrobially effective chlorhexidine containing chewing gums with

acceptable taste can be made (108), but consumer hesitance remains to exist and in

certain countries chlorhexidine containing chewing gums are likely to only be available on

prescription (59,109). Application of chlorhexidine in chewing gum reduces planktonic

levels of mutans streptococci directly after chewing (96), but also reduces oral biofilm

formation. Incorporation of chlorhexidine in chewing gum inhibited oral biofilm growth in a

four day study when only two pieces of gum were chewed per day in absence of other oral

hygiene measures (109). When used for one year, chlorhexidine containing chewing gum

showed a stronger reduction in gingival index and amount of oral biofilm formed than a

xylitol containing gum (110,111), but concerns remain about long-term consumption of

potent antimicrobial agents.

Similar to chlorhexidine, xylitol also resulted in reduction of salivary mutans

streptococcal numbers when used for five weeks, but this was too short to result in a

change in composition of oral biofilm (89,112). Also, in combination with regular brushing,

no effects of xylitol containing gum on biofilm and gingivitis scores were observed

compared to chewing gum base only (113). Six months chewing of xylitol containing gum

caused a decrease in the acidogenicity of oral biofilm (60), indicative of a change in biofilm

composition. In general, oral health care benefits of xylitol on oral biofilm are still subject to

debate and it is not clear whether effects of xylitol containing gum are solely due to

increased salivation or to the addition of xylitol as well (87,114,115).

Of the other ingredients mentioned above to lower planktonic levels of bacteria,

only mastic and eucalyptus containing chewing gum were hinted to successfully reduce

oral biofilm formation better than a control gum under the artificial condition of refraining

from other oral hygiene measures (116–118).


17

2

CHAPTER 2

18

2

Table 1 – Overview of active ingredients used in different chewing gums and oral health benefits

reported in the literature.

Activ

e in

gred

ient

Ef

fect

R

emar

ks

EFSA

su

ppor

t R

efer

ence

s

Non

e (b

asic

effe

cts

of

regu

lar s

ugar

-free

gu

m)

Red

uctio

n of

ora

l dry

ness

Che

win

g of

gum

can

relie

f dry

mou

th s

ympt

oms

durin

g ch

ewin

g Ye

s (2

3,24

)

C

lear

ance

of i

nter

dent

al

debr

is

Che

win

g gu

m in

crea

ses

rate

of c

lear

ance

of f

ood

debr

is, i

t rem

oves

sou

rces

of f

erm

enta

ble

suga

rs

-

(25,

26)

In

hibi

tion

of e

xtrin

sic

toot

h st

ain

Che

win

g at

leas

t 4 ti

mes

15

min

per

day

for 4

wee

ks o

r lon

ger r

educ

es e

xtrin

sic

toot

h st

ain

- (1

3,39

)

R

educ

tion

of V

SCs

Che

win

g gu

m te

mpo

raril

y fre

shen

s br

eath

by

redu

cing

VSC

s af

ter c

hew

ing

- (1

4)

N

eutra

lizat

ion

of b

iofil

m p

H

Incr

ease

d sa

livat

ion

neut

raliz

es b

iofil

m p

H

Yes

(24,

56–5

8)

R

emin

eral

izat

ion

of e

nam

el

Stim

ulat

ed s

aliv

a is

sat

urat

ed w

ith m

iner

als

incr

easi

ng re

min

eral

izat

ion

Yes

(9,2

4,68

)

R

educ

tion

of o

ral b

iofil

m

Prev

entin

g bi

ofilm

regr

owth

at o

cclu

sal s

urfa

ces

whe

n re

frain

ing

from

ora

l hyg

iene

for 4

day

s. N

o bi

ofilm

re

duct

ion

at o

ther

toot

h su

rface

s N

o (1

02,1

03,1

05)

C

arie

s pr

even

tion

Red

uced

car

ies

inci

denc

e ov

er 2

-3 y

ears

whe

n us

ing

2-3

g of

gum

for 2

0 m

in d

irect

ly a

fter m

eal

Yes

(24,

58,6

6–68

)

Bica

rbon

ate

Neu

traliz

atio

n of

bio

film

pH

Bi

ofilm

pH

ele

vate

d fa

ster

by

chew

ing

bica

rbon

ate

gum

afte

r suc

rose

cha

lleng

e -

(61,

62)

Cal

cium

R

emin

eral

izat

ion

of e

nam

el

In s

itu s

tudi

es s

how

sho

rt te

rm in

crea

sed

rem

iner

aliz

atio

n. N

o ad

ditio

nal c

arie

s pr

even

tive

effe

ct

No

(69,

70,7

3)

Car

bam

ide

Neu

traliz

atio

n of

bio

film

pH

C

once

ntra

tion

depe

nden

t ris

e in

bio

film

pH

. At l

east

20

mg

per p

iece

for 2

0 m

in d

irect

ly a

fter a

mea

l. N

o ad

ditio

nal c

arie

s pr

even

tive

effe

ct

Yes

(63–

65)

C

hito

san

Red

uctio

n of

pla

nkto

nic

bact

eria

in s

aliv

a R

educ

es to

tal n

umbe

r of o

ral b

acte

ria in

sal

iva

dire

ctly

afte

r che

win

g -

(97)

Chl

orhe

xidi

ne

Red

uctio

n of

pla

nkto

nic

bact

eria

in s

aliv

a

Dec

reas

e of

leve

ls o

f sal

ivar

y m

utan

s st

rept

ococ

ci d

irect

ly a

fter c

hew

ing

- (9

6)

R

educ

tion

of o

ral b

iofil

m

Bi

ofilm

gro

wth

was

inhi

bite

d w

hen

refra

inin

g fro

m o

ral h

ygie

ne fo

r 4 d

ays

-

(109

,110

)

In

crea

sed

ging

ival

hea

lth

Si

gnifi

cant

redu

ctio

n in

gin

giva

l ind

ex c

ompa

red

to c

ontro

l gum

afte

r 1 y

ear u

se

- (1

10,1

11)


19

2

CPP

-AC

P R

emin

eral

izat

ion

of e

nam

el

In

situ

stu

dies

sho

w s

hort

term

incr

ease

d re

min

eral

izat

ion

- (7

6,77

)

C

arie

s pr

even

tion

Larg

e sc

ale

2 ye

ar s

tudy

sho

ws

18%

less

cha

nce

of to

oth

surfa

ce p

rogr

essi

ng to

car

ies

whe

n ch

ewin

g C

PP-A

CP

3 tim

es p

er d

ay c

ompa

red

to c

ontro

l gum

-

(78)

Euca

lypt

us

Red

uctio

n of

bac

teria

pr

oduc

ing

VSC

s

Dec

reas

e in

org

anol

eptic

sco

re a

nd V

SCs

afte

r 14

wee

k us

e co

mpa

red

to c

ontro

l gum

-

(54)

R

educ

tion

of o

ral b

iofil

m

Bi

ofilm

form

atio

n w

as s

igni

fican

tly re

duce

d co

mpa

red

to c

ontro

l gum

whe

n re

frain

ing

from

ora

l hyg

iene

for 4

day

s -

(116

)

In

crea

sed

ging

ival

hea

lth

Bene

ficia

l effe

ct o

n pr

obin

g de

pth

and

ging

ival

ble

edin

g af

ter 1

2 w

eeks

use

-

(118

)

Fluo

ride

Rem

iner

aliz

atio

n of

ena

mel

G

ener

al fl

uorid

e cl

aim

s al

so a

pply

on

fluor

idat

ed c

hew

ing

gum

. Effe

ctiv

e in

cas

e ot

her w

ays

of a

dmin

istra

ting

fluor

ide

are

not p

ossi

ble

Yes

(82–

84)

Mag

nolia

bar

k ex

tract

R

educ

tion

of p

lank

toni

c ba

cter

ia in

sal

iva

Red

uctio

n of

tota

l sal

ivar

y ba

cter

ia a

nd m

utan

s st

rept

ococ

ci d

irect

ly a

fter c

hew

ing

and

afte

r 30

days

of u

se

- (5

3,88

)

R

educ

tion

of b

acte

ria

prod

ucin

g VS

Cs

Red

uctio

n of

VSC

s di

rect

ly a

fter c

hew

ing

and

in v

itro

effe

ctiv

enes

s ag

ains

t bac

teria

resp

onsi

ble

for o

ral m

alod

or

- (5

3,55

)

Mas

tic

Red

uctio

n of

pla

nkto

nic

bact

eria

in s

aliv

a

Red

uctio

n of

tota

l sal

ivar

y ba

cter

ia a

nd s

aliv

ary

mut

ans

stre

ptoc

occi

dire

ctly

afte

r che

win

g co

mpa

red

to c

ontro

l gu

m

- (9

8,11

7)

R

educ

tion

of o

ral b

iofil

m

Inhi

bitio

n of

bio

film

form

atio

n w

hen

refra

inin

g fro

m o

ther

ora

l hyg

iene

for 7

day

s co

mpa

red

to c

ontro

l gum

-

(117

)

Poly

ols -

Xyl

itol

Red

uctio

n of

pla

nkto

nic

bact

eria

in s

aliv

a

Whe

n us

ed fo

r 5 w

eeks

xyl

itol g

um is

mor

e ef

fect

ive

in re

duci

ng s

aliv

ary

mut

ans

stre

ptoc

occi

than

a c

ontro

l gum

. N

o ch

ange

in b

iofil

m c

ompo

sitio

n w

as o

bser

ved

- (8

9,11

4)

N

eutra

lizat

ion

of b

iofil

m p

H

N

eutra

lizat

ion

of b

iofil

m p

H w

as o

bser

ved

afte

r lon

g te

rm u

se (6

mon

ths)

N

o (6

0)

C

arie

s pr

even

tion

Valid

whe

n 10

0% x

ylito

l gum

is u

sed

thre

e tim

es p

er d

ay in

hig

h do

se (≥

6 g

per d

ay)

Yes

(92,

93)

Poly

phos

phat

es

Inhi

bitio

n of

cal

culu

s fo

rmat

ion

Cal

culu

s re

duct

ion

supr

agin

giva

lly, n

ot a

t site

s w

hich

mat

ter m

ost f

or g

ingi

val h

ealth

(gin

giva

l mar

gin,

in

terp

roxi

mal

spa

ces)

N

o (3

6,37

)

In

hibi

tion

extri

nsic

toot

h st

ain

Stai

n in

hibi

tion

with

in tw

o da

ys w

hen

chew

ed 8

tim

es p

er d

ay. W

ith 3

tim

es p

er d

ay s

tain

inhi

bitio

n w

as s

how

n af

ter s

ix w

eeks

- (4

1,42

,45)

Vita

min

C

Inhi

bitio

n of

cal

culu

s fo

rmat

ion

Red

uctio

n of

sup

ragi

ngiv

al c

alcu

lus

form

atio

n co

mpa

red

to n

o ch

ewin

g w

hen

used

5 ti

mes

per

day

for t

hree

m

onth

s -

(35)

Zinc

R

educ

tion

of V

SCs

Red

uctio

n of

VSC

s di

rect

ly a

fter c

hew

ing

com

para

ble

to z

inc

mou

thrin

se

- (5

2)

CHAPTER 2

20

2

Future outlook: improving the performance of chewing gum as an oral health promoting nutraceutical

Over the last decades chewing gum developed from a candy towards an oral health

promoting nutraceutical to be used as an adjunct to regular oral hygiene. The basic

beneficial effects of the chewing of gum on oral health have been well documented and are

mostly officially approved by the EFSA (see Fig. 1 and Table 1). The same cannot be said

about many active ingredients that are incorporated in chewing gum to enhance the oral

health benefits perceived, mainly because most effects aimed for by chewing gum

additives are overshadowed by effects of increased salivation and mastication as readily

achieved by the chewing of regular, sugar-free gum.

The main hurdle in demonstrating oral health care benefits of active ingredients

added to chewing gums is the same as with many other nutraceuticals: their potency is

generally low. This implies that when evaluated in vitro, nutraceuticals will always do

significantly less well than the “positive controls”, that are often used therapeutically. The in

vitro comparison of nutraceuticals, including chewing gum additives, with a therapeutic

drug however, is not a valid one, as nutraceuticals are seldom or never used

therapeutically but most prophylactically.

Owing to the low potency of their active ingredients, chewing gums with active

ingredients incorporated as a nutraceutical, may and must be used several times a day

and for prolonged periods of time to demonstrate clinical efficacy. It has been proposed

that such studies should preferably last more than one year to map clinical effects of

chewing gums on oral health (66,78,119), which adds a major cost factor to the translation

of new additives to the market. Unfortunately, demonstration of clinical oral health care

benefits is easily clouded by other factors, particularly since the oral cavity is under the

influence of many environmental factors that are hard to control over longer periods of

time. As an alternative, it might be proposed that for nutraceuticals, such as chewing gums

with active ingredients with a low potency, significance levels greater than p < 0.05 should

be adopted with respect to a control in one and the same volunteer group.

The low antimicrobial potency of many chewing gum additives might turn into an

advantage when viewed with respect to gradually changing the composition of oral biofilm

into a less pathogenic one (120). Gradual is the preferred way of changing a microbiome in

order to make changes lasting (121) and to maintain a symbiotic relation with the host

(120). Chewing of gum has already been demonstrated to yield non-specific removal of

around 108 oral bacteria with a single chew (86) either from the planktonic, salivary or

biofilm microbiome. Although removal of 108 bacteria with a single chew may be


21

2

considered low compared to the total microbial load in the oral cavity, chewing gum can be

modified to specifically bind pathogenic bacteria and remove them from the oral cavity, for

instance by adding porous type calcium carbonate (122). Also inclusion of additives that

increase the surface hydrophobicity of specific bacteria may facilitate their removal from

the oral cavity by the subsequent administration of hydrophobic ligands, as has also been

demonstrated for the use of a triclosan containing toothpaste in combination with a

mouthrinse based on essential oils (123). As a final option to advance chewing gum further

to a nutraceutical that drives the oral microflora into a healthy direction, probiotics such as

lactobacilli can be added (124,125) to compete for a position on oral surfaces with oral

pathogens, similar to the events occurring in the gastro-intestinal tract between probiotics

and other members of the gastro-intestinal microbiome (126,127).

In summary, whereas evidence for oral health care benefits of chewing gum

additives is hard to obtain viz a viz additives in advanced toothpaste formulations or

mouthrinses due to their relatively low concentrations and rapid wash-out, the basic

benefits of the long-term chewing of sugar-free gum due to increased mastication and

salivation are mostly beyond dispute. Given the fact that the chewing of gum non-

specifically removes bacteria from the oral cavity by entrapment in gum with only temporal

effects, it seems feasible to construct gum formulations that do so in a more specific

fashion. Therewith long-term use of such gums may aid to restore and maintain a more

healthy oral microbiome, further contributing to the recognition of chewing gum as a

nutraceutical.

Conflict of interest This work was funded by Wm. Wrigley Jr. Co, Chicago, USA and SASA BV, Thesinge, NL.

Authors were employed by their own organizations. HJB is also director-owner of a

consulting company SASA BV. AM and MD are employees of the Wm. Wrigley Jr.

Company. Opinions and assertions contained herein are those of the authors and are not

meant to be construed as necessarily representing views of the organizations to which the

authors are affiliated.

CHAPTER 2

22

2

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37. EFSA Panel on Dietetic Products Nutrition and Allergies (NDA). Scientific opinion on the substantiation of health claims related to sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation ( ID 1309 ) pursuant to Article 13 (1) of Regulation (EC) No 1924/2006. EFSA J. 2011; 9(6):2268.

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106. Imfeld T. Chlorhexidine-containing chewing gum. Schweiz Monatsschr Zahnmed. 2006; 116:476–83.

107. Cosyn J, Verelst K. An efficacy and safety analysis of a chlorhexidine chewing gum in young orthodontic patients. J Clin Periodontol. 2006; 33(12):894–9.

108. Kolahi J, Soolari A. formulated chlorhexidine gluconate chewing gum that gives both anti-plaque effectiveness and an acceptable taste: a double blind, randomized, placebo-

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30

Effects of chewing gum with and

without active ingredients on oral

biofilm viability and composition

Chapter 3

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Abstract

Objectives

Chewing gum has developed into an oral care agent with multiple oral health benefits.

Active ingredients can be incorporated in chewing gum to improve the oral health benefits

of chewing gum. The aim of this study was to evaluate two chewing gums with widely

different active ingredients (Magnolia bark extract (MBE) and sodium hexametaphosphate

(SHMP)) for four weeks in vivo with respect to the total number of bacteria in oral biofilms

and their viability as well as with respect to the composition of the biofilm.

Methods

Ten healthy volunteers chewed gum with and without MBE or SHMP three times per day

for four weeks, during which oral biofilm was collected. Subsequently, the total number,

viability and composition of bacteria in the biofilm collected were determined.

Results

During four weeks of chewing gum use, both gums with and without active ingredients

yielded no significant decreases in the total numbers of bacteria and their viability in oral

biofilm. A trend of increasing diversity of the bacterial composition of the biofilms collected

was observed for all gums, including control gums without active ingredients added.

Conclusions

The chewing of sugar-free gum on a daily basis for a prolonged period of time can slowly

shift the bacterial composition of the oral biofilm in a more diverse and therewith healthy

direction, regardless of the addition of active ingredients such as MBE or SHMP.

EFFECTS OF CHEWING GUM ON ORAL BIOFILM VIABILITY AND COMPOSITION

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Introduction

Oral health and disease centers around the formation and removal of oral biofilm as the

main cause of oral diseases such as caries, gingivitis or periodontitis. Planktonic bacteria

in saliva adhere to the salivary conditioning films on oral soft and hard surfaces and form a

matrix of extracellular polymeric substances which gives structure to the biofilm and shields

its inhabitants from environmental influences (1). During oral health, the bacterial

composition of the oral microbiome is diverse and in symbiosis with the host (2).

Pathogenicity arises when a unbalance in symbiosis occurs causing a shift in the

composition of the oral microbiome, in which pathogenic strains dominate (3). For instance,

strains that use environmental sugars to produce acids, lower the pH and demineralize the

enamel causing caries. Periodontopathogens cause an inflammatory response in the

gingiva, leading to gingivitis or in more severe cases periodontitis. In case of caries it has

been shown that as the disease progresses, the overall diversity of the oral microbiome

decreases (4). Maintaining oral health largely involves preventing a shift in oral microbiome

composition towards a pathogenic direction and is mainly achieved by regularly performing

mechanical procedures to remove the oral biofilm not targeted towards specific pathogenic

strains or species, such as toothbrushing, flossing and the use of toothpicks. Additionally

special dentifrice formulations, mouthrinses and chewing gums have been promoted to

enhance oral health (5,6). However, the majority of the studies on oral microbiome

composition focus on saliva and not on oral biofilm, despite the fact it is particularly the

biofilm and not the salivary microbiome that stimulates the development of diseases (7).

Chewing gum has developed in the past centuries from a candy into an oral

health care agent and functional food product. Sugar-free gum has multiple oral health

benefits mainly by increasing salivary flow (8), washing away food debris (9) and

neutralizing oral biofilm pH (10). Daily use of sugar-free chewing gum for one year or more,

especially when consumed after a meal, is effective in reducing the incidence of caries

(11,12).

The main component of chewing gum is the gum base, consisting of a mixture of

elastomers, like polyvinylacetate or polyisobutylene. The gum base is complemented with

softeners, texturizers, emulsifiers and plasticizers. Based on desired functionality,

formulation of the latter ingredients is adapted, for instance to generate a gradual release

profile of active ingredients from chewing gum into the oral cavity. Prolonged presence

and substantive action in the oral cavity make chewing gum a good vehicle to deliver active

ingredients that promote oral health (13,14). Highly diverse ingredients have been added to

chewing gum for various purposes. Magnolia bark extract (MBE) applied in chewing gum

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reduces the total number of salivary bacteria, including the number cariogenic

Streptococcus mutans (15,16). MBE is also advocated for a broad range of disorders such

as coughing, fever, pain relief and diarrhea (15,17). Its active components include

magnolol and honokiol which both possess antimicrobial properties (18). Another totally

different active ingredient applied in chewing gum is sodium hexametaphosphate (SHMP).

SHMP is a surface active agent, inhibiting the formation of extrinsic tooth stain when

supplemented in chewing gum (19,20) and creating a more hydrophilic enamel surface in

vivo (21). The main hurdle in demonstrating efficacy of active ingredients applied in

chewing gum is the overriding effect of the chewing action itself, stimulating salivary flow.

Moreover, since all added ingredients released by the chewing of gum are swallowed, only

low concentrations of added ingredients can be applied and as a result effects of a single

chew are relatively small compared to effects of the single use of a toothbrush. Any effects

of the chewing of gum should therefore be evaluated over a period of at least several

weeks.

The aim of this study was to evaluate two chewing gums with widely different

active ingredients (MBE and SHMP) for a prolonged period of time in vivo with respect to

the viability and composition of the oral microbiome adhering to enamel surfaces. To this

end, ten healthy volunteers chewed gum with and without MBE and SHMP three times per

day for four weeks, during which oral biofilm was collected weekly for analysis.

Materials and methods

Subjects and inclusion criteria Ten healthy volunteers (5 females and 5 males, aged between 24 and 57 years)

participated in this study. The Medical Ethical Testing Committee of the University Medical

Center Groningen (METc 2011/330) approved this study and all subjects agreed to sign a

declaration of informed consent. All volunteers had a dentition with at least 16 natural

elements and considered themselves in good health. Use of antibiotics up to three months

prior to the study or use of a mouthrinse in the month preceding the study led to exclusion

from participation. During the study, use of mouthrinse, antibiotics, mints or other chewing

gum was not allowed.

Treatment and schedule Two weeks prior to the start of the study and continuing during the entire study, volunteers

brushed their teeth with a standard fluoridated toothpaste without antimicrobial claims


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(Prodent Softmint, Sara Lee Household & Bodycare, The Hague, The Netherlands)

according to their habitual routine.

After two weeks of regular brushing, at the beginning of the third week volunteers

were asked to come to the laboratory after breakfast without their morning brushing for the

collection of oral biofilm. This marked the start of four weeks of chewing two pellets of gum

three times per day. Gums with and without the active ingredients were packaged with a

code only to be disclosed after full analysis of all data at the end of the evaluation period.

Gums were randomly assigned to each volunteer. Volunteers were instructed to chew the

gum for ten minutes, with the three chewing points in time evenly spread over the day but

preferably after breakfast, lunch and dinner. The laboratory visits were repeated after one,

two and four weeks of the start of the study. After finishing the four week period with one of

the gums received, a four week washout period was taken into account during which the

volunteers solely had to brush with the standard, fluoridated toothpaste provided.

Subsequently, volunteers were given another coded batch of chewing gum and the

schedule was repeated.

Chewing gum Chewing gums were provided by Wm. Wrigley Jr. Company (Chicago, IL, USA). Active

ingredients were added to 1.5 g pellet shaped gums. MBE (3 mg, Honsea Sunshine

Biotech Co., Ltd, Guangzhou, China) was added to the coating of a gum containing:

gumbase, sorbitol, flavors, sweeteners and coolants. SHMP (7.5 % w/w Sigma Aldrich, St.

Louis, MO, USA) was added to a gum containing: gumbase, sorbitol, xylitol, glycerol,

flavors, sweeteners and coolants. Respective gums without the active ingredient added

were used as control gums.

Biofilm collection After instructions, volunteers collected oral biofilm themselves from the left upper quadrant

of the dentition (buccal, palatal, occlusal and interproximal sides of the dentition) using a

sterile hook and a cotton swab. Biofilm was suspended in 1 ml sterile Reduced Transport

Fluid (RTF) (22) and stored on ice immediately after collection. Next, biofilm samples were

sonicated 10 sec at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT,

USA) in RTF to suspend bacterial clumps. The bacterial suspension was partly used for

bacterial viability analysis. The remainder of the suspension was stored at -20 °C for later

bacterial composition analysis.

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Bacterial viability analysis The total number of bacteria in suspension was determined using a Bürker Türk counting

chamber and expressed as the total number of bacteria collected from a volunteer at each

time of collection. Bacterial viability was determined using 20 µL of sonicated biofilm

suspension. The suspension was spread on a glass microscope slide, stained with 15 µL

of LIVE/DEAD staining solution (BacLight™, Molecular Probes Europe BV) and covered by

a coverslip. Next, images were collected using a fluorescence microscope (Leica DM 4000

B, Leica Microsystems Heidelberg GmbH, Heidelberg, Germany). At least three images

per suspension were taken at random spots, with a minimum of 100 visible bacteria. Live,

green fluorescent and dead, red fluorescent bacteria were counted and results are

expressed as the percentage of the total number of bacteria counted. Subsequently, using

the total number of bacteria and the percentage of live/dead bacteria, the total number of

live and dead bacteria per suspension was calculated and averaged over all volunteers for

each time of collection.

Bacterial composition – Denaturing Gradient Gel Electrophoresis (DGGE) To determine bacterial composition, DGGE was performed. First, frozen bacterial

suspensions were thawed and centrifuged at 18000 g for 10 min, after which the pellet was

washed by resuspension in 200 µL tris-ethylenediaminetetraacetic-acid (TE) buffer (10 mM

Tris HCl, pH 7.5, 1 mM EDTA) and centrifuged again for 10 min. Isolation of chromosomal

DNA from the biofilm bacteria was done as has been described earlier (23). The DNA

concentration was measured using a NanoDrop® Spectrophotometer (ND-100, NanoDrop

Technologies Inc., Wilmington, DE, USA) at 230 nm. Polymerase Chain Reaction (PCR)

was performed with 100 ng of DNA on a T-gradient thermocycler (Bio-rad I-Cycler, GENO-

tronics BV, USA) to amplify the universal V3 region of the 16S rRNA gene in all samples

with the F357-GC forward primer and R-518 as the reverse primer (24). Products of the

PCR were applied on an 8% (w/v) polyacrylamide gel in 0.5 x TAE buffer (20 mM Tris

acetate, 10 mM sodium acetate, 0.5 mM EDTA, pH 8.3). The denaturing gradient gel

ranged from 30 – 80% urea made from a stock solution (100% denaturant equals 7 M urea

and 37% formamide). A stacking gel without denaturant was added on top. Electrophoresis

was started 200 V for the first 10 min, and adjusted for overnight electrophoresis to 120 V

at 60 °C. Gels were subsequently stained using a silver nitrate solution (0.2% AgNO3 (w/v))

until maximum staining intensity was observed.

Gelcompar II (v6.5 Applied Maths, Sint-Martens-Latem, Belgium) was used for gel

analysis. A reference lane with known bacterial species was used on every gel to align and

compare separate gels. The presence of a band was taken as indicative of the presence of


37

3

a bacterial strain or species in the sample, regardless of the staining intensity. Dice’s

similarity coefficient was calculated according to band-based matching with 0.5%

optimization and 0.5% band tolerance as accuracy settings.

Statistical analysis Data was tested for normality using probability plots, Kolmogorov-Smirnov and Shapiro-

Wilk tests (P<0.05). Subsequently, data was assessed for an effect within subjects during

four weeks of use. In case of normality, data was analyzed using a repeated measures

ANOVA followed by a Bonferroni test for pairwise comparison otherwise, when data was

not normally distributed, using the Friedman test (P < 0.05) followed by a Wilcoxon signed

rank test to identify pairwise differences. All statistical tests were performed using SPSS

v20.0 (IBM inc., Chicago, USA)

Results

On average, volunteers collected between 4 x 108 and 8 x 108 bacteria from their left upper

quadrants. Chewing of the MBE control gum yielded lower numbers of bacteria than

chewing of the SHMP control gum, although differences were small (Fig. 1). Unfortunately

due to experimental problems, one and two week data for the MBE and its control gum are

missing. Overall, no significant decrease in numbers of bacteria collected was seen over

the four weeks period of chewing MBE gum nor its control. This is similar to what is seen

for the SHMP gum and where the control gum yielded similar bacterial numbers over the

evaluation period, use of the SHMP gum gave a small downward trend over the first two

weeks, however going up again after four weeks of use of the gum. Whether or not this

trend would also have existed in the MBE gum group, cannot be concluded from the data

available. The trends in the total numbers of bacteria collected coincided fully with changes

in the number of live bacteria collected (see also Fig. 1).

CHAPTER 3

38

3

Figure 1

The total number of bacteria in biofilm collected from the upper left side of the dentition of a volunteer,

including the number of live bacteria and the percentage viability of the biofilms. Total number of

bacteria displayed represent the start of the chewing period (week 0) and after 1, 2 and 4 weeks of

chewing. Errors bars indicate standard errors of the mean total number of bacteria over all ten

volunteers. No significant differences exist between any of the data for the MBE and SHMP groups.

The compositional similarity of the biofilms collected over time as obtained from

DGGE profiles, decreased irrespective of gum type(Fig. 2A), concurrent with an increase in

the number of bands identified (Fig. 2B). This indicates that the oral biofilms become more

diverse upon the chewing of gum. During the washout period, the number of bands

decreased to low numbers in week 0 of all experimental periods, indicating that this

diversity is lost again during the washout period.

74%±2

73%±3

0.E+00

2.E+08

4.E+08

6.E+08

8.E+08

1.E+09

Week 0 Week 4

Tota

l num

ber o

f bac

teria

co

llect

edMBE control gum

Dead

Live

72%±3

68%±2

0.E+00

2.E+08

4.E+08

6.E+08

8.E+08

1.E+09

Week 0 Week 4

Tota

l num

ber o

f bac

teria

co

llect

ed

MBE gum

69% ±4

63% ±4

64% ±3

61% ±5

0.E+00

2.E+08

4.E+08

6.E+08

8.E+08

1.E+09

Week 0 Week 1 Week 2 Week 4

SHMP control gumDead

Live

73%±3

66%±4

71%±5

67%±3

0.E+00

2.E+08

4.E+08

6.E+08

8.E+08

1.E+09

Week 0 Week 1 Week 2 Week 4

SHMP gum


39

3

Figure 2

A: The percentage of similarity of DGGE profiles of biofilms collected in different groups of volunteers.

Profile comparisons of 1, 2 and 4 weeks of chewing were made with the DGGE profile before chewing

(week 0). Error bars indicate standard errors of the mean over all ten volunteers.

B: The number of DGGE bands, representative of a bacterial strain/species in biofilms collected in

different groups of volunteers. Errors bars denote standard errors of the mean over all ten volunteers.

Discussion

In this paper we aimed to demonstrate the effects of chewing gum with and without the

active ingredients MBE and SHMP on oral biofilm in vivo. Differences in the total number of

bacteria collected from biofilm of volunteers after chewing control gums or gums

supplemented with MBE or SHMP, showed a minor downward trend for all gums during

four weeks of chewing, that was however far less than one log-unit irrespective of the

active ingredient. Also decreases in live bacteria in biofilm were extremely small and

generally less than 5%. Overall, decreases in the total number and viability of bacteria

collected from biofilm of volunteers after chewing different gums must be considered too

789

1011121314151617

SHMP control gum SHMP gum

Week 0Week 1Week 2Week 4

789

1011121314151617

MBE control gum MBE gum

No.

of b

ands

B. Week 0Week 1Week 2Week 4

40

50

60

70

80

90

100

SHMP control gum SHMP gum

Similarity week 0 vs. 1 (%)Similarity week 0 vs. 2 (%)Similarity week 0 vs. 4 (%)

40

50

60

70

80

90

100

MBE control gum MBE gum

Sim

ilarit

y (%

)A. Similarity week 0 vs. 1 (%)

Similarity week 0 vs. 2 (%)Similarity week 0 vs. 4 (%)

CHAPTER 3

40

3

small to be clinically relevant. This conclusion coincides with previous reports stating that

the chewing of regular sugar-free gum is not effective in reducing oral biofilm (25,26).

Nevertheless, DGGE profile analysis did reveal a trend that biofilm composition

became more diverse during four weeks of chewing, irrespective of the gum type. This

effect was observed to be only temporal, since the biofilm composition returned to a less

diverse state during the washout period, which does not necessarily mean that bacterial

composition is the same as before four weeks of chewing. An increase in bacterial diversity

in oral biofilm is considered to be important in the maintenance of a healthy oral biofilm and

various studies have shown that progression of disease is related to the diversity of the

oral microbiome (3,4,27). For instance, the oral biofilm composition of children who did not

experience caries was more diverse than that of children who suffered from severe early

childhood caries (28). Bacterial diversity is similarly related to the occurrence of symptoms

in root canal infections and halitosis (3,27,29).

No differences were observed between the two widely different active ingredients

in the gums studied, which may reflect that the concentration of active ingredients in the

gums evaluated was too low or the length of the evaluation period too short. Alternatively, it

is likely that the effects observed in this study are solely related to the basic effects of

chewing sugar-free gum; mastication and stimulation of salivary flow. As food debris is

washed away during chewing (9), it also takes away sources of nutrients for bacteria, likely

affecting the composition of the oral biofilm.

In summary we have demonstrated that chewing two pieces of gum, three times

per day for four weeks does not significantly affect the total number and viability of bacteria

in oral biofilm, but that it creates a trend of increasing bacterial diversity in oral biofilm.

Since no differences between two widely different active ingredients were observed, this is

likely caused by the basic effects of chewing: mastication and stimulation of saliva. Disclosure statement This work was funded by Wm. Wrigley Jr. Co, Chicago, USA and SASA BV, Thesinge, NL.


consulting company SASA BV, AM, MWJD are employees of Wm. Wrigley Jr. Company.

Opinions and assertions contained herein are those of the authors and are not meant to be

construed as the representing views of the organizations to which the authors are affiliated.

Acknowledgements We would like to thank all volunteers for their participation in the study.


41

3

References

1. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000. 2011; 55(1):16–35.

2. Marsh PD, Head DA, Devine DA. Ecological approaches to oral biofilms: Control without killing. Caries Res. 2015; 49(Suppl 1):46–54.


4. Gross EL, Leys EJ, Gasparovich SR, Firestone ND, Schwartzbaum JA., Janies DA., et al. Bacterial 16S sequence analysis of severe caries in young permanent teeth. J Clin Microbiol. 2010; 48(11):4121–8.

5. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res. 2003; 82(3):206–11.

6. Otten MPT, Busscher HJ, Van der Mei HC, Abbas F, Van Hoogmoed CG. Retention of antimicrobial activity in plaque and saliva following mouthrinse use in vivo. Caries Res. 2010; 44(5):459–64.

7. Marsh PD. Dental plaque as a biofilm and a microbial community - implications for health and disease. BMC Oral Health. 2006; 6(Suppl 1):S14.

8. Bots CP, Brand HS, Veerman ECI, Van Amerongen BM, Nieuw Amerongen AV. Preferences and saliva stimulation of eight different chewing gums. Int Dent J. 2004; 54(3):143–8.

9. Fu Y, Li X, Ma H, Yin W, Que K.

Assessment of chewing sugar-free gums for oral debris reduction: a

randomized controlled crossover clinical trial. Am J Dent. 2012; 25(2):118–22.

10. Dodds MWJ, Chidichimo D, Haas MS.

Delivery of active agents from chewing gum for improved remineralization. Adv Dent Res. 2012; 24(2):58–62.


12. EFSA Panel on Dietetic Products Nutrition and Allergies (NDA). Scientific opinion on the substantiation of a health claim related to sugar free chewing gum and reduction of tooth demineralisation which reduces the risk of dental caries pursuant to Article 14 of Regulation (EC) No 1924/2006. EFSA J. 2010; 8(10):1775.

13. Chaudhary SA, Shahiwala AF.

Medicated chewing gum - a potential drug delivery system. Expert Opin Drug Deliv. 2010; 7(7):871–85.

14. Imfeld T. Chlorhexidine-containing chewing gum. Schweiz Monatssch Zahnmed. 2006; 116:476–83.

15. Greenberg M, Urnezis P, Tian M. Compressed mints and chewing gum containing magnolia bark extract are effective against bacteria responsible for oral malodor. J Agric Food Chem. 2007; 55(23):9465–9.


17. Hu Y, Qiao J, Zhang X, Ge C. Antimicrobial effect of Magnolia officinalis extract against Staphylococcus aureus. J Sci Food Agric. 2011; 91(6):1050–6.

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18. Ho K, Tsai C, Chen C. Antimicrobial activity of honokiol and magnolol isolated from Magnolia officinalis. Phyther Res. 2001; 141:139–41.


20. Porciani P, Grandini S. Whitening effect by stain inhibition from a chewing gum with sodium hexametaphosphate in a controlled twelve-week single-blind trial. J Clin Dent. 2006; 17(1):14–6.

21. Van der Mei HC, Kamminga-Rasker HJ,De Vries J, Busscher HJ. The influence of a hexametaphosphate-containing chewing gum on the wetting ability of salivary conditioning films in vitro and in vivo. J Clin Dent. 2003; 14(1):14–8.

22. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbiol. 1972; 24(4):638–44.

23. Ferreira AVB, Glass NL. PCR from fungal spores after microwave treatment. Fungal Genet Newsl. 1996; 43:25–6.

24. Muyzer G, De Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Env Microb. 1993; 59(3):695–700.

25. EFSA Panel on Dietetic Products Nutrition and Allergies (NDA). Scientific opinion on the substantiation of health claims related to sugar free chewing gum and reduction of dental plaque (ID 3084) pursuant to Article 13 (1) of Regulation (EC) No 1924/2006. EFSA J. 2010; 8(2):1480.

26. Mouton C, Scheinin A, Mäkinen K.

Effect on plaque of a xylitol-containing chewing-gum: A clinical and biochemical study. Acta Odontol Scand. 1975; 33:33–40.

27. He J, Li Y, Cao Y, Xue J, Zhou X. The

oral microbiome diversity and its relation to human diseases. Folia Microbiol (Praha). 2014; 60:69–80.

28. Li Y, Ge Y, Saxena D, Caufield PW. Genetic profiling of the oral microbiota associated with severe early-childhood caries. J Clin Microbiol. 2007; 45(1):81–7.

29. Kazor CE, Mitchell PM, Lee AM, Stokes LN, Dewhirst FE, Paster BJ, et al. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J Clin Microbiol. 2003; 41(2):558–63.

43

Effects of chewing gum on tooth

surface wettability and

mouthfeel perception

Chapter 4

CHAPTER 4

44

4

Abstract

Objectives

The use of sugar-free chewing gum is known to contribute to oral health, mainly through

stimulating salivary flow, washing away food debris and neutralizing the pH of the oral

biofilm. Adsorbed salivary conditioning films on tooth surfaces are important determinants

for tooth surface wettability and mouthfeel. The objective of this paper is to investigate the

effects of chewing gum, with and without active ingredients added, on tooth surface

wettability and mouthfeel perception in volunteers.


Ten healthy volunteers chewed two different gums both with and without active ingredients

(magnolia bark extract or sodium hexametaphosphate) for four weeks three times per day.

During four weeks of chewing, mouthfeel was assessed using a questionnaire and intra-

oral water contact angles were measured, both before, directly after and up to 60 min after

chewing.

Results

After using chewing gum, mouthfeel scores were significantly better than before chewing

lasting up to 60 min. Concurrently, intra-oral water contact angles decreased significantly

directly after chewing, creating a more hydrophilic tooth surface.

Conclusions

The chewing of gum, with or without active ingredients added, significantly increases tooth

hydrophilicity. Mouthfeel scores increased with increasing tooth hydrophilicity.

Clinical relevance

A positive subjective mouthfeel experience may constitute a stimulus for people to chew

gum and therewith stimulate salivary flow and wash away food debris with associated oral

health benefits.

EFFECTS OF CHEWING GUM ON TOOTH SURFACE WETTABILITY AND MOUTHFEEL

45

4

Introduction

The oral cavity is constantly under influence of environmental challenges such as food and

drink intake, frictional forces during mastication and changes in salivary flow rate and

composition throughout the day (1). These environmental challenges affect the

physiological functions and the perception of mouthfeel. Physiologically, salivary

conditioning films physically protect the enamel surface against acid challenges (2).

Specific salivary proteins adsorbed on the enamel surface facilitate the presence of

calcium and phosphate in the adsorbed film to help maintain a balance between re- and

demineralization (e.g. statherins). Other proteins have antibacterial properties (e.g.

lysozymes, IgA, lactoferrin) (3).

Salivary conditioning films also facilitates lubrication of oral surfaces to enable speech and

mastication (4) through the presence of adsorbed glycosylated proteins such as mucins.

Therewith, the salivary conditioning film is important for the mouthfeel as perceived by

consumers. Considerable research has been done over the past decades to evaluate

mouthfeel in consumers after use of specific toothpastes, mouthrinses or toothbrushes (5–

8). A positive mouthfeel not only reflects cleanliness of the dentition, but often relates with

the perception of fresh breath. Therewith a positive mouthfeel increases consumer

acceptance and constitutes a stimulus for people to perform oral healthcare procedures.

In the past decennia, chewing gum has developed more towards a nutraceutical

and oral health product. Sugar-free gum promotes oral health mainly by increasing salivary

flow (9), clearing food debris (10) and neutralizing oral biofilm pH (11). The chewing of gum

has been shown to ameliorate both the feelings of dry mouth and fresh breath (3,12–15).

Easy incorporation and gradual release, combined with prolonged contact with the oral

cavity (16) make chewing gum a suitable carrier for active ingredients. This has led to the

incorporation of antimicrobial agents such as magnolia bark extract (MBE) with a reported

efficacy in reducing the prevalence of sulfur producing bacteria (1) responsible for bad

breath (12,17) or sodium hexametaphosphate (SHMP), known to inhibit tooth stain

formation (18,19).

Considering the importance of mouthfeel perception as a stimulus in oral health

care maintenance, we here aim to determine the influence of the chewing of gum with

either MBE or SHMP incorporated on the hydrophilicity of oral hard surfaces and relate the

hydrophilicity to the mouthfeel perception in a group of volunteers. Ten volunteers chewed

gum, with and without MBE or SHMP added, after which they were asked to fill out a

mouthfeel questionnaire and intra-oral water contact angles were measured.

CHAPTER 4

46

4


Chewing gum Chewing gums were provided by Wm. Wrigley Jr. Company (Chicago, IL, USA). Active

ingredients were added to 1.5 g pellet shaped gums. MBE (3 mg, Honsea Sunshine

Biotech Co., Ltd, Guangzhou, China) was added to the coating of a gum containing:

gumbase, sorbitol, flavors, sweeteners and coolants. SHMP (7.5 % w/w Sigma Aldrich, St.

Louis, MO, USA) was added to a gum containing: gumbase, sorbitol, xylitol, glycerol,

flavors, sweeteners and coolants. Respective gums without the active ingredient added

were used as control gums.

Participants and inclusion criteria Ten healthy volunteers (five females and five males, aged between 25 to 57 years)

participated in this study. Volunteers gave their written informed consent to the study

design that was approved by the Medical Ethical Testing Committee of the University

Medical Center Groningen (METc 2011/330). Inclusion criteria described that volunteers

should consider themselves in good health and had a dentition with at least 16 natural

elements including the central incisors. Volunteers were excluded from the study in case

antibiotics were used up to three months prior to the study or a mouthrinse in the month

preceding the study. Also, the use of antibiotics, mouthrinses, mints or chewing gums other

than the prescribed chewing gum was not permitted during the entire study.

Treatment and schedule Two weeks prior to the start of the study and continuing during the entire study, volunteers

brushed their teeth with a standard fluoridated toothpaste without antimicrobial claims

(Prodent Softmint, Sara Lee Household & Bodycare, The Hague, The Netherlands)

according to their habitual routine.

After two weeks of regular brushing, at the beginning of the third week volunteers

were asked to come to the laboratory after breakfast without their morning brushing, which

marked the start of four weeks of chewing two pellets of gum three times per day. Gum

was chewed for 10 min, evenly spread over the day and preferably after breakfast, lunch

and dinner. Gums with or without active ingredients were double blind and randomly

assigned to the volunteers. Laboratory visits were repeated after one, two and four weeks

of chewing. Subsequently, after a four week washout period, during which no gum was

chewed, gum with active ingredients or control gum were switched among volunteers and

the cycle of laboratory visits was repeated.


47

4

During a laboratory visit, volunteers filled out a mouthfeel questionnaire and intra-

oral water contact angles were measured before and after chewing gum at the following

time-points; prior to chewing, directly after 10 min of chewing, 30 and 60 min after chewing.

Mouthfeel questionnaire Prior to chewing, directly after 10 min of chewing, as well as after 30 and 60 min after

chewing, volunteers were asked to fill out a questionnaire to evaluate their perceived

mouthfeel. The following questions needed to be scored on a “mouth condition likert-scale”

ranging from -2 (dislike extremely) to 2 (like extremely): - How clean do your teeth feel?

- How fresh is your breath? - How moist is your mouth?

- How smooth are your teeth?

- Overall: How does your mouth feel?

Mouthfeel scores were averaged for all time-points per visit over all volunteers.

Intra-oral water contact angles Directly after filling out the mouthfeel questionnaire at each time point, tooth surface

wettability was assessed. To this end, volunteers were seated in a dental chair in

horizontal position wearing a mouth-opening device to expose the maxillary central

incisors. These surfaces were dried to a plateau level by drawing ambient air over the tooth

surface for 30 s. Next, a droplet (approximately 1 µl) of ultrapure water (Sartorius Arium

611, Göttingen, Germany) was placed in the middle of the labial surface of a maxillary

central incisor and photographed (Canon EOS D30, Tokyo, Japan) 1 s after placement. A

minimum of 3 droplets for every time-point was photographed after which water contact

angles (θ) were determined using software according to

𝜃𝜃 = 2 tan−1 2ℎ𝑏𝑏

in which h is height and b the width of the baseline of the droplet. Intra-oral water contact

angles were averaged for each time point over all volunteers (20,21).

Statistics Mouthfeel questionnaire data was treated as interval data. To assess the effect within

individuals during four weeks use of chewing gum data were analyzed using the Friedman

test (P < 0.05). Since no effect on the mouthfeel experience was found over four weeks of

CHAPTER 4

48

4

use, data of all visits were pooled per gum and analyzed for short term changes up to 60

min after chewing and for differences between gums with and without active ingredients.

Again the Friedman test was used followed by a Wilcoxon signed rank test to identify

differences between groups.

Intra-oral water contact angle data was tested for normality using probability plots,

Kolmogorov-Smirnov and Shaprio-Wilk tests (P < 0.05). Subsequently, data were

assessed for an effect within individuals during four weeks of use employing a repeated

measures ANOVA. Since no changes in intra-oral water contact angles were observed

over four weeks, data of all visits were pooled per gum and analyzed for short term

changes up to 60 min after chewing and for differences between gums with and without

active ingredients. Again a repeated measures ANOVA was performed, followed by a

Bonferroni test for pairwise comparison.

To assess the relationship between intra-oral water contact angles and mouthfeel

perception, intra-oral water contact angles were averaged per mouthfeel score for all

mouthfeel questions together and displayed in a scatterplot for all gum types. All statistical

analyses were conducted with SPSS v23.0 (IBM corp., Armonk, USA).

Results

Directly after chewing, volunteers experienced a significantly better mouthfeel (Fig. 1).

Feelings of cleanliness, freshness of breath and overall mouthfeel were experienced

significantly better up to 60 min after chewing. The perceived moistness of the mouth and

smoothness of the teeth were also found to be better directly after chewing. However, this

effect did not always last up to 60 min. There were no significant differences between

gums with and without an active ingredient. However, when comparing MBE and control

gum to SHMP and control gum, it was noticed that in general there was a better mouthfeel

experience for the MBE and control gum (Fig. 1).

Concurrently, intra-oral water contact angles significantly decreased directly after

chewing, irrespective of the type of gum chewed, indicating increased tooth surface

hydrophilicity. Although this decrease in water contact angle became smaller over time, it

remained significant up to 60 min after chewing for the MBE and control gum, whilst for the

SHMP and control gum this decrease disappeared after 30 min (Fig. 2). However directly

after chewing, the SHMP gum showed the lowest intra-oral contact angle (48 ± 3 degrees),

opposite to the MBE gum, which gave rise to the


49

A-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Control gum (MBE)

MBE gum

*

**

-1.5

-1

-0.5

0

0.5

1

1.5

-10 0 10 20 30 40 50 60

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Mou

thfe

el sc

ore

Control gum (SHMP)

SHMP gum

**

* *

-1.5

-1

-0.5

0

0.5

1

1.5

-10 0 10 20 30 40 50 60

Mou

thfe

el sc

ore

*

**

*

**

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Mou

thfe

el sc

ore

*

**

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Mou

thfe

el sc

ore

** *

How clean do your teeth feel?

How fresh is your breath?

How moist is your mouth?

How smooth are your teeth?

4

highest intra-oral water contact angle (51 ± 2 degrees). No statistically significant

differences were observed between gums with and without active ingredients.

CHAPTER 4

50

45

50

55

60

65

70

-10 0 10 20 30 40 50 60

Cont

act a

ngle

(deg

rees

)

Time (min)

Control gum (SHMP)

SHMP gum

*

*45

50

55

60

65

70

-10 0 10 20 30 40 50 60Time (min)

Control gum (MBE)

MBE gum

***

*

*

*

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Time (min)

-1

-0.5

0

0.5

1

-10 0 10 20 30 40 50 60

Mou

thfe

el sc

ore

Time (min)

*

* *

Overall: How does your mouth feel?

4

Figure 1 (continued from previous page) Average mouthfeel scores as a function of time after chewing (min) for the different gum types

evaluated, as averaged over all four visits. The time points -10 min and 0 min indicate evaluations

immediately prior to chewing and directly after chewing, respectively. Asterisks (*) indicate significant

differences compared to prior to chewing. Error bars denote standard errors over ten volunteers.

Figure 2 Intra-oral water contact angles (θ) as a function of time after chewing (min) for the different gum types

evaluated, as averaged over all four visits. The time points -10 min and 0 min indicate evaluations

immediately prior to chewing and directly after chewing, respectively. Asterisks (*) indicate significant

differences compared to prior to chewing. Error bars denote standard errors over 10 volunteers.

A decrease in intra-oral water contact angle was accompanied by an improved

mouthfeel experience for the majority of the mouthfeel questions (Figs. 1 and 2). Upon

averaging the intra-oral water contact angles for every mouthfeel score for all questions, a

clear relationship between intra-oral water contact angles and mouthfeel score was

revealed (Fig. 3), indicating that a more hydrophilic tooth surface is generally preferred.


51

Mouthfeel score210-1-2

65

60

55

50

Control g MBE

M C

M C

R2 Li

MBE gumControl gum (MBE)

MBE gumControl gum (MBE)

R2 = 0.98R2 = 0.90

Mouthfeel score210-1-2

65

60

55

50

Control SH

SHMP gumControl gum (SHMP)SHMP gumControl gum (SHMP)

R2 = 0.36R2 = 0.89

Aver

age

cont

act a

ngle

(deg

rees

)

4

Figure 3 Intra-oral water contact angles (θ) as a function of mouthfeel scores averaged for all mouthfeel

questions. Linear relations between the intra-oral contact angles and the mouthfeel experience include

95% confidence intervals of the mean, indicated by the two outer lines.

Discussion

In this study we investigated the effects of chewing gum with and without active ingredients

on hydrophilicity of oral hard surfaces and mouthfeel perception in a group of volunteers.

Directly after chewing, irrespective of the type of gum chewed, intra-oral water

contact angles decreased significantly, indicating increased hydrophilicity of the tooth

surface. Since this was irrespective of the type of gum chewed, it is assumed that this

effect results mainly from increased salivation and the good lubricating properties of saliva

(22). As both the presence of water and glycosylated proteins increases during chewing

(1,23), more water molecules are retained at the surface, increasing lubrication and

hydrophilicity (24). A small part of the change in hydrophilicity can probably be attributed to

the active ingredients in the gums, as the SHMP-containing gum showed a slightly more

hydrophilic surface than the MBE-containing gum, which yielded the most hydrophobic

tooth surface. This is in line with earlier reports that chewing gum and dentifrices

containing SHMP create a more hydrophilic tooth surface (25,26) by desorbing

hydrophobic proteins from the conditioning film (24,27) and creating a phosphate-rich,

highly negatively charged conditioning film that is more open and penetrable for fluids (28).

The more hydrophobic conditioning film after chewing of MBE-containing gum is probably

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due to adsorption of the hydrophobic MBE components honokiol and magnolol (29,30). We

here propose that hydrophilicity of tooth surfaces may reflect the vulnerability of the

underlying enamel surface to acid attacks. Since directly after chewing, the hydrated

conditioning film retains minerals that protect and recover the tooth enamel (31,32), this

provides a secure environment which is translated in a reduced caries incidence (33).

Irrespective of active ingredients, the mouthfeel after chewing gum was perceived

better than before, often lasting up to 60 min. We demonstrated a clear correlation

between the overall mouthfeel and hydrophilicity of the tooth surface (Fig. 3). Especially

perception of oral moistness and smoothness are likely to be closely related to the surface

hydrophilicity and oral lubrication, since both are dependent on water retention at the

surface by glycosylated proteins. As the hydrophilicity of the surface decreases again

between 30 and 60 min after chewing, the perception of moistness and smoothness is also

lost more easily (Fig. 1 and 2). Feelings of cleanliness and freshness of breath can also be

connected to tooth surface hydrophilicity, but are more likely related to specific gum

ingredients. For instance, coolants, such as menthol or menthonone, cause an oral cooling

sensation (34,35) and are traceable in saliva up to 60 min after chewing (36). Also xylitol is

known to cause a longer lasting and stronger sensation of sweetness than sorbitol (37).

Since there were some differences between the gum used for MBE and the gum used for

SHMP it may explain the difference in mouthfeel perception by volunteers.

Previous studies showed that the chewing of gum created the subjective feeling of

a healthy oral cavity (14,38). Similar to after toothbrushing and use of mouthrinses, also

chewing gum causes and improved mouthfeel perception after use and is likely to stimulate

people to perform additional oral health care by the chewing of gum and benefit from the

oral health care advantages resulting from the chewing of gum, although these could not

be related to the incorporation of either MBE or SHMP in the gum.

Disclosure statement This work was funded by Wm. Wrigley Jr. Co, Chicago, USA and SASA BV, Thesinge, NL.


consulting company SASA BV, AM, MWJD are employees of Wm. Wrigley Jr. Company.

Opinions and assertions contained herein are those of the authors and are not meant to be

construed as the representing views of the organizations to which the authors are affiliated. Acknowledgements We would like to thank all volunteers for their participation in the study.


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References 1. Edgar M, Dawes C. Saliva and oral

health. 3rd ed. London: BDJ Books; 2004. 146 p.

2. Meurman J, Frank R. Scanning electron microscopic study of the effect of salivary pellicle on enamel erosion. Caries Res. 1991; 25(1):1–6.

3. Mathews SA, Kurien BT, Scofield RH. Oral manifestations of Sjögren’s syndrome. J Dent Res. 2008; 87(4):308–18.

4. Berg ICH, Rutland MW, Arnebrant T. Lubricating properties of the initial salivary pellicle - an AFM study. Biofouling. 2003; 19(6):365–9.

5. Shabir QSA, Skaria CS, O Brien HOB, Loni A, Barnett C, Canham L. Taste and mouthfeel assessment of porous and non-porous silicon microparticles. Nanoscale Res Lett. 2012; 7:407.

6. Kerr A, Corby P, Kalliontzi K, McGuire J, Charles C. Comparison of two mouthrinses in relation to salivary flow and perceived dryness. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2015; 119(1):59–64.

7. Klukowska M, Grender J, Conde E, Goyal C, Qagish J. A six-week clinical evaluation of the plaque and gingivitis efficacy of an oscillating-rotating power toothbrush with a novel brush head utilizing angled CrissCross bristles versus a sonic toothbrush. J Clin Dent. 2014; 25(2):6–12.

8. Gürgan CA, Zaim E, Bakirsoy I, Soykan E. Short-term side effects of 0.2% alcohol-free chlorhexidine mouthrinse used as an adjunct to non-surgical periodontal treatment: A double-blind clinical study. J Periodontol. 2006; 77(3):370–84.

9. Bots CP, Brand HS, Veerman ECI, Van

Amerongen BM, Nieuw Amerongen A V. Preferences and saliva stimulation of eight different chewing gums. Int Dent J. 2004; 54(3):143–8.

10. Fu Y, Li X, Ma H, Yin W, Que K. Assessment of chewing sugar-free gums for oral debris reduction: a randomized controlled crossover clinical trial. Am J Dent. 2012; 25(2):118–22.

11. Dodds MWJ, Chidichimo D, Haas MS. Delivery of active agents from chewing gum for improved remineralization. Adv Dent Res. 2012; 24(2):58–62.


13. Tanaka M, Toe M, Nagata H, Ojima M, Kuboniwa M, Shimizu K, et al. Effect of eucalyptus-extract chewing gum on oral malodor: a double-masked, randomized trial. J Periodontol. 2010; 81(11):1564–71.

14. Simons D, Baker P, Knott D, Rush S, Briggs T, Kidd EA, et al. Attitudes of carers and the elderly occupants of residential homes to antimicrobial chewing gum as an aid to oral health. Br Dent J. 1999; 187(11):612–5

15. Furness S, Worthington H. Interventions for the management of dry mouth: topical therapies. Cochrane Database Syst Rev. 2011; 12:1–106.


17. Campus G, Cagetti MG, Cocco F, Sale S, Sacco G, Strohmenger L, et al. Effect of a sugar-free chewing gum containing

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magnolia bark extract on different variables related to caries and gingivitis: a randomized controlled intervention trial. Caries Res. 2011; 45(4):393–9.

18. Sensabaugh RC, Sagel M. Stannous fluoride dentifrice with sodium hexametaphosphate: Review of laboratory, clinical and practice-based data. J Dent Hyg. 2012; 83(2):1–18.


20. Perdok JF, Weerkamp A, Van Dijk L, Arends J, Busscher H. Clinical determination of contact angles on tooth surfaces in vivo. J Dent Res. 1988; 67:701–2.

21. Van der Mei HC, White DJ, Busscher HJ. On the wettability of soft tissues in the human oral cavity. Arch Oral Biol. 2004; 49(8):671–3.

22. Selway N, Stokes JR. Soft materials deformation, flow, and lubrication between compliant substrates: impact on flow behavior, mouthfeel, stability, and flavor. Annu Rev Food Sci Technol. 2014; 5:373–93.

23. Payment SA, Liu B, Soares RV, Offner GD, Oppenheim FG, Troxler RF. The effects of duration and intensity of stimulation on total protein and mucin concentrations in resting and stimulated whole saliva. J Dent Res. 2001; 80(6):1584–7.

24. Veeregowda DH, Busscher HJ, Vissink A, Jager DJ, Sharma PK, Van der Mei HC. Role of structure and glycosylation of adsorbed protein films in biolubrication. PLoS One. 2012; 7(8):1–8.

25. Van der Mei HC, Kamminga-Rasker HJ, De Vries J, Busscher HJ. The influence of a hexametaphosphate-containing

chewing gum on the wetting ability of salivary conditioning films in vitro and in vivo. J Clin Dent. 2003; 14(1):14–8.

26. Van der Mei HC, White DJ, Kamminga-Rasker HJ, Knight J, Baig AA, Smit J, et al. Influence of dentifrices and dietary components in saliva on wettability of pellicle-coated enamel in vitro and in vivo. Eur J Oral Sci. 2002; 110(6):434–8.

27. Rykke M, Rölla G. Desorption of acquired enamel pellicle in vivo by pyrophosphate. Eur J Oral Sci. 1990; 98(3):211–4.

28. Busscher HJ, White DJ, Kamminga-Rasker HJ, Poortinga AT, Van der Mei HC. Influence of oral detergents and chlorhexidine on soft-Layer electrokinetic parameters of the acquired enamel pellicle. Caries Res. 2003; 37(6):431–6.

29. Zhang L, Wang X. Hydrophobic ionic liquid-based ultrasound-assisted extraction of magnolol and honokiol from cortex Magnoliae officinalis. J Sep Sci. 2010; 33(13):2035–8.

30. William Wrigley Jr. company. Application for the approval of magnolia bark supercritical carbon dioxide extract (MBSE) from Magnolia officinalis. Regulation (EC) No 258/97 of the European parliament and the council of 27th January 1997 concerning novel foods and novel food ingredients. 2009; 258: 79p.

31. Wu W, Nancollas GH. The relationship between surface free-energy and kinetics in the mineralization and demineralization of dental hard tissue. Adv Dent Res. 1997; 11(4):566–75.

32. Lendenmann U, Grogan J, Oppenheim

FG. Saliva and dental pellicle - a review. Adv Dent Res. 2000; 14(1):22–8.

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Bezerra AC, Cruvinel V. Sugar-free chewing gum and dental caries: a systematic review. J Appl Oral Sci. 2007; 15(2):83–8.

34. McKemy DD, Neuhausser WM, Julius

D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature. 2002; 416(6876):52–8.

35. Eccles R. Role of cold receptors and menthol in thirst, the drive to breathe and arousal. Appetite. 2000; 34(1):29–35.

36. Haahr AM, Bardow A, Thomsen CE, Jensen SB, Nauntofte B, Bakke M, et al. Release of peppermint flavour compounds from chewing gum: effect of oral functions. Physiol Behav. 2004; 82(2-3):531–40.

37. Ovejero-López I, Bro R, Bredie WLP. Univariate and multivariate modelling of flavour release in chewing gum using time-intensity: a comparison of data analytical methods. Food Qual Prefer. 2005; 16(4):327–43.

38. Hashiba T, Takeuchi K, Shimazaki Y, Takeshita T, Yamashita Y. Chewing xylitol gum improves self-rated and objective indicators of oral health status under conditions interrupting regular oral hygiene. Tohoku J Exp Med. 2015; 235(1):39–46.

56

Adhesion forces and composition

of planktonic and adhering

microbiomes

Stefan W. Wessel, Yun Chen, Amarnath Maitra, Edwin R. van den Heuvel,


Journal of Dental Research, 2014; 93(1): 84-88

Reprinted with permission from SAGE Publishing

Chapter 5

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Abstract

The oral microbiome consists of a planktonic microbiome as residing in saliva and an

adhering microbiome; the biofilm adhering to oral hard and soft tissues. Here we

hypothesize, that possible differences in microbial composition of the planktonic and

adhering oral microbiome on teeth can be related to the forces by which different bacterial

species are attracted to the tooth surface. The relative presence of seven oral bacterial

species in saliva and biofilm collected from ten healthy human volunteers was determined

twice in each volunteer using denaturing gradient gel electrophoresis. Analysis of both

microbiomes showed complete separation of the planktonic from the adhering oral

microbiome. Next, adhesion forces of corresponding bacterial strains with saliva coated

enamel surfaces were measured using atomic force microscopy. Species that were found

predominantly in the adhering microbiome had significantly higher adhesion forces to

saliva coated enamel (-0.60 to -1.05 nN) than species mostly present in the planktonic

microbiome (-0.40 to -0.55 nN). It is concluded that differences in composition of the

planktonic and the adhering oral microbiome are due to small differences in the forces by

which strains adhere to saliva coated enamel, providing an important step in understanding

site- and material-specific differences in composition of biofilms in the oral cavity.

ADHESION FORCES AND COMPOSITION OF PLANKTONIC AND ADHERING MICROBIOMES

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Introduction

The oral microbiome is a highly diverse and intricate system, consisting of more than 700

bacterial strains and species (1), although remarkably higher estimates of the microbial

diversity in the oral cavity have been reported as well (2). The oral microbiome is different

for every individual and specific to different sites and materials in the oral cavity. Minor

disturbances in the delicate balance of the oral microbiome can lead to the onset of

disease, such as caries and periodontitis (3). Oral bacteria reside either in a planktonic state, as in saliva or in an adhering

state, as in a biofilm on oral hard and soft surfaces. Although the oral microbiome is site-,

material- and subject-specific, there is the concept of a general core microbiome, that plays

an important role in the formation of oral biofilm (1,4). The formation of an oral biofilm is

initiated by the adhesion of initial colonizers (5–7) to adsorbed salivary conditioning films.

As such, oral biofilm can be regarded as a transition of bacteria from the planktonic

microbiome to the adhering microbiome on oral hard and soft surfaces. This transition is

mediated by an interplay between ligand-receptor interactions and non-specific Lifshitz-

Van der Waals, acid-base and electrostatic forces.

Bacterial probe atomic force microscopy (AFM) has enabled the measurement of

the forces by which bacteria are attracted to surfaces and relatively small differences in

adhesion forces have been demonstrated to have major implications with respect to the

ability of bacteria to adhere to a surface (8). Such observations lead us to hypothesize that

differences in the composition of the planktonic and adhering oral microbiome on oral hard

surfaces can be related to the force by which different bacterial strains and species are

attracted to the tooth surface.

The aim of this study is to verify the hypothesis that differences in the composition

of the planktonic microbiome and adhering oral microbiome on oral hard surfaces can be

related to the force by which different bacterial strains and species are attracted to the

tooth surface. To this end, we first determined the strains and species predominantly

present in the planktonic and adhering microbiomes of ten healthy volunteers using

Denaturing Gradient Gel Electrophoresis (DGGE) and related their prevalence to the

average adhesion forces of strain representatives for different species to saliva coated

enamel measured with bacterial probe AFM.

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Subjects and inclusion criteria Ten healthy volunteers (6 females and 4 males, aged between 24 to 57 years) participated

in this study. The study design was approved by the Medical Ethical Testing Committee of

the University Medical Center Groningen (METc 2011/330) and all subjects signed a

declaration of informed consent. All volunteers considered themselves in good health and

had a dentition with at least 16 natural elements. Volunteers having used antibiotics up to

three months prior to the study or having used a mouthrinse in the month prior to the study

were excluded. Two weeks before entering the study, volunteers were requested to brush

their teeth according to their habitual oral hygiene, but the use of a standard, fluoridated

toothpaste without antimicrobial claims (Prodent Softmint, Sara Lee Household &

Bodycare, The Hague, The Netherlands) was imposed, as during the entire study.

Sample collection Biofilm

Volunteers were asked to come to the laboratory for biofilm collection immediately after

breakfast, without having brushed their teeth. Biofilm was collected using a sterile hook

and a cotton swab from the left upper quadrant of the dentition (buccal, palatal, occlusal

and interproximal sides of the dentition). Biofilm was suspended in 1 ml sterile Reduced

Transport Fluid (RTF) (9) and all biofilm samples were immediately put on ice after

collection and sonicated 10 sec at 30 W (Vibra Cell model 375, Sonics and Materials Inc.,

Danbury, CT, USA) to suspend bacterial clumps. The samples were stored in RTF at -20

°C for subsequent DGGE analysis.

Saliva

Volunteers were requested to collect 2 ml of unstimulated whole saliva. Saliva samples

were put directly on ice after collection and sonicated two times 10 sec at 30 W.

Subsequently, samples were centrifuged at 18000 g for 5 min (Eppendorf Centrifuge

5417R, Hamburg, Germany), supernatant was removed and the pellet was resuspended in

200 µL TE buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA) and stored at -20 °C for later

DGGE analysis.

Biofilm and saliva samples were taken twice from each volunteer with a six weeks

interval in between and treated as separate samples.


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DGGE analyses of biofilm and saliva samples DNA extraction, PCR and DGGE analysis are described in Appendix I. In order to compare

the gels, reference markers were added, containing various species representing the oral

microbiome in health and disease namely: Streptococcus mutans ATCC10449,

Streptococcus sanguinis ATCC10556, Streptococcus sobrinus ATCC33478, Streptococcus

salivarius HB, Streptococcus mitis ATCC9811, Streptococcus oralis ATCC35307 and an

isolate of Lactobacillus sp.

Gelcompar II (v6.5 Applied Maths) was used for gel analysis. Presence of

reference species in all samples was analyzed using band-based matching with 0.5%

optimization and 0.5% band tolerance as accuracy settings. Presence of a band was taken

indicative of the presence of the reference species in the sample, regardless of the staining

intensity. Dice’s similarity coefficient was used to construct a similarity matrix. A

dendrogram was calculated based on the non-weighted pair group method with arithmetic

averages as clustering algorithm (10). Atomic force microscopy measurements AFM was used to measure adhesion forces of selected bacterial strains to saliva coated

enamel surfaces. Nine oral bacterial strains, comprising a combination of different

laboratory strains and clinical isolates, representative for the oral microbiome were

included in AFM force measurements: S. mutans ATCC700610, S. mutans NS, S.

sanguinis ATCC10556, S. sobrinus HG1025, S. salivarius HB, S. mitis BMS, S. mitis

ATCC9811, S. oralis J22, Lactobacillus acidophilus JP and a Lactobacillus isolate. Bacteria

were grown on blood agar plates for 24 h at 37 °C from frozen DMSO stock and inoculated

in 10 ml Todd-Hewitt broth (Oxoid, Basingstoke, UK) for 24 h at 37 °C in ambient air. The

pre-culture was used to inoculate a main culture which was grown for 16 h. Bacterial

harvesting was done by centrifugation (5000 g, 5 min). Subsequently, the pellet was

washed twice and resuspended in demineralized water. Bacterial clumps were suspended

by sonicating 3 x 10 sec at 30 W.

Human whole saliva, stimulated by chewing parafilm, was collected of both

genders in ice cooled beakers. All volunteers gave their informed consent agreeing with

the rules as stated by the Medical Ethical Testing Committee of the University Medical

Center Groningen (letter 06-02-2009). The saliva was pooled, centrifuged to remove

particulate debris, dialyzed against demineralized water and subsequently lyophilized for

storage. Lyophilized saliva was reconstituted at a concentration of 1.5 g/l in adhesion

buffer (50 mM potassium chloride, 2 mM potassium phosphate, 1 mM calcium chloride, pH

6.8).

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Enamel slabs were cut (0.6 x 0.6 x 0.2 cm) from the buccal surfaces of bovine

incisors, grinded and polished, as described in Appendix II. Salivary conditioning films were

created by immersing the enamel slabs into reconstituted saliva for 16 h at 4 °C. Before

use in AFM experiments, the slabs were dipped three times in demineralized water to

remove excess saliva.

Tipless AFM cantilevers (MP-010, Bruker, Billerica, USA) with immobilized

bacteria were prepared and AFM was performed (BioScope Catalyst AFM Bruker, Billerica,

USA), as described in Appendix III. For each combination of bacterial strains, at least 40

force-distance curves were recorded at randomly chosen spots with two to four bacterial

probes and bacteria from at least two different cultures of each strain (for an example of a

force-distance curve, see Appendix Fig. 1).

Statistics DGGE profiles were assessed on predominant presence of bacterial species in the

planktonic or adhering microbiome. Therefore a three way mixed effects ANOVA model

was fitted to the bacterial presence among volunteers to investigate the effect of the

microbiome for each species separately at a significance level of 0.05. The relative

presence of species (adhering microbiome minus planktonic microbiome) in the model was

taken random to be able to overcome the sparse data for the relative large number of

species and address possible correlations, the microbiome and period were taken as fixed

effects. The relative presence of species was estimated and accompanied with a 95%

confidence interval. The residuals were investigated to evaluate the model fit.

Adhesion forces of individual bacterial strains displayed a skewed distribution

(Shapiro-Wilk test, P < 0.01) and are presented as medians. Species averaged adhesion

forces were calculated as weighted averages over the different strains used in AFM to

represent a given species and presented as means with standard errors. SAS v9.3 (SAS

institute inc., Cary, USA) and SPSS v20.0 (IBM Corp., Armonk, USA) were used to

conduct statistical analysis.

Results

The planktonic and adhering oral microbiomes of nine out of all ten volunteers separated at

the main branch of the clustering tree (Fig. 1) of their DGGE profiles (Appendix Fig. 2) at

36% similarity to each other. Samples taken on different occasions from each volunteer

showed remarkably high similarities, on average 75%, indicating no periodic carry over

effects.


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This difference in microbial composition between both microbiomes was further

analyzed by determining the species predominantly present in the adhering and planktonic

microbiomes (Fig. 2). S. mutans, S. sanguinis and Lactobacillus strains were

predominantly present in the adhering microbiome of the volunteers. S. mutans was not

detected in saliva samples of any of the volunteers, but present in biofilm samples of 50%

of all volunteers. S. sanguinis was present in 30% of all saliva samples, while found in 85%

of all biofilm samples. No Lactobacillus strains were found in any of the saliva samples and

only in 30% of all biofilm samples.

Figure 1 Clustering tree from the DGGE profiles of the adhering and planktonic oral microbiomes, indicated as

“biofilm” and “saliva” respectively as taken from ten healthy volunteers, on two different occasions from

each volunteer (individual volunteers are indicated by numbers). Clustering tree was based on a band-

based percentage similarity matrix.

Bacteria significantly more present in saliva samples were identified as S.

sobrinus (60% in saliva samples versus 35% in biofilm samples) and S. salivarius (90% in

saliva samples versus 35% in biofilm samples). In nine out of ten volunteers, S. mitis and

S. oralis were found in both microbiomes.

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Figure 2 The percentage of volunteers carrying specific bacterial species in their adhering and planktonic oral

microbiomes, indicated as “biofilm” and “saliva” respectively. Error bars denote SD over ten

volunteers, each measured on two occasions. Statistically significant differences in species

occurrence in saliva and biofilm (P < 0.05) according to three way mixed effects ANOVA model,

corrected for periodic effects, are indicated by an asterisk (*). The residuals of the model do not

demonstrate outliers and the distribution is close to a normal distribution implying an appropriate

description of the observed bacterial presence.

The relative presence (percentage of volunteers with the species present in the

adhering microbiome minus the percentage occurrence in the planktonic microbiome) of

different species toward the adhering microbiome increased with their adhesion forces to

saliva coated enamel (Fig. 3). For species predominantly present in the adhering

microbiome, species averaged adhesion forces varied from -0.60 nN to -1.05 nN. S.

sanguinis and Lactobacillus sp. showed the highest adhesion forces followed by S.

mutans. Bacteria predominantly present in the planktonic microbiome, S. salivarius and S.

sobrinus, had lower species averaged adhesion forces between -0.40 and -0.55 nN.

Bacterial species without a predominant presence in either microbiome, S. mitis and S.

oralis, had adhesion forces in the range of -0.60 nN to -0.80 nN. Note that in Appendix Fig.

3, we present the adhesion forces of the individual strains used in AFM to represent a

given species and obtain species averaged adhesion forces.


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Figure 3 The relative presence of different species in human volunteers as a function of the species averaged

adhesion force to saliva coated enamel surfaces, separated with respect to their predominant

occurrence in the planktonic (saliva) or adhering (biofilm) microbiome. Adhesion forces are displayed

as mean values over all representatives of a given species used in the AFM part of this study with

standard errors. Linear regression was significant at P < 0.05 and 95% confidence intervals of the

mean is indicated by two outer lines.

Discussion

This paper demonstrates the validity of our hypothesis that differences in the composition

of the planktonic and adhering oral microbiome on oral hard surfaces are related to the

forces by which different bacterial species adhere to the tooth surface, i.e. saliva coated

enamel. Bacterial adhesion forces were higher for species predominantly residing in the

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adhering microbiome than for species mostly present in the planktonic microbiome. To the

best of our knowledge, this is the first time that such an influence of bacterial adhesion

forces on the composition of oral biofilm versus bacterial composition in saliva is

demonstrated. As an important additional observation, residence in the planktonic or

adhering microbiome appears to be dictated by species averaged adhesion forces that

range between -0.40 and -1.05 nN. Species with adhesion forces stronger than -0.55 nN

end up in the adhering microbiome, while species with smaller adhesion forces become

member of the planktonic microbiome. A very similar adhesion force value of -0.5 nN has

been suggested to dictate whether E. coli would become a predominant member of the

adhering urogenital microbiome (8). It is amazing how such small differences in bacterial

adhesion forces can select strains from saliva to become member of the adhering

microbiome. Earlier, it has already been found that initial colonizers of dental hard surfaces

possess adhesion forces to saliva coated enamel that are only 0.1 nN stronger than of later

colonizing, more cariogenic strains (11). With respect to Staphylococcus aureus strains, a

difference in adhesion force of 0.28 nN appears to dictate whether a strain can invade

mammalian cells or not (12). Such observations endorse the concept that bacteria adhere

to a surface according to their own specific characteristics, such as their specific hydrogen

bonding capability and ability to form ligand-receptor bonds (13,14). In a first instance, it

appears as a limitation of our study that other interactions between bacteria, such as co-

adhesion between different strains and species, are not included in the analysis. However,

selection of appropriate co-adhesion partners also seems governed by the magnitude of

the adhesion forces between the co-adhesion partners (15) similar as to how the adhering

microbiome arises from adhesion force governed selection from the planktonic

microbiome, as we show in this paper.

The complete separation of the adhering and planktonic oral microbiomes as

found in our study (Fig. 1) is in line with literature results, showing that biofilm and saliva

samples display statistically significant clustering profiles with no significant differences

between PCR-DGGE profiles of males and females (16). S. mitis and S. oralis were found

in 90% of our volunteers (Fig. 2) and are indeed known to account for a major proportion of

all Streptococcus spp. (17) in the oral cavity. Note that DNA analysis in PCR-DGGE can

yield multiple and overlapping bands (18,19), as in the present case for S. mitis and S.

oralis, making it impossible to discriminate between these two species (Appendix Fig. 2).

Levels of S. mutans and S. sobrinus in the adhering microbiome relative to each other are

in line with literature, where the latter is rarely found in higher numbers than S. mutans

(20,21). Finally, the complete absence of sub-gingival species, like Porphyromonas

gingivalis in all samples (Appendix Fig. 2) is supported by literature as well (1). This


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agreement with literature data demonstrates that the group of volunteers used can be

considered as a representative one and justifies its use to support evidence for our

hypothesis that the composition of the adhering microbiome is determined by the adhesion

force values of different strains and species.

Interestingly, some species (S. mutans, Lactobacillus sp.) were only found in the

adhering microbiome on oral hard surfaces, whereas the planktonic microbiome naturally

constitutes the species’ origin. This attests to our conclusion that the ability of a strain to

adhere determines its presence in either the adhering or planktonic microbiome. Evidently,

in case of strains present below the detection limit of DGGE (22), stronger adhesion forces

lead to selection followed by a detectable presence of these strains in the adhering

microbiome on oral hard surfaces. These observations support ongoing discussion in

literature that the microbial composition of saliva is of minimal value for the prediction of

dental caries and should only be used as an association instead of a causal relationship

(21,23–25).

The correlation between adhesion forces and the predominant presence of

bacterial strains and species in either the salivary or adhering microbiome demonstrated in

this research, is highly new and provides an important step in understanding the site- and

material-specific differences in composition of biofilms in the oral cavity.

Acknowledgements We would like to thank all volunteers for their cooperation in this study. This work was

funded by Wrigley Co, Chicago, USA/SASA BV, Thesinge, NL. Authors were employed by

their own organizations. The authors declare no potential conflicts of interest with respect

to authorship and/or publication of this article. Opinions and assertions contained herein

are those of the authors and are not construed as necessarily representing views of the

funding organizations.

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13. Gibbons RJ, Etherden I, Moreno EC. Contribution of stereochemical interactions in the adhesion of Streptococcus sanguis C5 to experimental pellicles. J Dent Res. 1985; 64(2):96–101.

14. Nobbs AH, Jenkinson HF, Jakubovics NS. Stick to your gums: mechanisms of oral microbial adherence. J Dent Res. 2011; 90(11):1271–8.

15. Postollec F, Norde W, De Vries J, Busscher HJ, Van der Mei HC. Interactive Forces between co-aggregating and non-co-aggregating oral bacterial pairs. J Dent Res. 2006; 85(3):231–4.

16. Ling Z, Kong J, Jia P, Wei C, Wang Y, Pan Z, et al. Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing. Microb Ecol. 2010; 60(3):677–90.

17. Diaz PI, Dupuy a K, Abusleme L, Reese B, Obergfell C, Choquette L, et al. Using high throughput sequencing to explore the biodiversity in oral bacterial communities. Mol Oral Microbiol. 2012; 27(3):182–201.

18. Janse I, Bok J, Zwart G. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J Microbiol Meth. 2004; 57(2):279–81.


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19. Kušar D, Avguštin G. Optimization of the DGGE band identification method. Folia Microbiol. 2012; 57(4):301–6.

20. Mäkinen K, Isotupa KP, Mäkinen P. Six-month polyol chewing-gum programme in kindergarten-age children: a feasibility study focusing on mutans streptococci and dental plaque. Int Dent J. 2005; 55:81–8.

21. Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Comm Dent Oral. 2005; 33(4):248–55.

22. Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol. 1996; 11(4):266–73.

23. Van Houte J. Microbiological predictors of caries risk. Adv Dent Res. 1993; 7(2):87–96.

24. Van Palenstein Helderman WH, Mikx FH, Van’t Hof MA, Truin G, Kalsbeek H. The value of salivary bacterial counts as a supplement to past caries experience as caries predictor in children. Eur J Oral Sci. 2001; 109(5):312–5.

25. Gamboa F, Estupiñan M, Galindo A. Presence of Streptococcus mutans in saliva and its relationship with dental caries: Antimicrobial susceptibility of the isolates. Univ Sci. 2004; 9:23–7.

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Appendices

Adhesion forces and composition

of planktonic and adhering

microbiomes

Stefan W. Wessel, Yun Chen, Amarnath Maitra, Edwin R. van den Heuvel,


Journal of Dental Research, DOI: 10.1177/0022034513511822

Reprinted with permission from SAGE Publishing

Appendices chapter 5

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Appendix I. DNA Extraction, PCR and DGGE

DNA was isolated from thawed samples and centrifuged at 18000 g for 10 min. The pellet

was washed by resuspension in 200 µL TE buffer and centrifuged again for 10 min.

Isolation of chromosomal DNA for both biofilm and saliva samples was done as been

described earlier (1). The DNA concentration was measured using a NanoDrop®

Spectrophotometer (ND-100, NanoDrop Technologies Inc., Wilmington, DE, USA) at 230

nm. PCR was performed with 100 ng of DNA on a T-gradient thermocycler (Bio-rad I-

Cycler, GENO-tronics BV, USA) to amplify the universal V3 region of the 16S rRNA gene

in all samples with the F357-GC forward primer and R-518 as the reverse primer (2). The

PCR products were applied on an 8% (w/v) polyacrylamide gel in 0.5 x TAE buffer (20 mM

Tris acetate, 10 mM sodium acetate, 0.5 mM EDTA, pH 8.3). The denaturing gradient had

a range of 30–80% and was made with a stock solution (100% denaturant equals 7 M

urea and 37% formamide). A 10 ml stacking gel was made without denaturant and added

on top. Electrophoresis conditions were set at 200 V for the first 10 min, and then set to

120 V at 60 °C overnight. Gels were stained for at least 10 min using a silver nitrate

solution (0.2% AgNO3 (w/v)) until maximal staining intensity was observed.

Appendix Figure 1 Example of a force distance curve between a bacterial probe (S. salivarius HB) and saliva coated

enamel. The grey line shows the approach curve towards the surface, while the black line shows the

retraction curve, with the maximum adhesion force occurring around 200 nm.

Appendix II. Enamel preparation Enamel slabs were cut from the buccal surface of bovine incisors into pieces of 0.6 x 0.6 x

0.2 cm under running tap water and grinded with 220 to 1200 grit sandpaper. The surface

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was micropolished for 3 min using wet polishing pad with 0.05 µm alumina particles

(Buehler Ltd., Lake Bluff, IL, USA) and subsequently cleaned with demineralized water

and 2 min of sonication in a 35 kHz bath (Transsonic TP 690-A, Elma, Germany). Saliva

conditioning films were created by immersing the enamel slabs into reconstituted saliva for

16 h at 4 °C. Before use in AFM experiments the slabs were dipped three times in

demineralized water.

Appendix Figure 2 Dendrogram analysis of bacterial DGGE profiles of saliva (green) and biofilm samples (red). Numbers

indicate different volunteers and first (A) or second (B) sampling moment. Positions of reference

strains, used to identify bacterial species presence, are displayed above the DGGE profiles.

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Appendix III. Preparation of bacterial probes for AFM For the immobilization of bacteria on tipless AFM cantilevers (MP-010, Bruker, Billerica,

USA), cantilevers were first immersed for 1 min in a drop 0.01 % (w/v) poly-L-lysine

(Sigma, Poole, UK). After air drying for 2 min, the tip of the cantilever was dipped in a

bacterial suspension for 1 min to immobilize the bacteria to the tip. All cantilevers were

used immediately after preparation for experiments. AFM measurements were initiated in

contact mode on a BioScope Catalyst AFM (Bruker, Billerica, USA) in adhesion buffer at

room temperature, with a scan rate of 0.5 Hz, a ramp size of 1.5 µm and a trigger threshold

of 3 nN.

Bacterial adhesion forces were retrieved from force distance curves. To this end,

the cantilevers spring constant, determined for each experiment, was used to convert the

cantilever deflection into force values (nN). In order to verify that a bacterial probe enabled

a single contact with the enamel surface, a scanned image in AFM contact mode with a

loading force of 1-2 nN was made at the onset of each experiment and examined for

double contour lines, which are indicative of multiple bacteria on the probe in contact with

the enamel surface. Any probe exhibiting double contour lines was discarded. To ensure

that a bacterial probe was not affected by previous measurements, force curves at 0 sec

surface delay on clean glass were regularly compared to five initially measured control

force curves on glass. If a continued measurement differed by more than 0.2 nN from the

initial control force, data were discarded and a new probe prepared.

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Appendix Figure 3 Median adhesion forces (nN) of individual bacterial strains to saliva coated enamel used to determine

species average adhesion forces. Error bars denote SE, while colors indicate a predominant presence

of a given species in a particular microbiome, as based on DGGE analysis.

References


2. Muyzer G, de Waal EC, Uitterlinden a G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Env Microb. 1993; 59(3):695–700.

76

Quantification and qualification

of bacteria trapped

in chewed gum

Stefan W. Wessel, Henny C. van der Mei, David Morando, Anje M Slomp,

Betsy van de Belt-Gritter, Amarnath Maitra and Henk J. Busscher

PLoS One., 2015; 10(1): e0117191

Reprinted with permission from PLoS

Chapter 6

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Abstract

Chewing of gum contributes to the maintenance of oral health. Many oral diseases,

including caries and periodontal disease, are caused by bacteria. However, it is unknown

whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize

that chewing of gum can trap bacteria and remove them from the oral cavity. To test this

hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed

gum. In the first method, known numbers of bacteria were finger-chewed into gum and

chewed gums were molded to standard dimensions, sonicated and plated to determine

numbers of colony-forming-units incorporated, yielding calibration curves of colony-

forming-units retrieved versus finger-chewed in. In a second method, calibration curves

were created by finger-chewing known numbers of bacteria into gum and subsequently

dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid

(TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction

(qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in.

Next, five volunteers were requested to chew gum up to 10 min after which numbers of

colony-forming-units and total numbers of bacteria trapped in chewed gum were

determined using the above methods. The qPCR method, involving both dead and live

bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable

bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a

slow decrease over time up to 10 min was observed. Around 108 bacteria were detected

per gum piece depending on the method and gum considered. The number of species

trapped in chewed gum increased with chewing time. Trapped bacteria were clearly

visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel

methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is

confirmed that chewing of gum can trap and remove bacteria from the oral cavity.

QUANTIFICATION AND QUALIFICATION OF BACTERIA TRAPPED IN CHEWED GUM

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Introduction Descriptions of the first use of chewing gum date back to the ancient Greek, who used tree

resins from the mastic tree to quench thirst and refresh their breath. The first commercial

chewing gum was not successfully marketed until the late 19th century, when the rubbery

tree sap of the Sapodilla tree formed the basis for gum manufacturing (1). In the late 20th

century, chewing gum is not only regarded as a symbol of lifestyle, but also effects on

cognitive performance, mood, alertness and appetite control have been reported (2–5).

Moreover, chewing gum has developed more and more towards an oral care and

functional food product (“nutraceutical”), as it provides an easily applicable drug delivery

vehicle with potential benefits for oral health (1). High consumption rates, up to 2.5 kg per

person per year, have made it into a billion dollar industry (6,7).

Most chewing gums consist of a mixture of food grade synthetic elastomers, like

polyvinyl acetate or polyisobutylene, generally referred to as the gumbase (1). Important

requirements to gumbase materials are that they do not dissolve in the oral cavity and can

be chewed for long periods of time without undergoing compositional and structural

changes. In most commercially available chewing gums, the gumbase is supplemented

with sweeteners, flavors and other bulking agents, while nowadays sugar is frequently

replaced by artificial sweeteners such as sorbitol, xylitol or mannitol (6,7).

The inclusion of xylitol and other artificial sweeteners has been described to

reduce the formation of oral biofilms on teeth (8,9). Oral biofilms are causative to the

world’s most wide-spread infectious diseases, namely dental caries and periodontal

disease (10). Caries arises from an unbalance between naturally occurring de- and

remineralization of dental enamel. Demineralization occurs when the pH of oral biofilm

drops below 5.5 (11) due to the fermentation of carbohydrates by specific bacterial strains

in oral biofilms on teeth. Most artificial sugars are not or barely fermented by oral bacteria

and therewith do not lower the pH (12). Moreover, chewing gum yields enhanced

mastication that stimulates salivation, which clears fermentable carbohydrates, dislodges

loosely bound oral bacteria from oral surfaces (13) and increases the concentrations of

calcium and phosphates in the oral cavity required for remineralization (14). Fluorides have

been added to commercial gums to prevent enamel demineralization and stimulate

remineralization (15). It is tempting to regard the chewing of gum as an addendum to daily

oral hygiene procedures, especially since most people are unable to maintain a level of

oral biofilm control required to prevent disease through daily toothbrushing and other

conventional oral hygiene measures. This has led to the incorporation of antimicrobials like

chlorhexidine (16) and herbal extracts (17) to chewing gums and gums have indeed been

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demonstrated successful in preventing re-growth of oral biofilm (18). It is also known that

chewing of gum aids in the removal of interdental debris (19). To increase the cleaning

power of chewing gum, detergents like polyphosphates (20) have been added to gums.

However, it is unclear whether chewing of gum itself will actually remove bacteria from the

oral cavity. Especially the preferential removal in sizeable numbers of disease-causing

microorganisms like acid-producing Streptococcus mutans or species that are regarded as

initial colonizers of tooth surfaces by chewing gum would turn chewing gum into a valuable

addendum to daily oral hygiene.

Therefore, the aim of this study is firstly to develop methods to quantify the

number of bacteria that are trapped into a gum after chewing, and secondly to qualitatively

determine the bacterial composition of bacteria trapped in chewed gums. The first method

is based on measuring the number of colony-forming units (CFUs) that can be retrieved

from pieces of gum, chewed by different volunteers. The method relies on finger-chewing

known numbers of different oral bacterial strains into commercially available spearmint

gums and retrieving bacteria from the gums by sonication followed by agar-plating of the

bacterial suspension to yield a calibration curve. By comparing it to the number of bacteria

retrieved from pieces of gum chewed by volunteers, the number of CFUs trapped in pieces

of chewed gum can be calculated. In the second method, pieces of chewed gum are

dissolved and the amount of bacterial genomic DNA is quantitated using quantitative

Polymerase-Chain-Reaction (qPCR) and converted to numbers of bacteria trapped in the

chewed gums using a calibration curve, also obtained by finger-chewing. The composition

of the different bacterial species trapped in chewed gum was compared with the

composition of the salivary microbiome and the microbiome adhering to teeth using

Denaturing Gradient Gel Electrophoresis (DGGE). Finally, we demonstrate bacterial

presence in chewed gum using Scanning Electron Microscopy (SEM).


Chewing gum Two commercially available spearmint chewing gums were used in this study:

Gum A – (commercially available spearmint gum, 1.5 g tabs). Composition in descending

order of predominance by weight: Sorbitol, gumbase, glycerol. Natural and artificial flavors;

less than 2% of: Hydrogenated starch hydrolysate, aspartame, mannitol, acesulfame K,

soy lecithin, xylitol, beta-carotene, blue 1 lake and butylated hydroxytoluene.

Gum B – (commercially available spearmint gum, 1.5 g tabs.). Composition in descending

order of predominance by weight: Sorbitol, gumbase, glycerin, mannitol, xylitol. Natural and


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artificial flavors; less than 2% of: Acesulfame K, aspartame, butylated hydroxytoluene, blue

1 lake, soy lecithin and yellow 5 lake. Both gums were similarly hydrophobic with water

contact angles on sectioned pieces of gum of 69 and 74 degrees for gum A and B,

respectively.

Method 1: Enumeration of bacteria trapped in chewed gums using sonication of gum molded to standard dimensions Basics of the method and preparation of a calibration curve

In this method, four different bacterial strains were used for the preparation of a calibration

curve that relates the numbers of CFUs retrieved from a piece of gum to the numbers of

CFUs incorporated in the gum for coccus-shaped Streptococcus oralis J22, Streptococcus

mutans ATCC 25175, Streptococcus mitis ATCC 9811 and rod-shaped Actinomyces

naeslundii T14V-J1. S. oralis and A. naeslundii are considered initial colonizers of tooth

surfaces in vivo (21,22), while S. mutans is causative to dental caries (23) and S. mitis is

an abundantly present species in the oral cavity (24). Streptococci were grown aerobically

in Todd Hewitt Broth (THB) at 37 °C and actinomyces anaerobically in Schaedler broth.

Bacteria were first grown on THB agar or blood agar plates from a frozen stock in

dimethylsulfoxide for 24 h after which one colony was inoculated in 10 ml of the

appropriate culture medium and incubated for 24 h. A main culture was prepared with a

1:10 dilution in fresh medium for 16 h. Main cultures were sonicated for 1 x 10 s at 30 W

(Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT, USA) to suspend bacterial

aggregates. The bacterial concentration was determined using the Bürker Türk counting

chamber, while percentage viability of the suspended bacteria was determined after serial

dilution and agar-plating. Next, concentrations were adjusted to 104, 105, 107 and 109

bacteria per ml. Since viability of the cultures was near 100%, these numbers are

equivalent to 4, 5, 7 and 9 log-units of CFUs per ml.

For each strain, known numbers of CFUs were finger-chewed into gum pieces by

adding 1.5 g chewing gum together with 200 µl of a bacterial suspension into the finger of

a sterile latex glove (Powder-Free Latex Examination Gloves, VWR international, Radnor,

USA). Next, bacteria were finger-chewed into the gum in a water bath at 37 °C for 5 min.

After finger-chewing, the gum was removed from the glove, dipped once in 10 ml sterile

water and put into a Teflon mold (15 x 15 x 1 mm) with a sterile pair of tweezers to create

reproducible gum dimensions (15 x 15 x 4 mm) and surface area (690 mm2).

Subsequently, the gum was inserted in sterile polystyrene cups with 5 ml filter sterile

Reduced Transport Fluid (RTF) (25). Bacteria were removed from the gum surface layer by

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sonication for 60 s in a water bath sonicator (ELMA Transsonic TP690, Elma GmbH & Co,

Germany). Sonication times up to 60 s did not affect bacterial viability (26,27). Finally, the

resulting suspension was serially diluted, plated on THB agar or blood agar plates (Blood

agar base no. 2, 40 g/l, hemin 5 mg/l, menadion 1 mg/l, sheep blood 50 ml/l) and incubated

at 37 °C for 48 h after which the number of CFUs retrieved were counted. Accordingly,

since different numbers of bacteria were finger-chewed into the gums, a calibration curve

was made of the numbers of CFUs retrieved from each gum for the different bacterial

strains versus the numbers of CFUs finger-chewed into the gum. To account for possible

loss of bacteria due to adhesion to the inner surface of the glove, the glove finger was

turned inside out after removal of the gum and sonicated in 10 ml filter sterile RTF for 60 s

and serial dilutions plated on agar plates as described above after which the number of

CFUs lost were determined. Similarly, the water in which the finger-chewed gums were

dipped (see above) was analyzed for bacterial losses. Calibration curves were made in

triplicate for each chewing gum and bacterial strain.

Application of the method in human volunteers

Volunteers included in this study were five healthy members of the department of

Biomedical Engineering (1 male, 4 females, aged 27 to 56 years). All experiments were

performed according to the rules as set out by the Medical Ethics Committee of the

University Medical Center Groningen, and they approved this study (approval METc

2011/330). Volunteers gave their written informed consent. Inclusion criteria described that

all volunteers should be in good health and have at least 16 natural elements. Exclusion

criteria were the use of antibiotics or mouth rinses in the month prior to the study or the use

of antibiotics, mouth rinses and additional chewing gum during the study. Furthermore,

volunteers were requested to brush their teeth twice a day, according to their habitual

routines.

On separate days, volunteers were asked to chew 1.5 g (one serving size) of

each chewing gum once a day at the same time for 0.5, 1, 3, 5 or 10 min according to their

own personal routine without specific instructions for chewing. Chewing time and gum

types (A or B) were randomly assigned to the volunteers over the experimental period.

After chewing, the gum was spit in a polystyrene cup with 10 ml sterile water, after which

the chewed gum was put into the Teflon mold and sonicated, as described above.

Resulting suspensions were serial diluted, agar-plated and the numbers of CFUs were

determined after incubation for 7 days at 37 °C under anaerobic conditions (5% H2, 10%

CO2, 85% N2) (Concept 400 anaerobic workstation, Ruskinn Technology Ltd., Pencoed,

UK). Finally, the numbers of CFUs retrieved from the gums after different chewing times


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and for both types of gum were converted to the total number of CFUs trapped in chewed

gums using the calibration curve obtained from finger-chewing known numbers of bacteria

into the gums. Note that this requires the assumption that bacterial viability is equally

maintained in finger-chewed gum as in gum chewed by volunteers. All experiments were

carried out in duplicate for each volunteer, gum type and time point.

Method 2: Enumeration of bacteria trapped in chewed gums using qPCR and microbial composition

Basics of the method and preparation of a calibration curve

Similar to method 1, a calibration curve was made by finger-chewing known numbers of S.

oralis J22, S. mutans ATCC 25175, S. mitis ATCC 9811 or A. naeslundii T14V-J1 in the

different spearmint gums. Bacterial concentrations were adjusted using the Bürker Türk

counting chamber to 107, 109 and 1010 bacteria per ml, in which the latter concentration

was achieved by centrifugation (5 min, 5000 g at 10 °C). After finger-chewing as described

above, the gum was removed from the glove, dipped once in 10 ml sterile water and

subsequently dissolved in a mixture of 5 ml chloroform (67-66-3, Fisher Scientific,

Waltham, USA) and 3 ml tris-ethylenediaminetetraacetic-acid (TE) buffer (AM9849,

Ambion® - LifeTechnologies™, Carlsbad, USA) in a sterile centrifuge tube. The gum was

dissolved in 45 min by shaking horizontally. The resulting suspension was centrifuged for

10 min at 1500 g to remove large particles and gumbase from the aqueous TE buffer top

layer.

For qPCR, 17.5 µl master mix was used for every sample consisting of 10 µl

PCR - mix (iQ5™ SYBR® Green Supermix, Bio-rad, Hercules, USA), 5 µl DNA free water

(95284, Sigma, St. Louis MO, USA) and 2.5 µl primer mix (300 nM). To amplify the

universal V3 region of the 16S rRNA gene in all samples F357-GC was used as the

forward primer and R-518 (28) as the reverse primer. In a 384-well PCR plate (HSP-3805,

Bio-rad, Hercules, USA), 2.5 µl of sample dilutions (1x, 10x, 100x), taken from the

centrifuged aqueous TE buffer top layer, was mixed with 17.5 µl of master mix.

Subsequently, a qPCR was performed on a thermocycler (CFX384, Bio-rad, Hercules,

USA), according to a 3 step amplification (95.0 °C for 45 s, 58.0 °C for 45 s, 72 °C for 60 s)

of 39 cycles. A calibration curve was obtained by relating threshold cycle (Ct) at fixed

relative fluorescence units to the number of bacteria chewed-in the gum (29,30).

Calibration curves were obtained for both gums in triplicate for all four bacterial strains.

DNA free water and a piece of unchewed gum, dissolved as described above, were used

as negative controls.

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Application of the method in human volunteers

Five healthy members of the department of Biomedical Engineering chewed each type of

chewing gum, as described above. Chewed gum was spit in a polystyrene cup with 10 ml

sterile water after which the gum was dissolved in a sterile centrifuge tube with the mixture

of chloroform and TE buffer. After centrifugation, qPCR was performed using the aqueous

TE buffer top layer (see above). The total number of bacteria trapped in the gum was

determined using the calibration curve. Part of each dissolved gum TE-buffer sample was

stored in -80 °C for later DGGE analysis.

Determination of the bacterial composition using DGGE

The composition of the different species trapped in pieces of chewed gum was determined

using DGGE and compared to the bacterial compositions of the planktonic, salivary

microbiome and the microbiome adhering to tooth surfaces. After 10 min of chewing,

volunteers were asked to donate 1 ml of unstimulated saliva and collect oral biofilm from

their entire dentition using a cotton swab and a sterile hook in 1 ml RTF. Both saliva and

biofilm samples were centrifuged at 18000 g for 5 min (Eppendorf Centrifuge 5417R,

Hamburg, Germany), DNA was isolated (31), after which the samples were resuspended in

50 µl TE buffer.

The DNA concentration of saliva, biofilm and dissolved gum samples were

measured with the Nanodrop® Spectrophotometer (ND-110, NanoDrop Technologies Inc.,

Wilmington, DE, USA). A PCR was performed with 100 ng DNA using the primers and

amplification program as described above. The products of the PCR were applied on a

polyacrylamide gel (8% w/v) in 0.5 TAE buffer (20 mM Tris acetate, 10 mM sodium

acetate, 0.5 mM EDTA, pH 8.3). Using a 100% stock solution (7 M urea, 37% formamide)

a denaturing gradient was made with the range of 30-80%. A stacking gel without

denaturant was added on top and equal amounts of sample were applied to the gel.

Electrophoresis was performed overnight at 60 °C and 120 V. Silver nitrate solution (0.2%

AgNO3) was used until maximal staining intensity was reached.

Gels were scanned and transferred to analysis software BioNumerics (v7.1

Applied Maths, Sint-Martens-Latem, Belgium). Gels were normalized to reference markers

that were added to every gel. Presence of a band on the gel was taken as the presence of

a bacterial species or strain in the sample. The similarity of bands was determined

according to the band-based matching module in the software (0.5% optimization, 1%

band tolerance).


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Scanning electron microscopy In order to visualize bacteria trapped in chewed gum, a 5 min chewed gum piece was spit

into liquid nitrogen, kept immersed for 2 min and broken into multiple pieces, which were

subsequently examined in a SEM (JEOL JSM-6301F, Akishima, Japan). Gum pieces were

fixed directly for 24 h in 2.0% glutaraldehyde at 4.0 °C, washed with 0.1 M cacodylate

buffer and incubated for 1 h in 1.0% OsO4 in 0.1 M cacodylate buffer at room temperature.

After washing with water, samples were dehydrated with an ethanol series (30, 50 and

70%) each for 15 min and 3 times 30 min with 100% ethanol. Fracture surfaces of the

chewed gum were examined for the presence of bacteria at a magnification of 7.500x with

an acceleration voltage of 2.0 kV and 39.0 mm working distance.

Statistics Data was evaluated for normality using Shapiro-Wilk and Kolmogorov-Smirnov test (p <

0.05) and in case of a normal distribution equality of means was tested using an ANOVA

followed by Tukey-HSD post hoc test (p < 0.05). In case no normal distribution of data was

observed, a non-parametric Kruskal-Wallis test was used (p < 0.05). SPSS v20.0 (IBM

Corp., Armonk, USA) to conduct all statistical analysis.

Results

Bacteria of the four different strains were finger-chewed into the two different types of

chewing gums in order to obtain a relation between the number of bacteria trapped in a

gum piece and the number of CFUs or total bacteria that can be retrieved from a gum by

agar-plating or qPCR, respectively. On average, 0.05 log-units of CFUs were lost due to

adhesion to the surface of the glove in which gums were finger-chewed, while A. naeslundii

adhered in slightly higher numbers to the glove surface than streptococcal strains.

Bacterial losses due to dipping the finger-chewed gum pieces in water were much smaller

and amounted on average 0.004 log-units of CFUs.

Accounting for these losses, linear relations were obtained for both methods (Fig.

1). For CFUs, the calibration lines were independent of the gum type involved. Lines were

generally independent of the bacterial strains involved, apart from a small but statistically

significant difference (p < 0.05) between A. naeslundii and S. mitis at the highest bacterial

concentration (Fig. 1A). As sonication can only release bacteria trapped in a gum from the

outer surface, the number of bacteria retrieved was roughly 1.5 log-units less than chewed-

in. The qPCR method yielded small but statistically significant differences (p < 0.05) in Ct

values for the different bacterial strains (Fig. 1B). However, neglecting these strain-related

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differences, average linear calibration lines could be obtained that were independent of the

gum type involved.

Figure 1 Calibration curves for bacterial trapping in finger-chewed gums. Calibration curves for bacterial trapping after finger-chewing known numbers of bacteria into gum.

Results are obtained from three independent experiments with separately cultured bacteria. Data are

corrected for losses of bacteria due to adhesion to the glove-finger and during water rinsing. Error bars

denote the standard deviation over triplicate experiments and linear relations are presented by the

equations with their corresponding correlation coefficients.

A. The number of CFUs retrieved as a function of the numbers of CFUs finger-chewed in a

gum piece for the four different bacterial strains, obtained by sonication of chewed pieces of

gum, molded into a standard dimension and followed by sonication and agar-plating.

B. The number of threshold cycles (Ct) at fixed relative fluorescence units as a function of

the total number of bacteria finger-chewed in a gum piece for the four different bacterial

strains, obtained after dissolving the gum in chloroform and TE buffer and performing qPCR.

Next, volunteers were asked to chew the two types of chewing gums for varying

amounts of time up to 10 min and the number of bacteria chewed-in was determined in

terms of CFUs after sonication and agar-plating or in terms the total number of bacteria, as


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obtained after dissolving the gum and performing qPCR on bacterial DNA. Agar plating

indicates that most CFUs are trapped (approximately 7.8 log-units) within the first minute,

regardless of the gum involved, while approximately 1 log-unit less CFUs remained

trapped in a gum piece after prolonged chewing (Fig. 2A). qPCR yields higher numbers of

bacteria retrieved than agar-plating (Fig. 2B), but displays only a minor decrease in total

number of bacteria trapped in time for both types of chewing gums.

Figure 2 Bacteria trapped in two different types of spearmint gums chewed by human volunteers as function of time.

The number of bacteria trapped in chewed gums for two types of spearmint gums as a function of the

chewing time. Error bars denote the standard deviation over a group of five volunteers, with each

volunteer having chewed the same gum twice for all time points.

A. CFUs trapped per gum piece obtained after molding, sonication and agar-plating.

B. Total number of bacteria trapped per gum piece obtained after dissolving the gum and

performing qPCR

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Figure 3 Diversity of bacterial strains and species trapped in chewed gum in comparison with the bacterial diversity in the salivary microbiome and the micobiome adhering to tooth surfaces.

A. The number of bands in DGGE gels in bacterial DNA obtained from pieces of chewed

gum as a function of the chewing time. Error bars denote the standard deviation over a

group of five volunteers. No statistically significant differences were observed.

B. Percentage of species detected in the microbiome adhering to tooth surfaces or in the

salivary microbiome relative to the number of species found in chewed gum (10 min of

chewing) set at 100%. Error bars denote the standard deviation over a group of five

volunteers. No statistically significant differences were observed.

C. Percentage of species found in chewed gum based on origin, i.e. found in chewed gum

and the adhering microbiome, chewed gum and the salivary microbiome and found in gum

and both microbiomes. The category “other origin” indicates species that were solely found

in chewed gum and below detection in the salivary and in the adhering microbiome.

The number of species detected in chewed gum increases with increasing

chewing time for both types of chewing gums (Fig. 3A), while after 10 min of chewing 50-

70% of the detected species in the salivary and adhering microbiome are ultimately

detected in the chewed gum piece (Fig. 3B). A more elaborate analysis of the origin of

bacterial species found in chewed gum indicated that 9% and 16% of the species found in


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chewed gum were solely detected in the adhering oral microbiome for gum A and B,

respectively, while a relatively similar percentage of approximately 15% of the detected

species chewed-in were solely found in the salivary microbiome (Fig. 3C). Remaining

percentages of species found in chewed gum could either be attributed to the salivary or

the adhering microbiome or their origin could not be detected, suggesting the tongue,

gums or oral mucosal surfaces as an origin.

Considering the numbers of bacteria found in chewed gum and the field of view

and depth of focus of SEM, it can be appreciated that microscopic imaging of trapped

bacteria in chewed gum is like looking for a needle in a haystack. Yet after extensive

searching, a scanning electron micrograph could be taken of a chewed gum piece showing

an open and porous structure (Fig. 4) in which trapped bacteria can be observed as direct

evidence of the ability of chewing gum to trap bacteria during chewing.

Figure 4 SEM visualization of bacteria trapped in a piece of chewed gum. Scanning electron micrograph of a bacterium (indicated by white arrow) trapped in a chewed gum

piece of gum A. The scale bar indicates 1 µm.

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Discussion

In this paper we provide evidence that bacteria are trapped inside gum pieces chewed by

human volunteers and therewith may contribute to the maintenance of oral health. The

number of bacteria trapped in chewed gums were determined using two distinctly different

methods. Finger-chewing and subsequent sonication and agar-plating demonstrated that

approximately 1 – 1.5 log-units less than the number of bacteria chewed-in could be

retrieved, regardless of the type of gum or bacterial strain involved, i.e. coccus- or rod-

shaped microorganisms (Fig. 1A). Although this recovery is confined to the surface layer of

the gums amenable to sonic removal of chewed-in bacteria and therefore relatively low, it

allows to culture the bacteria retrieved and express them in terms of CFUs. Compared to

qPCR, which requires chemical dissolution of the gum and bacterial lysis to determine the

presence of genomic DNA from bacteria trapped in chewed gums, agar-plating yields lower

numbers of trapped bacteria, likely because qPCR includes both dead and live bacteria

(32) while agar-plating only reports viable ones. Whereas agar plating yielded results that

were independent of the bacterial strain involved, Ct values obtained in qPCR were

somewhat strain-dependent (Fig. 1B), possibly due to differences in efficacy of lysis of the

different strains and the relative efficiencies of the primer pairs used. However, since

calibration curves are applied to bacterial samples of unknown composition, the small

strain-dependent differences in Ct values were neglected and average calibration curves

were calculated and employed.

Both methods indicate a slow but significant decrease in bacterial trapping with

increasing chewing time in human volunteers after an initial maximum, regardless of the

type of gum involved. Whereas the initial gum bases are thus most adhesive to oral

bacteria (Fig. 2) continued chewing changes the structure of the gums, decreasing the

hardness of the gum due to uptake of salivary components (33) and release of water

soluble components. This presumably affects the adhesion of bacteria to the gum (34),

causing a release of initially trapped, more weakly adhering bacteria from the gum. Such a

change in composition of trapped bacteria is supported by the observation that the diversity

of species trapped in chewed gum increases with chewing time (Fig. 3A).

Despite an increasing diversity in species developing over time in chewed gums,

there is a gradual decrease in the number of bacteria trapped in chewed gum over time.

This can be attributed to a decrease in bacterial concentration in saliva during chewing,

shown in earlier reports (13). However, alternative explanations exist as well, especially

since this decrease is far more prominent for the numbers of CFUs retrieved than for the

total numbers of bacteria found by qPCR in chewed gum. This difference in decrease


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suggests that bacteria are killed during their entrapment in the gum by sweeteners like

xylitol, food preservatives or flavoring agents like spearmint and peppermint, which are

reported to have antimicrobial properties (9,35–37).

Numbers of bacteria trapped in a chewed piece of gum amount around 108

depending on the time of chewing and retrieval method. Although this number may be

considered low, it shows that when gum is chewed on a daily basis, it may contribute on

the long-term to reduce the bacterial load in the oral cavity, which is supported by

observations that long-term studies on the use of chewing gum cause a reduction in the

amount of oral biofilm (38). Bacteria trapped in chewed gum can originate either from the

salivary microbiome or the adhering microbiome on teeth, but also from the tongue, gums

or oral mucosal surfaces from which we did not sample. No DNA was detected in

unchewed gum pieces. Saliva harbors up to 109 microorganisms per ml before chewing

(11,39). Assuming a volume of saliva of around 1 ml in the oral cavity, our results indicate

that chewing of one piece of gum removes around 10% of the oral microbial load in saliva.

However, as our DGGE results pointed out, saliva does not necessarily have to be the

source of the bacteria found trapped in chewed gum. Making the alternative assumption

that all bacteria trapped in chewed gum come from the adhering microbiome, we can place

this number in further perspective by comparing it to the number of bacteria removed by

toothbrushing. Using a new, clean toothbrush without any toothpaste reportedly removes

around 108 CFUs per brush (39,40), which would put chewing of gum on par with the

mechanical action of a toothbrush. Moreover, also the mechanical action of floss wire

removes a comparable number of bacteria from the oral cavity than does chewing of a

single piece of gum, as we established in a simple pilot involving 3 human volunteers who

used 5 cm of floss wire (unpublished). Chewing however, does not necessarily remove

bacteria from the same sites of the dentition as does brushing or flossing, therefore its

results may be noticeable on a more long-term than those of brushing or flossing (7,19,41).

Our findings that chewing of gum removes bacteria from the oral cavity, may

promote the development of gum that selectively removes specific disease-related bacteria

from the human oral cavity, for instance by using porous type calcium carbonate (42). It is

known that the key to oral health is a balanced and diverse composition of the oral

microbiome, although the exact composition of what is tentatively called “the oral

microbiome at health” is not known. Removal of specific pathogens however, is directly in

line with the general notion arising in dentistry that oral diseases develop when the oral

microbiome shifts its composition into a less diverse direction (43). In this respect, a

gradual removal of bacteria from the oral cavity through regular removal of low numbers of

pathogens by chewing gum is preferable to sudden ecological shifts that can change the

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relationship between the oral microbiome and the host as another potential cause of

disease (43).

Acknowledgements

We would like to thank all volunteers for their cooperation in this study.

References

1. Fritz D. Formulation and production of chewing and bubble gum. Mestres J, Estruch RA, editors. Cambridge: Woodhead Publishing Ltd.; 2006. 340 p.

2. Hetherington MM, Regan MF. Effects of chewing gum on short-term appetite regulation in moderately restrained eaters. Appetite. 2011; 57(2):475–82.

3. Scholey A. Chewing gum and cognitive performance: a case of a functional food with function but no food? Appetite. 2004; 43(2):215–6.

4. Smith A. Effects of chewing gum on cognitive function, mood and physiology in stressed and non-stressed volunteers. Nutr Neurosci. 2010; 13(1):7–16.

5. Johnson AJ, Jenks R, Miles C, Albert M, Cox M. Chewing gum moderates multi-task induced shifts in stress, mood, and alertness. A re-examination. Appetite. 2011; 56(2):408–11.

6. Ly K, Milgrom P, Rothen M. The potential of dental-protective chewing gum in oral health interventions. J Am Dent Assoc. 2008; 139(5):553–63.


8. Birkhed D. Cariologic aspects of xylitol and its use in chewing gum: a review. Acta Odontol Scand. 1994; 52(2):116–27.

9. Milgrom P, Ly KA, Roberts MC, Rothen M, Mueller G, Yamaguchi DK. Mutans streptococci dose response to xylitol chewing gum. J Dent Res. 2006; 85(2):177–81.

10. Balakrishnan M, Simmonds RS, Tagg JR. Dental caries is a preventable infectious disease. Aust Dent J. 2000; 45(4):235–45.


12. Burt B. The use of sorbitol-and xylitol-sweetened chewing gum in caries control. J Am Dent Assoc. 2006; 127:190–6.

13. Dawes C, Tsang RW, Suelzle T. The effects of gum chewing, four oral hygiene procedures, and two saliva collection techniques, on the output of bacteria into human whole saliva. Arch Oral Biol. 2001; 46(7):625–32.



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15. Sjögren K, Ruben J, Lingström P, Lundberg A, Birkhed D. Fluoride and urea chewing gums in an intra-oral experimental caries model. Caries Res. 2002; 36(1):64–9.



18. Hanham A, Addy M. The effect of chewing sugar-free gum on plaque regrowth at smooth and occlusal surfaces. J Clin Periodontol. 2001; 28(3):255–7.

19. Kakodkar P, Mulay S. Effect of sugar-free gum in addition to tooth brushing on dental plaque and interdental debris. Dent Res J (Isfahan). 2011; 7(2):64–9.

20. Van der Mei HC, Kamminga-Rasker HJ, De Vries J, Busscher HJ. The influence of a hexametaphosphate-containing chewing gum on the wetting ability of salivary conditioning films in vitro and in vivo. J Clin Dent. 2003; 14(1):14–8.

21. Dige I, Raarup MK, Nyengaard JR, Kilian M, Nyvad B. Actinomyces naeslundii in initial dental biofilm formation. Microbiology. 2009; 155:2116–26.


23. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986; 50(4):353–80.

24. Diaz PI, Dupuy a K, Abusleme L, Reese B, Obergfell C, Choquette L, et al. Using high throughput sequencing

to explore the biodiversity in oral bacterial communities. Mol Oral Microbiol. 2012; 27(3):182–201.


26. Pitt WG, Ross SA. Ultrasound increases the rate of bacterial cell growth. Biotechnol Prog. 2003; 19(3):1038–44.

27. Drakopoulou S, Terzakis S, Fountoulakis MS, Mantzavinos D, Manios T. Ultrasound-induced inactivation of Gram-negative and Gram-positive bacteria in secondary treated municipal wastewater. Ultrason Sonochem. 2009; 16(5):629–34.

28. Muyzer G, de Waal EC, Uitterlinden a G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Env Microb. 1993; 59(3):695–700.

29. Lyons S, Griffen A, Leys E. Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria. J Clin Microbiol. 2000; 38(6). 2362–5

30. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, et al. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans , Porphyromonas gingivalis , Prevotella intermedia , tetQ gene and total bacteria. FEMS Immunol Med Microbiol. 2003; 39(1):81–6.


32. Weiger R, Ohle C. Vital microorganisms in early supragingival dental plaque and in stimulated human saliva. J Periodontal Res. 1997; 32:233–40

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33. Rosenhek M, Macpherson LM, Dawes C. The effects of chewing-gum stick size and duration of chewing on salivary flow rate and sucrose and bicarbonate concentrations. Arch Oral Biol. 1993; 38(10):885–91.

34. Stinson M, Levine M. Modulation of intergeneric adhesion of oral bacteria by human saliva. Crit Rev Oral Biol Med. 1993; 4:309–14.

35. Al-Ahmad A, Wiedmann-Al-Ahmad M, Auschill TM, Follo M, Braun G, Hellwig E, et al. Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro. Arch Oral Biol. 2008; 53(8):765–72.

36. Chaudhari LKD, Jawale BA, Sharma S, Sharma H, Kumar CDM, Kulkarni PA. Antimicrobial activity of commercially available essential oils against Streptococcus mutans. J Contemp Dent Pract. 2012; 13(1):71–4.

37. Rasooli I, Shayegh S, Astaneh S. The effect of Mentha spicata and Eucalyptus camaldulensis essential oils on dental biofilm. Int J Dent Hyg. 2009; 7(3):196–203.

38. Keukenmeester RS, Slot DE, Putt MS, Van der Weijden GA. The effect of

sugar-free chewing gum on plaque and clinical parameters of gingival inflammation: a systematic review. Int J Dent Hyg. 2013; 11(1):2–14.

39. Quirynen M, de Soete M, Pauwels M, Goossens K, Teughels W, van Eldere J, et al. Bacterial survival rate on tooth- and interdental brushes in relation to the use of toothpaste. J Clin Periodontol. 2001; 28(12):1106–14.

40. Quirynen M, De Soete M. Can toothpaste or a toothbrush with antibacterial tufts prevent toothbrush contamination? J Periodontol. 2003; 74:312–22.

41. Mouton C, Scheinin A, Mäkinen K. Effect on plaque of a xylitol-containing chewing-gum: A clinical and biochemical study. Acta Odontol Scand. 1975; 33:33–40.



Societal versus Scientific impact

of "Quantification and

qualification of bacteria trapped

in chewed gum"

Appendix chapter 6

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Research output can be assessed in various ways. Within the academic world, research

impact is stooled upon publication in a high impact factor journal and the number of

citations in other research papers. Outside academia, impact is harder to assess but can

be measured by their influence on society, e.g. the general public, business and

government (1). As the article “Quantification and qualification of bacteria trapped in

chewed gum” received a substantial amount of attention in the press, we here describe the

societal impact our article has had and compare the societal impact with its scientific one.

The research article was published online in PLoS ONE (Impact Factor 3.234,

ranked no. 8 in the field of multidisciplinary sciences (2)) on January 20th 2015. Despite

that PLoS ONE is a decent journal, statistically based on the journals impact factor, the

number of citations of this article, the main judgement of academic success, are unlikely to

reach high levels. This is mainly because research in dentistry, especially on oral health

care benefits of chewing gum, is a relatively small field and very little research papers

concerning chewing gum are published. This is reflected in the number of citations up until

this date (26-10-2015), which is 0.

However, directly after publication online, the article did receive a lot of attention

outside academia. It was picked up almost immediately on an internet blog that reported

quite accurately on the outcome of the article stating “Chewing gum removes up to 100

million bacteria” (see Fig. 1, this Appendix) (3). From here on multiple news sites started

reporting this message among which: FoxNews.com, DailyMail.co.uk and

ScienceAlert.com (4–6), all greatly aided by Twitter and Facebook causing it to reach other

parts of the world like China and India (see

Fig. 2) (7–10). In The Netherlands it was

covered by De Telegraaf.nl, nu.nl and

HPdeTijd.nl (11–13).

Figure 1

Scienceblog www.realclearscience.com reports

on January 21st, 2015.

APPENDIX: SOCIETAL VERSUS SCIENTIFIC IMPACT

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Figure 2

Written press coverage in foreign media.

A: Zaobao Singapore

B: Yahoo Taiwan

C: The times of India

Besides written press coverage, radio stations (3FM, RTV Noord) showed interest

in the research results and a couple of video coverages on the internet were made, among

which a video of DNews (a Discovery.com news medium), which showed an accurate

report (14).

Unfortunately, in the process of more media reporting on the research, the actual

content of the article was sometimes misinterpreted or construed in the wrong manner.

Especially the comparison in the discussion section of the article between the number of

bacteria trapped in a piece of chewing gum and a the number found on a used piece of

floss wire, was misconstrued as “chewing gum is equal or better than flossing”. This

comparison in the article was made solely to put the number of trapped bacteria in gum in

perspective and was most certainly not meant as a comparison of effectiveness of both

techniques. Chewing gum is and will not be a replacement of flossing or brushing, as was

A B

C

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insinuated by some media. Luckily, after having our own press release, websites of dental

practices, dental magazines and most written newspapers, influential on the behavior of

patients and dental professionals, reported correctly with the side note that chewing gum is

not a replacement of flossing or brushing (see Fig. 3) (15,16).

The attention in the press, can be ascribed to a couple of factors. First of all, an

open-access journal might have accelerated news coverage, allowing anyone without

subscription to access the full text. Secondly, the main subject of this research, a common

daily life product such as chewing gum relates to many people, opposite to more

fundamental research topics.

Opposite to the number of citations, the number of views of the article on the

PLoS ONE website is very high, 10.332 views up until 26-10-2015. This clearly indicates

that research with societal impact does not necessarily mean scientific impact, and vice

versa. Smith (17) clearly illustrates this with the following example: “scientists would think

of the original work on apoptosis (programmed cell death) as high quality, but 30 years

after it was discovered there has been no measurable impact on health. In contrast,

research that is unlikely to be judged as high quality by scientists — say on the cost

effectiveness of different incontinence pads — may have immediate and importance social

benefits” (17).

One important difference between scientific impact and

societal impact is the way of measuring impact. The

academic world has clearly defined grading systems,

such as ISI Web of Science or institutional requirements

to publish in top 25% journals in the research field.

Consequently, more and more research is driven

towards achieving a high ranking in these areas.

However this could have consequences for the impact

on society. Mostert et al. (18) strikingly state: “… high

scientific quality of research groups is not necessarily

related to communication with society, and that in order

to increase societal quality of research groups, additional

activities are needed.

Figure 3 Dutch magazine for dental professionals (Nederlands

Tandartsenblad)


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Therefore societal quality is not simply the consequence of high scientific quality.

Obviously, in a university medical center, scientific quality prevails, and is a prerequisite,

which cannot be replaced by aiming instead for high societal quality.” Here it is also

suggested that assessment of societal quality should be evaluated more synergistically

with scientific quality and should have a more important role in the evaluation of research

organizations (18).

Societal impact or quality is not as clearly defined and more difficult to measure

than scientific impact (19) , although it is receiving more emphasis and new methods for

assessment are being developed. Mostert et al. (18) proposed an assessment system of

societal impact of health research in The Netherlands based on outreach to the different

stakeholders; the general public, healthcare professionals and the private sector according

to various indicators (see Table 1).

Table 1

Societal quality indicators (Mostert et al. (18))

In this system our article “Quantification and qualification of bacteria in chewed

gum” will mainly contribute to knowledge production for the general public (see Table 1).

Overall it raised awareness on the oral health benefits of sugar-free chewing gum.

Unfortunately, it also raised a false general consensus due to misinterpretation in some

media, insinuating the false notion that chewing gum would be better than flossing. It is

regrettable that this occurred, although it can be easily understood in the view of fast acting

media trying to comply with high demand for news with headlines that contain quick

catchphrases. Next to the general public, there was knowledge production for healthcare

professionals in magazines or websites for dental health professionals (15,16) and

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knowledge exchange on a dental research conference (20). Lastly, as this project is in

collaboration with industry, in the private sector a patent was filed for the developed

methods and more importantly the research is a contribution to the development of a new

product. Obviously ways of measuring societal impact vary, and can include current

internet media such as Researchgate, Google Scholar or even Facebook (21).

Regardless of the specifics of the measurement system, the media attention for

“quantification and qualification of bacteria in trapped in chewed gum” invoked an important

realization that besides the classic assessment of academic research also societal impact

should be taken into account for the evaluation of research organizations. If not only to shift

research goals more towards practical uses for society, also to aid researchers in certain

fields in receiving grants in the competitive world of research funding.

References

1. Maximizing the impacts of your research: a handbook for social scientists. Biometrika. 2011; 62(9):1–298.

2. ISI Web of knowledge - Journal citation reports. Available from: http://www.webofknowledge.com

3. Pomeroy R. RealClearScience Journal Club - Chewing gum removes up to 100 million bacteria. 2015; Available from: http://www.realclearscience.com/journal_club/2015/01/21/chewing_gum_removes_bacteria_from_your_mouth_109038.html

4. Seamons K. Fox News - How chewing gum improves your mouth’s health. 2015; Available from: http://www.foxnews.com/health/2015/01/21/how-chewing-gum-improves-your-mouth-health/

5. O’Callaghan J. Dailymail.co.uk - Is gum better than flossing? 10 minutes of chewing can remove 100 million bacteria from your mouth, study claims. 2015; Available from: http://www.dailymail.co.uk/sciencetech/article-2923385/Is-GUM-better-flossing-10-minutes-chewing-remove-100-MILLION-bacteria-mouth-study-claims.html

6. Crew B. Science Alert - Chewing sugar-free gum removes as much oral bacteria as flossing. 2015; Available from: http://www.sciencealert.com/chewing-sugar-free-gum-removes-as-much-oral-bacteria-as-flossing

7. Zaobao.sg. 2015; Available from: http://www.zaobao.com.sg/wencui/technology/story20150124-439036

8. Yahoo Taiwan. 2015; Available from: http://bit.ly/1NpQgr0

9. The times of India. 2015; Available from: http://timesofindia.indiatimes.com/home/science/Chewing-gum-helps-fight-oral-bacteria/articleshow/46025099.cms

10. People.cn. 2015; Available from: http://health.people.com.cn/n/2015/0127/c14739-26459842.html

11. Telegraaf.nl. 2015; Available from: http://www.telegraaf.nl/gezondheid/23602187/__Kauwgom_net_zo_effectief_als_flossen___.html

12. Nu.nl - Kauwgom kauwen even effectief als flossen. 2015; Available from:


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http://www.nu.nl/gezondheid/3979810/kauwgom-even-effectief-als-flossen.html

13. Voorn J. HP de Tijd - Onderzoek: 30 seconden kauwgom kauwen verwijdert 100 miljoen bacteriën. 2015; Available from: http://www.hpdetijd.nl/2015-01-29/onderzoek-30-seconden-kauwgom-kauwen-verwijdert-100-miljoen-bacterien/

14. DNews - Is Chewing Gum Better Than Flossing? 2015; Available from: https://www.youtube.com/watch?v=hOOv2pwqsPw

15. Nederlands tandartsenblad. Suikervrije kauwgom is gezond voor de mond. 2015; 3:6

16. Mughal R. Dr. Bicuspid - Chewing gum may trap as much oral bacteria as flossing. 2015; Available from: http://www.drbicuspid.com/index.aspx?sec=ser&sub=def&pag=dis&ItemID=317255

17. Smith R. Measuring the social impact of research. BMJ. 2001; 323:528.

18. Mostert SP, Ellenbroek SP, Meijer I, van Ark G, Klasen EC. Societal output and use of research performed by health research groups. Health Res Policy Syst. 2010; 8:30

19. Bornmann L. What is societal impact of research and how can it be assessed? A literature survey. J Am Soc Inf Sci Technol. 2013; 64(2):217–33.

20. Wessel S, Van der Mei H, Morando D, Slomp A, Van de Belt-Gritter B, Maitra A, et al. Novel Method for quantitation of bacteria trapped in chewed gum. 92nd IADR General Session and Exhibition - June 25-28 - Cape Town. 2014; Available from: https://iadr.confex.com/iadr/14iags/webprogram/Paper190839.html

21. Ringelhan S, Wollersheim J, Welpe IM. I Like, I Cite? Do Facebook likes predict the impact of scientific work? PLoS One. 2015; 10(8):e0134389.

102

MBE INCREASES ADHESION OF ORAL GRAM-NEGATIVE BACTERIA TO A HYDROPHOBIC LIGAND

103

Magnolia bark extract increases

adhesion of oral Gram-negative

bacteria to a hydrophobic ligand

Stefan W. Wessel, Henny C. van der Mei, Amarnath Maitra,

Michael W.J. Dodds and Henk J. Busscher

Submitted to Journal of Agricultural and Food Chemistry

Chapter 7

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Abstract

Cell surface hydrophobicity of oral bacteria can be altered by adsorption of components

from oral health care products. A more hydrophobic cell surface enhances bacterial

removal by hydrophobic ligands from the oral cavity. Here we investigate whether

exposure of oral Gram-positive and Gram-negative bacteria to magnolia-bark-extract alters

their hydrophobicity to enhance their removal from an aqueous suspension by adhesion to

hexadecane. Eleven oral bacterial strains were exposed to aqueous solutions containing

different concentrations of magnolia-bark-extract. Subsequently their removal from the

aqueous phase by hexadecane was measured using the kinetic “Microbial Adhesion To

Hydrocarbons” assay. Exposure of bacteria to magnolia-bark-extract in aqueous solution

yielded changes in hydrophobicity of Gram-negative oral bacteria in a dose responsive

manner, enhancing removal by hexadecane. This suggests that a combination of

magnolia-bark-extract and hydrophobic ligands may find applications in nutraceuticals to

reduce the prevalence of Gram-negative bacteria and associated oral diseases in the oral

cavity.


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Introduction

Maintenance of oral health is largely achieved by regular toothbrushing, the use of

mouthrinses, dental floss or other interdental cleaning devices. In addition, chewing of gum

has been promoted as an adjunct to regular oral hygiene (1,2). The oral cavity is

comprised of more than 700 bacterial species (3) many of which contribute to oral health

(4) rather than to disease. Yet, conventional maintenance of oral health is geared toward

removal of as many bacteria as possible, irrespective of whether they are known

contributors to oral health or disease.

The composition of the oral microbiome is not only of importance in maintaining

oral health but also relates with the occurrence of several other, more general diseases

such as diabetes mellitus, cardiovascular disease, preterm birth and obesity (4–6). Key to

prevention of disease is symbiosis of bacteria with the host (7) and preventing overgrowth

by specific oral pathogens. Accordingly, alternative oral hygiene measures that aim toward

the removal of specific oral pathogens from the oral cavity rather than untargeted bacterial

removal are currently looked for more than ever (8–11). As early as 1967, it was already

demonstrated that the composition of the oral microbiome could be shifted towards a

composition of solely Gram-negative bacteria by rinsing with vancomycin (12). Goldberg

and Rosenberg described that of various mouthrinses tested, only rinses that contain

hydrophobic ligands in aqueous formulations, can effectively bind and remove bacteria

from aqueous suspensions or desorb bacteria from solid surfaces (13,14). A sufficiently

high bacterial cell surface hydrophobicity is crucial for bacterial removal by hydrophobic

ligands and these observations suggest that making oral bacteria more hydrophobic will

facilitate their removal from the oral cavity by hydrophobic ligands. Exposure of oral

bacteria to cetylpyridinium chloride and chlorhexidine (13) as well as low concentrations of

amoxicillin, penicillin, metronidazole (15) changed the cell surface hydrophobicity of oral

bacterial strains such as Porphyromonas gingivalis and Fusobacterium nucleatum. Also

triclosan, a common oral antibacterial, has been demonstrated to make bacteria more

hydrophobic (16). An oral health care regimen consisting of brushing with a triclosan

containing toothpaste followed by rinsing with a mouthrinse containing hydrophobic ligands

yielded a significant reduction in the prevalence of Streptococcus mutans on orthodontic

retention wires after only 1 week of use (17). The reduction in the prevalence of specifically

S. mutans was attributed to a slight increase in the cell surface hydrophobicity of S. mutans

strains relative to other members of the oral microbiome upon exposure to triclosan,

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although others (18) could not demonstrate effects of exposure to triclosan on S. mutans

cell surface hydrophobicity.

There is a rising interest worldwide in natural products for the maintenance of

health. Magnolia bark extract (MBE) has been widely used in medicine for 2,000 years,

and lately MBE received scientific attention as an anti-inflammatory, anti-platelet and even

chemopreventive agent (19–23). Moreover, inhibitory effects of MBE on the growth of

specific oral bacterial strains have been reported (24). When applied in a chewing gum,

this resulted in a reduction of mutans streptococcal prevalence in saliva and decreased

biofilm acidogenicity after 30 days of use (25). Also in candy applications, such as

compressed mints, incorporation of MBE was effective in reducing the total amount of

bacteria in saliva (26). MBE is harvested from the Magnolia officinalis tree and its two

active components, magnolol and honokiol, are hydrophobic (19,27).

In this study we aim to investigate whether exposure of different oral Gram-

positive and Gram-negative bacteria to MBE alters their hydrophobicity in a direction that

enhances their removal from an aqueous suspension by a hydrophobic ligand. To this end,

a wide array of both Gram-positive and Gram-negative oral strains was exposed to

aqueous solutions with different concentrations of MBE and subsequently their removal by

a hydrophobic ligand was determined using the MATH (Microbial Adhesion To

Hydrocarbon) assay (28) in its kinetic mode (29).


Preparation of bacterial strains A total of 11 oral bacterial strains were used in this study. All strains were grown on blood

agar plates from frozen stocks in dimethylsulfoxide and subsequently inoculated in a 10 ml

pre-culture. Next, 100 µl of the pre-culture was used to inoculate 100 ml of a main culture.

S. mutans ATCC 25175, Streptococcus oralis J22, Streptococcus mitis ATCC 9811,

Streptococcus salivarius HB, Streptococcus sanguinis ATCC 10556 and Streptococcus

sobrinus HG 1025 were grown aerobically at 37 °C in Todd-Hewitt broth (Oxoid,

Basingstoke, UK). Actinomyces naeslundii T14V-J1, P. gingivalis ATCC 33277, Prevotella

intermedia ATCC 43046, Veillonella parvula BME1 and F. nucleatum BME1 were grown

anaerobically at 37 °C in Brain Heart Infusion broth (Oxoid) supplemented with sterile 0.5%

hemin and 0.1% menadione. Bacteria were harvested by centrifugation at 1700 g for 10

min and washed twice in sterile buffer (1 mM calcium chloride, 2 mM potassium

phosphate, 50 mM potassium chloride, pH 6.8) with an ionic strength and composition

similar to saliva (30) and suspended to a concentration of 5 x 108 bacteria/ml.


107

7

Magnolia bark extract A 1% (w/v) solution of MBE (95% magnolol (5,5′-di-2-propenyl-(1,1′biphenyl)-2,2′-diol), 5%

honokiol (3′,5-di-2-propenyl-(1,1′-biphenyl)-2,4′diol), Honsea Sunshine Biotech Co. Ltd.,

Guangzhou, China) in 100% ethanol was mixed with sterile buffer to yield concentrations of

MBE of 25, 50, 100 and 200 µg/ml. Buffer without MBE and with an equal amount of

ethanol added as in the solution containing 200 µg/ml MBE, were used as a control. During

the course of the experimental period, MBE was stored at -20 °C and all solutions were

freshly prepared for each experiment.

Microbial adhesion to hydrocarbons Microbial Adhesion to Hydrocarbons (MATH) measures the removal of microorganisms

from aqueous suspensions by quantifying their adhesion to a hydrophobic ligand after

mixing (28). As the original MATH assay was criticized for not being sufficiently

quantitative, we here use the MATH assay in its kinetic mode (31). First, 3 ml of bacterial

suspension was combined with MBE solutions for 10 min and its optical density (A0)

measured (Spectronic 20 Genesys, Thermo Scientific, Waltham MA, USA). Subsequently,

150 µl of hexadecane was added to each glass tube and mixed for 10 s using a vortex

mixer set at a fixed rotation speed. The resulting emulsion was allowed to settle for 10 min

for phase separation before the optical density of the aqueous phase was measured again

(At). This process was repeated 6 times yielding a total mixing time of 60 s. Next, log(At/A0

x 100) was plotted against mixing time and the initial removal rate (R0) was calculated from

the tangent of the curve at time zero. R0 accordingly represents bacterial removal rate per

min from the suspension by hexadecane. All experiments were performed in triplicate for

each bacterial strain.

Statistics Removal rates of each bacterial strain were averaged for the different concentrations of

MBE applied, analyzed for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests (p

< 0.05) and compared using an ANOVA followed by LSD post-hoc analysis to identify

differences between MBE concentrations (p < 0.05). Next, removal rates of all Gram-

negative and Gram-positive strains were averaged at the different MBE concentration,

again analyzed for normality and equality of means was compared using an ANOVA

followed by LSD post-hoc analysis to identify differences between MBE concentrations (p <

0.05). Statistical analysis was performed using SPSS v20.0 (IBM Corp., Armonk, USA).

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7

Results

As an example, Fig. 1 presents the decrease in the optical density as a function of time

during mixing with hexadecane of bacterial suspensions prior to and after bacterial

exposure to different concentrations of MBE. Clearly, exposure of Gram-positive S.

sobrinus to MBE has no significant effect on its removal by a hydrophobic ligand, while P.

gingivalis removal increases with increasing MBE concentration.

Figure 1

Removal by hexadecane, expressed as log(At/A0 x 100) as a function of the mixing time for a Gram-

positive (S. sobrinus HG 1025) and Gram-negative (P. gingivalis ATCC 33277) oral bacterial strain

after exposure to different concentrations of MBE. Note: bacterial removal rates R0 (min-1) are derived

from the initial linear part of the curves. Error bars represent standard error of the mean over three

experiments with separately grown bacteria.

Initial removal rates R0, as derived from the graphs such as presented in Fig. 1,

are summarized in Fig. 2 for all strains and MBE concentrations. Initial removal rates prior

to exposure to MBE vary ten-fold across the strains and are relatively low for S. mutans

ATCC 25175, S. oralis J22 and P. intermedia ATCC 43046, whilst being high for S. mitis

ATCC 9811 and S. salivarius HB. Initial removal rates of F. nucleatum were extremely high

compared to the other strains. In general initial removal rates increase upon exposure to

solutions with increasing concentrations of MBE and increases are statistically significant

compared to 0 µg MBE/ml at 200 µg MBE/ml for all Gram-negative strains with the

exception of F. nucleatum. Initial removal rates of F. nucleatum also increase with

increasing concentrations of MBE in the exposure solution, but a maximal removal rate is

reached at 100 µg MBE/ml after which a minor decrease sets in.

1.6

1.7

1.8

1.9

2.0

0.0 0.2 0.4 0.6 0.8 1.0

Mixing time (min)

P. gingivalis ATCC 33277

1.6

1.7

1.8

1.9

2.0

0.0 0.2 0.4 0.6 0.8 1.0

log

(At/

A 0 *

100)

Mixing time (min)

S. sobrinus HG 10250 µg/ml

25 µg/ml

50 µg/ml

100 µg/ml

200 µg/ml


109

7

Figure 2

Initial removal rate by hexadecane (R0) for 11 oral bacterial strains prior to and after exposure to

aqueous solutions of MBE at different concentrations. Bacterial names indicated in bold are Gram-

negative strains. Asterisks (*) indicate a significant difference compared to 0 µg MBE/ml, i.e. prior to

exposure to MBE. Error bars denote standard error of the mean over three experiments with

separately grown bacteria.

Next, initial removal rates were averaged for all Gram-negative (with the exception

of F. nucleatum as this would yield a skewed data distribution) and Gram-positive strains

(Fig. 3), revealing that the initial removal rates of Gram-negative bacteria significantly

increase by up to a factor of four with increasing concentrations of MBE, while removal of

Gram-positive bacteria is not affected at all by the exposure to MBE.

* *

*

CHAPTER 7

110

7

Figure 3

Initial removal rates averaged for the Gram-positive and Gram-negative bacterial strains included in

this study prior to and after exposure to aqueous solutions of MBE at different concentrations. The

removal rate of Gram-negative strains increases with higher concentrations of MBE. Asterisks (*)

indicate a significant difference compared to 0 µg MBE/ml, i.e. prior to exposure to MBE. Error bars

denote standard deviation over the different strains included. Note that among the Gram-negative

strains, F. nucleatum has not been included in this graph as this would yield a skew data distribution

due to its extremely high removal rate.

Discussion

The development of new oral health care products is currently aimed towards maintaining

the oral microbiome at health, targeting at the removal of oral pathogens (8,10). Here we

demonstrate that bacterial exposure to MBE in aqueous solution yields changes in

hydrophobicity of Gram-negative oral bacteria that enhance their removal by a hydrophobic

ligand but not of Gram-positive strains. This suggests that an oral health care regimen

consisting of exposure of the oral microbiome to MBE followed by exposure to a

hydrophobic ligand can selectively remove Gram-negative bacterial strains from the oral

cavity. Such a health care regimen may be used to reduce the prevalence of Gram-

negative associated oral diseases.

Gram-negative bacterial strains differ from Gram-positive strains by the

possession of a double lipid membrane. Every bacterial strain is decorated with a variety of

different surface appendages (32), but Gram-positive strains mainly expose a thick

peptidoglycan layer as their outer surface, whilst the outer surface layer of Gram-negative

bacteria mainly consists of the outer lipid membrane. Hydrophobic compounds adsorb

-1.0

-0.8

-0.6

-0.4

-0.2

0.00 25 50 100 200

Initi

al re

mov

al ra

te R

0 (m

in-1

)

MBE concentration (µg/ml)

Gram-positive-1.0

-0.8

-0.6

-0.4

-0.2

0.00 25 50 100 200

MBE concentration (µg/ml)

Gram-negative*

*


111

7

more readily to hydrophobic lipids than to more hydrophilic peptidoglycan, which explains

why magnolol and honokiol are known to bind more tightly to the surface of Gram-negative

bacteria (lipid content of cell wall 25% dry weight) than to the Gram-positive bacterial cell

surface (lipid content of cell wall 0-3% dry weight) (33). Accordingly, it can be understood

why bacterial exposure to solutions containing MBE enhances removal by a hydrophobic

ligand of Gram-negative strains and not of Gram-positive ones.

In general, anaerobic Gram-negative bacteria, such as P. intermedia, P. gingivalis

and F. nucleatum are considered causative in the etiology of halitosis (34,35) as well as in

oral diseases like gingivitis and periodontitis. Previously it was shown that a small, but

significant four-fold increase in removal rate of S. mutans after exposure to triclosan by

hydrophobic mouthrinse components could invoke a change in oral biofilm composition

(17). Our current study shows a similar potential of MBE to affect the Gram-negative

bacterial cell wall and enhance its removal from the oral cavity when applied in two-

component health care products to target Gram-negative bacteria associated oral

diseases. For experimental reasons, we could not expose bacteria for shorter times to

MBE solutions than the 10 min used, which does not necessarily imply that shorter

exposure times would have given different results. Nevertheless, mouthrinses or

toothpastes with typical applications times of less than 2 min, may not be the most suitable

vehicles for a two-component product suggested here. Chewing gums or candies typically

have residence times in the oral cavity around 10 min and may therefore be more suitable

to exploit the potential of MBE in two-component variants.

The current data provide proof of principle for an effect of a two-component oral

health care product containing MBE and a hydrophobic ligand on selective removal of

Gram-negative bacteria from the oral cavity. Subsequent clinical testing should be

conducted to establish whether such a product will also enhance bacterial removal from

enamel surfaces, leading to an in vivo reduction of the prevalence of Gram-negative

pathogens in the oral cavity, potentially associated with reductions in disease prevalence.

Concluding, in this study we demonstrate that exposure to MBE of Gram-negative

oral bacteria enhances their removal from an aqueous phase by a hydrophobic ligand,

while not affecting removal of Gram-positive oral bacterial strains. To the best of our

knowledge this is the first time that enhanced microbial adhesion to a hydrophobic ligand

for Gram-negative oral bacteria by MBE is demonstrated, showing the potential of MBE as

an active ingredient in oral care products and nutraceuticals that contain a hydrophobic

ligand.

CHAPTER 7

112

7

Acknowledgements We would like to thank Minne Koopmans for assisting and performing a part of the MATH

experiments.

Conflict of interest This work was funded by Wm. Wrigley Jr. Co, Chicago, USA and SASA BV, Thesinge, NL.

Authors were employed by their own organizations. HJB is also director-owner of SASA

BV, AM, MWJD are employees of the Wm. Wrigley Jr. Company. Opinions and assertions

contained herein are those of the authors and are not meant to be construed as the

representing views of the organizations to which the authors are affiliated. This study

relates to a pending patent application.

References


2. Crocombe L, Brennan D, Slade G, Loc D. Is self interdental cleaning associated with dental plaque levels, dental calculus, gingivitis and periodontal disease? J Periodontal Res. 2012; 47(1):188–97.

3. Aas J, Paster B, Stokes L. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005; 43(11):5721–32.


5. He J, Li Y, Cao Y, Xue J, Zhou X. The oral microbiome diversity and its relation to human diseases. Folia Microbiol (Praha). 2014; 60:69–80.

6. Han YW. Oral health and adverse pregnancy outcomes - what’s next? J Dent Res. 2011; 90(3):289–93.

7. Marsh PD, Head DA, Devine DA.

Ecological approaches to oral biofilms: Control without killing. Caries Res. 2015; 49:46–54.

8. Eckert R, Sullivan R, Shi W. Targeted Antimicrobial treatment to re-establish a healthy microbial flora for long-term protection. Adv Dent Res. 2012; 24(2):94–7.

9. Allaker RP, Ian Douglas C. Non-

conventional therapeutics for oral infections. Virulence. 2015; 6(3):196–207.

10. Li L, Guo L, Lux R, Eckert R,

Yarbrough D, He J, et al. Targeted antimicrobial therapy against Streptococcus mutans establishes protective non-cariogenic oral biofilms and reduces subsequent infection. Int J Oral Sci. 2010; 2(2):66–73.

11. Sullivan R, Santarpia P, Lavender S,

Gittins E, Liu Z, Anderson MH, et al. Clinical efficacy of a specifically targeted antimicrobial peptide mouth rinse: Targeted elimination of Streptococcus mutans and prevention of demineralization. Caries Res. 2011; 45(5):415–28.

12. Loe H, Theilade E, Jensen SB.

Experimental gingivitis in man. J


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Periodontol. 1967; 36:177–87. 13. Goldberg S, Konis Y, Rosenberg M.

Effect of cetylpyridinium chloride on microbial adhesion to hexadecane and polystyrene. Appl Environ Microbiol. 1990; 56(6):1678–82.

14. Goldberg S, Rosenberg M. Bacterial desorption by commercial mouthwashes vs two-phase oil: Water formulations. Biofouling. 1991; 3:193–8.

15. Lee SY, Hong J, Cheong YJ. Subinhibitory concentrations of antibiotics affect cell-surface hydrophobicity and morphology of Porphyromonas gingivalis and Fusobacterium nucleatum. 81st General Session of the International Association for Dental Research - June 25-28. 2003. Abstract #2157. Available from: https://iadr.confex.com/iadr/2003Goteborg/techprogram/abstract_33158.htm

16. Ellison ML, Champlin FR. Outer membrane permeability for nonpolar antimicrobial agents underlies extreme susceptibility of Pasteurella multocida to the hydrophobic biocide triclosan. Vet Microbiol. 2007; 124(3-4):310–8.

17. Jongsma MA, Van der Mei HC, Atema-

smit J, Busscher HJ, Ren Y. In vivo biofilm formation on stainless steel bonded retainers during different oral health-care regimens. Int J Oral Sci. 2015; 6:1–7.

18. Bedran TBL, Grignon L, Spolidorio DP,

Grenier D. Subinhibitory concentrations of triclosan promote Streptococcus mutans biofilm formation and adherence to oral epithelial cells. PLoS One. 2014; 9(2):e89059.

19. Zhang L, Wang X. Hydrophobic ionic liquid-based ultrasound-assisted extraction of magnolol and honokiol from cortex Magnoliae officinalis. J Sep Sci. 2010; 33(13):2035–8.

20. Lai CS, Lai YS, Kuo DH, Wu CH, Ho CT, Pan MH. Magnolol potently suppressed lipopolysaccharide-induced iNOS and COX-2 expression via downregulating MAPK and NF-kB signaling pathways. J Funct Food. 2011; 3(3):198–206.

21. Fuentes E, Palomo I. Antiplatelet effects of natural bioactive compounds by multiple targets: Food and drug interactions. J Funct Foods. 2014; 6(1):73–81.

22. Liou SF, Hua KT, Hsu CY, Weng MS. Honokiol from Magnolia spp. induces G1 arrest via disruption of EGFR stability through repressing HDAC6 deacetylated Hsp90 function in lung cancer cells. J Funct Foods. 2015; 15(510):84–96.

23. Chao LK, Liao PC, Ho CL, Wang EC, Chuang CC, Chiu HW, et al. Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein kinase, and the NF-kB pathway to reduce LPS-induced TNFalpha and NO expression. J Agric Food Chem. 2010; 58(6):3472–8.

24. Cheng L, Li J, He L, Zhou X. Natural

Products and caries prevention. Caries Res. 2015; 49(1):38–45.


26. Greenberg M, Urnezis P, Tian M.

Compressed mints and chewing gum containing magnolia bark extract are effective against bacteria responsible for oral malodor. J Agric Food Chem. 2007; 55:9465-9

27. William Wrigley Jr. company. Application for the approval of magnolia bark supercritical carbon dioxide

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extract (MBSE) from magnolia officinalis under regulation (EC) No 258/97 of the European parliament and of the council of 27th January 1997 concerning novel foods and novel food ingredients. 2009; 1–79

28. Rosenberg M. Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiol Lett. 1984; 22:289–95.

29. Lichtenberg D, Rosenberg M, Sharfman N, Ofek I. A kinetic approach to bacterial adherence to hydrocarbon. J Microbiol Methods. 1985; 4(3-4):141–6.


31. Lichtenberg D, Rosenberg M, Scharfman N, Ofek I. A kinetic approach to bacterial adherence to hydrocarbons. J Microbiol Methods. 1985; 4:141–6.

32. Nesbitt WE, Doyle RJ, Taylor KG. Hydrophobic interactions and the adherence of Streptococcus sanguis to hydroxylapatite . 1982; 38(2):637–44.

33. Greenberg M, Dodds M, Tian M. Naturally occurring phenolic antibacterial compounds show effectiveness against oral bacteria by a quantitative structure- activity relationship study. J Agric Food Chem. 2008; 56(23):11151–6.

34. Bollen CM, Beikler T. Halitosis: the multidisciplinary approach. Int J Oral Sci. 2012; 4(2):55–63.

35. Porter SR, Scully C. Oral malodour (halitosis). BMJ. 2006; 333:632–5.

General discussion

Chapter 8

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Towards targeting specific bacterial strains and species using chewing gum

Over the last decades, sugar-free chewing gum has established itself, alongside traditional

oral care products, as an oral health promoting agent. Especially after a meal, chewing

sugar-free gum is a valuable adjunct to regular oral hygiene, mainly by stimulating salivary

flow and washing away food debris. However, as was described in chapter 2, effects of

active ingredients incorporated in chewing gum remain difficult to prove next to these basic effects of regular sugar-free gum. This was confirmed in chapter 3, where we could not

observe differences between chewing gum with and without active ingredients but we did

observe a basic effect of chewing; increased diversity in bacterial composition of the oral

biofilm after chewing gum for four weeks. Effects of active ingredients in chewing gum are

often non-specific, targeting all bacterial strains and species present, providing equal

opportunities for all bacterial strains to colonize the surface again and form a biofilm (1,2).

Recent developments in preventive dentistry focus on specifically targeting pathogenic

bacteria. For instance, specifically removing and maintaining low levels of cariogenic

Streptococcus mutans in oral biofilm results in the protection from caries (1–3). Therefore,

the development of a chewing gum that can specifically target pathogenic bacteria seems

a feasible strategy to reduce the chances of establishment and overgrowth of pathogenic

bacteria in an oral biofilm. Using the results of this thesis as a framework, we expand on

this strategy, providing possibilities and experimental guidelines.

Within this strategy we can define two possible approaches. The first approach is

based on specific binding of pathogenic bacteria to chewing gum. The second approach

concerns release of active ingredients from chewing gum to target pathogenic bacteria.

Specific binding of pathogenic bacteria to chewing gum

For this approach, chapters 5 and 6 form the foundation; bacterial adhesion forces play an

important role in the oral cavity and bacteria can (non-specifically) be trapped in a piece of

chewed gum and thereby removed from the oral cavity. An interesting additional finding

was that the total numbers of trapped bacteria decreased as people chewed longer,

indicating that the adhesion forces of bacteria to the gum changed during chewing,

presumably due to salivary uptake. Furthermore, there was a small difference between the

two gum types that were evaluated. Both findings indicate that bacterial entrapment can be

influenced by components in chewing gum.

The next step is exploring the possibilities to promote specific binding of bacteria

to gum. Previously, in vitro research demonstrated that calcium carbonate incorporated in

GENERAL DISCUSSION

117

8

chewing gum promotes adsorption of cariogenic bacteria (4). Unfortunately, this study did

not assess whether calcium carbonate stayed in the gum during chewing, which is an

important requirement for binding bacteria to the gum. To avoid the problem of “leaching

out of ingredients”, it seems plausible to make adjustments to the insoluble core

component, the gumbase. Changing molecular weight of the elastomers and/or resins,

such as polyvinylacetate (PVA), the main components of gumbase, determines the

softening point and viscosity of the gum (5), which could be a feasible strategy to influence

bacterial adhesion.

Assessment of bacterial binding We performed a series of pilot experiments to facilitate the selection of the appropriate

gumbase for this approach. Properties of the gumbase can reflect the tendency for

bacterial adhesion forces. We used atomic force microscopy (AFM) to determine

differences in adhesion forces of specific bacteria towards different gumbases and water

contact angles to determine the hydrophobicity of the gumbase.

For both techniques, four types of ball-shaped gumbase samples (0.2 g per

sample) were cut in half to expose the interior of the gumbase (average surface roughness

300 nm). Bacterial probes of different bacterial species were prepared and AFM

measurements were performed on the interior of the gumbase (see chapter 5 for bacterial

probe preparation and AFM measurements). Depending on the gumbase and bacterial

strain, small but significant differences in adhesion forces were observed. Especially forces

towards gumbase A and B were higher, respectively lower than to the other gumbases, but

this is very strain dependent (Fig. 1A and Table 1A). Despite that this model still has to be

improved in order to better mimic the in vivo situation, for instance by applying a saliva

coating to the gumbase and testing more bacterial strains at various contact times, it

shows that gumbase properties can be adjusted to influence the adhesion forces of oral

bacteria.

Additionally, water contact angles were measured on the gumbase to determine

surface hydrophobicity (Fig. 1B). Here, the water contact angle of gumbase D was

significantly higher than of the other gumbases (Table 1B) and all tested gumbases displayed slightly higher contact angles than the chewing gums that were used in chapter 6 (69 and 74 degrees, respectively). Despite that no definitive conclusion could be drawn

from this pilot study, it shows that hydrophobicity of gumbase is an interesting parameter to

include in future experiments.

Although more research is necessary to determine suitable components for

specific bacterial binding to gumbase surfaces, once a product has been developed,

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118

0.0

0.5

1.0

1.5

2.0

2.5

3.0

S mutansATCC700610

S sobrinusHG1025

S sanguinisATCC10556

S mutans NS

Adhe

sion

For

ce (n

N)

Gumbase A Gumbase B Gumbase C Gumbase D

8

bacterial culturing on specific agar plates and RT-PCR with specific bacterial primers to determine bacterial entrapment in chewing gum (see also chapter 6) can be adapted and

applied to check whether such a gum actually enhances specific bacterial binding to the

gum in vivo compared to a regular sugar-free gum.

Figure 1

A: Adhesion forces of bacteria to different types of gumbase, error bars denote 95% confidence

intervals over at least 40 force curves measured at randomly chosen spots using two different

bacterial probes.

B: Water contact angles (θ) to the different gumbases, error bars denote standard deviations over at

least two gumbase samples each measured in triplicate..

Release of active ingredients from chewing gum that specifically target pathogenic bacteria

A second approach is focused on the release of active ingredients, aimed to decrease the

presence of specific pathogenic bacteria in the oral cavity. Active ingredients in chewing

gum, such as chlorhexidine (6) have been shown to reduce the total number of oral

bacteria. Currently ingredients are investigated that can specifically target pathogenic

bacteria.

-

0

20

40

60

80

100

120

Cont

act a

ngle

(deg

rees

)

Gumbase S mutans

ATCC 700610

S sobrinus

HG 1025

S sanguinis

ATCC 10556

S mutans

NS

-

-

-

-

-

GENERAL DISCUSSION

119

8

Especially the reduction of mutans streptococci, associated with a reduced risk of

caries incidence, received attention in literature. For example, antimicrobial peptides that

specifically kill Streptococcus mutans, without affecting other non-cariogenic bacteria (2)

might be suitable ingredients for chewing gum, although no long term clinical studies have

been performed yet to see whether this results in reduced incidence of caries.

Table 1 A: Statistical analysis of adhesion forces of bacteria to different types of gumbases (A, B, C and D)

using the Kruskal Wallis test, followed by a Mann-Whitney U test to identify pairwise difference.

B: Statistical analysis of water contact angles to the same types of gumbase using a Student T-test.

Asterisks (*) indicate a significant difference according to an adjusted significance level after a

bonferroni correction (P<0.008)

A.

S. mutans ATCC 700610

B. Contact angle

A B C D

A B C D

A X

X B 0.000* X

0.955 X

C 0.000* 0.072 X

0.016 0.016 X

D 0.000* 0.003* 0.679 X

0.005* 0.005* 0.14 X

S sobrinus HG 1025

A X B 0.000* X C 0.201 0.000* X D 0.196 0.001* 0.027 X

S sanguinis ATCC 10556

A X B 0.084 X C 0.001* 0.632 X D 0.001* 0.016 0.306 X

S mutans NS

A X B 0.000* X

Furthermore various other compounds can be introduced in chewing gum that

prevent adhesion of specific bacteria to the biofilm or enamel. For instance certain types of

glycoconjugates or plants lectins can specifically inhibit the biofilm forming properties of

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mutans streptococci (7,8), while also biosurfactants released by other oral bacteria have

been proposed as a tool to specifically prevent the adhesion of mutans streptococci (9).

Lastly, in Chapter 7 we proposed to use magnolia bark extract in combination

with an hydrophobic ligand to specifically remove Gram-negative bacteria from the oral

cavity. Similar to a two-phase mouthrinse (10), this could have interesting applications in

chewing gum helping to reduce diseases associated with Gram-negative oral bacteria,

such as halitosis.

The future of chewing gum

In this thesis we explored new possibilities to further develop the oral health benefits of

chewing gum. Based on the research in this thesis and the current developments in

preventive dentistry of specifically targeting pathogenic bacteria, we identified two

approaches for chewing gum to go along with these developments. The first approach is

based on specific binding of pathogenic bacteria to chewing gum and the second approach

concerns release of active ingredients from chewing gum to specifically target pathogenic

bacteria.

Although more research is needed, the findings of this thesis show that both

approaches are feasible and it can form the framework towards the development of such a

chewing gum.

References 1. Eckert R, Sullivan R, Shi W. Targeted

Antimicrobial treatment to re-establish a healthy microbial flora for Long-term Protection. Adv Dent Res. 2012; 24(2):94–7.

2. Guo L, McLean JS, Yang Y, Eckert R, Kaplan CW, Kyme P, et al. Precision-guided antimicrobial peptide as a targeted modulator of human microbial ecology. Proc Natl Acad Sci. 2015;112(24):7569-74.

3. Li L, Guo L, Lux R, Eckert R, Yarbrough D, He J, et al. Targeted antimicrobial therapy against Streptococcus mutans establishes protective non-cariogenic oral biofilms and reduces subsequent infection. Int J Oral Sci. 2010; 2(2):66–73.


5. Wacker Chemie. Info sheet Vinnapas ® Polyvinylacetate.


7. Oliveira MRTR, Napimoga MH, Cogo K, Gonçalves RB, Macedo MLR, Freire MGM, et al. Inhibition of bacterial adherence to saliva-coated through plant lectins. J Oral Sci. 2007; 49(2):141–5.

GENERAL DISCUSSION

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8. Schüler V, Lussi A, Kage A, Seemann R. Glycan-binding specificities of Streptococcus mutans and Streptococcus sobrinus lectin-like adhesins. Clin Oral Investig. 2012; 16(3):789–96.

9. Van Hoogmoed CG, Van der Mei HC, Busscher HJ. The influence of biosurfactants released by S. mitis BMS on the adhesion of pioneer strains and cariogenic bacteria. Biofouling. 2004; 20(6):261–7.

10. Kozlovsky A, Goldberg S, Natour I, Rogatky-Gat A, Gelernter I, Rosenberg M. Efficacy of a 2-phase oil: water mouthrinse in controlling oral malodor, gingivitis, and plaque. J Periodontol. 1996; 67(6):577–82.

122

Summary

Summary

124

Causative to most oral diseases, including caries, gingivitis and periodontitis, is the

formation of oral biofilm. Daily removal of the oral biofilm is required to prevent the onset of

these diseases. Traditional oral health care products include the toothbrush, mouthrinses,

toothpicks and dental floss. Since the 1970s, also chewing gum developed from a candy

into an oral care product. The main oral health benefits of chewing gum center around the

stimulation of saliva due to mastication, but also various active ingredients are incorporated

in chewing gum aiming to enhance the oral health benefits of chewing gum.

In chapter 1 we set out to explore new possibilities to further develop the oral

health benefits of chewing gum.

The evidence for oral health benefits of chewing gum, with emphasis on

identification of active ingredients in gum that facilitate prevention and removal of oral biofilm is reviewed in chapter 2. Here we found that chewing of sugar-free gum yields oral

health benefits that include clearance of interdental debris, reduction in oral dryness and

amount of occlusal oral biofilm. These basic effects of the chewing of gum are attributed to

increased mastication and salivation. Active ingredients incorporated in chewing gums aim

to expand these effects to inhibition of extrinsic tooth stain and calculus formation,

stimulation of enamel remineralization, reduction of the numbers of bacteria in saliva and

amount of oral biofilm, neutralization of biofilm pH, and reduction of volatile sulfur

compounds. However, clinical benefits of incorporating active ingredients are often hard to

prove, since they are frequently overshadowed by the effects of increased mastication and

salivation and require daily use of chewing gum for prolonged periods of time. We

concluded that chewing of sugar-free gum can most certainly contribute to oral health

when used on a daily basis, but clinical benefits of incorporating active ingredients into

chewing gum are hard to demonstrate over the beneficial effects of increased mastication

and salivation. In chapter 3 we evaluated the daily use of two chewing gums with two different

active ingredients; magnolia bark extract (MBE) and sodium hexametaphosphate (SHMP),

for a prolonged period of time in vivo with respect to the total number of bacteria in oral

biofilms and their viability as well as with respect to the composition of the biofilm. Ten

healthy volunteers chewed gum with and without MBE or SHMP three times per day for

four weeks, during which oral biofilm was collected. Subsequently, the total number,

viability and composition of bacteria in the biofilm collected were determined. During the

four weeks of chewing gum use, both gums with and without active ingredients yielded no

significant decreases in the total numbers of bacteria and their viability in oral biofilm.

However, a trend of increasing diversity of the bacterial composition of the biofilms

collected was observed for all gums, including control gums without active ingredients

SUMMARY

125

added. In conclusion, the chewing of sugar-free gum on a daily basis for a prolonged

period of time can slowly shift the bacterial composition of the oral biofilm in a more

diverse and therewith healthy direction, however we did not observe an effect of the

addition of active ingredients such as MBE or SHMP.

Adsorbed salivary conditioning films on tooth surfaces are important determinants

for tooth surface wettability and mouthfeel. Based on the same study as in chapter 3, we focused in chapter 4 on effects of chewing gum, with and without MBE and SHMP on

wettability and mouthfeel perception in volunteers. During the four weeks of chewing,

mouthfeel was assessed using a questionnaire and intra-oral water contact angles were

measured, both before, directly after and up to 60 min after chewing. It was found that after

using chewing gum, mouthfeel scores were significantly better than before chewing lasting

up to 60 min. Concurrently, intra-oral water contact angles decreased significantly directly

after chewing, creating a more hydrophilic tooth surface. Results were irrespective of the

addition of active ingredients. A positive subjective mouthfeel experience may constitute a

stimulus for people to chew gum and therewith stimulate salivary flow and wash away food

debris with associated oral health benefits.

In the next chapters we set out to explore other possibilities of improving the oral health benefits of chewing gum. Therefore in chapter 5 we go back to the basics of biofilm

formation, looking at the importance of adhesion forces of bacteria in the oral cavity and its

role in the eventual bacterial composition of the oral microbiome. The latter consists of a

planktonic microbiome as residing in saliva and an adhering microbiome; the biofilm

adhering to oral hard and soft tissues. We hypothesized, that possible differences in

microbial composition of the planktonic and adhering oral microbiome on teeth can be

related to the forces by which different bacterial species are attracted to the tooth surface.

The relative presence of seven oral bacterial species in saliva and biofilm collected from

ten healthy human volunteers was determined twice in each volunteer using denaturing

gradient gel electrophoresis. Analysis of both microbiomes showed complete separation of

the planktonic from the adhering oral microbiome. Next, adhesion forces of corresponding

bacterial strains with saliva coated enamel surfaces were measured using atomic force

microscopy. Species that were found predominantly in the adhering microbiome had

significantly higher adhesion forces to saliva coated enamel (-0.60 to -1.05 nN) than

species mostly present in the planktonic microbiome (-0.40 to -0.55 nN). It is concluded

that differences in composition of the planktonic and the adhering oral microbiome are due

to small differences in the forces by which strains adhere to saliva coated enamel,

providing an important step in understanding site and material specific differences in

composition of biofilms in the oral cavity.

126

In Chapter 6 we investigated whether a piece of chewing gum can trap oral

bacteria and thereby remove them from the oral cavity. To test this, we developed two

methods to quantify numbers of bacteria trapped in chewed gum. In the first method,

known numbers of bacteria were finger-chewed into gum and chewed gums were molded

to standard dimensions, sonicated and plated to determine numbers of colony-forming-

units incorporated, yielding calibration curves of colony-forming-units retrieved versus

finger-chewed in. In a second method, calibration curves were created by finger-chewing

known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of

chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was

analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration

curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were

requested to chew gum up to ten min after which numbers of colony-forming-units and total

numbers of bacteria trapped in chewed gum were determined using the above methods.

The qPCR method, involving both dead and live bacteria yielded higher numbers of

retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria

were maximal during initial chewing after which a slow decrease over time up to ten min

was observed. Around 108 bacteria were detected per gum piece depending on the method

and gum considered. The number of species trapped in chewed gum increased with

chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-

electron-microscopy. Summarizing, using novel methods to quantify and qualify oral

bacteria trapped in chewed gum, we were able to confirm that chewing gum can trap and

remove bacteria from the oral cavity.

In preventive dentistry there is rising interest in specifically targeting pathogenic

bacteria in the oral cavity. Since conventional maintenance of oral health is geared toward

removal of as many bacteria as possible, irrespective of whether they are known

contributors to oral health or disease. It is known that cell surface hydrophobicity of oral

bacteria can be altered by adsorption of components from oral health care products. A

more hydrophobic cell surface enhances bacterial removal by hydrophobic ligands from the oral cavity. In chapter 7 we investigated whether exposure of oral Gram-positive and

Gram-negative bacteria to MBE alters their hydrophobicity to enhance their removal from

an aqueous suspension by adhesion to hexadecane. Eleven oral bacterial strains were

exposed to aqueous solutions containing different concentrations of MBE. Subsequently

their removal from the aqueous phase by hexadecane was measured using the kinetic

“Microbial Adhesion To Hydrocarbons” assay. Exposure of bacteria to MBE in aqueous

solution yielded changes in hydrophobicity of Gram-negative oral bacteria in a dose

responsive manner, enhancing removal by hexadecane. This suggests that a combination

SUMMARY

127

of MBE and hydrophobic ligands may find applications such as chewing gum to reduce the

prevalence of Gram-negative bacteria and associated diseases in the oral cavity.

In chapter 8, we expand on the findings of the previous three chapters towards

new applications in chewing gum. Here we identified two approaches for developments in

chewing gum to specifically target pathogenic bacteria. The first approach is based on specific binding of pathogenic bacteria to chewing gum. For this approach, chapters 5 and 6 form the foundation; bacterial adhesion forces play an important role in the oral cavity

and bacteria can (non-specifically) be trapped in a piece of chewed gum and thereby

removed from the oral cavity. Despite that experiments should be expanded, in a series of

pilot experiments we showed that chewing gum properties can influence the adhesion

forces of oral bacteria to the gum and therefore it seems plausible use chewing gum to

specifically trap pathogenic bacteria. The second approach is an expansion of chapter 7, where we provide

possibilities for release of active ingredients from chewing gum that specifically target

pathogenic bacteria. Although more research is needed, the findings of this thesis show

that both approaches are feasible and it can form the framework towards the development

of such a chewing gum.

128

Dutch summary

Samenvatting

DUTCH SUMMARY

130

De vorming van een orale biofilm, ofwel tandplak, is de oorzaak van de meeste orale

ziekten, waaronder cariës, gingivitis en parodontitis. Om deze ziekten te voorkomen is het

noodzakelijk om de biofilm dagelijks te verwijderen. Traditionele

mondverzorgingsproducten zijn de tandenborstel, mondspoelmiddelen, tandenstokers en

flosdraad. Maar sinds de jaren 70 heeft ook kauwgom zich ontwikkeld van snoepgoed tot

een mondverzorgingsproduct. De voornaamste orale gezondheidsvoordelen van het

kauwen van suikervrije kauwgom centreren zich rond de stimulatie van de speekselvloed

door de kauwbeweging, tevens worden er ook verscheidene ingrediënten toegevoegd aan

kauwgom met als doel om de gezondheidsvoordelen verder uit te breiden. In hoofdstuk 1 stellen we als doel van dit proefschrift om nieuwe mogelijkheden

te ontdekken om de orale gezondheidsvoordelen van kauwgom verder uit te breiden. Hiertoe bespreken we allereerst in hoofdstuk 2 het huidige bewijs voor de

gezondheidsvoordelen van kauwgom, met de nadruk op het identificeren van toegevoegde

ingrediënten die preventie van de vorming en verwijdering van orale biofilm moeten

vergemakkelijken. Hierin werd gevonden dat het kauwen van suikervrije kauwgom

voordelen oplevert met betrekking tot het verwijderen van voedselresten tussen de tanden,

vermindering van een droge mond en de hoeveelheid biofilm op de kauwvlakken van de

tanden. Deze “basiseffecten” kunnen worden toegeschreven aan de kauwbeweging en de

gestimuleerde speekselvloed. Het doel van toegevoegde ingrediënten aan kauwgom is om

deze voordelen uit te breiden tot het voorkomen van extrinsieke tandverkleuring en

tandsteenvorming, stimulatie van de remineralisatie van tandglazuur, reductie van het

aantal bacteriën in speeksel en de hoeveelheid orale biofilm, neutralisatie van de pH in de

biofilm en tot slot een reductie van vluchtige zwavelverbindingen, die geassocieerd zijn met

een slechte adem. Echter, de klinische voordelen van deze ingrediënten zijn vaak moeilijk

te bewijzen, aangezien de effecten gemakkelijk overschaduwd worden door de

“basiseffecten” van kauwen en bovendien het dagelijks gebruik van kauwgom op een

langere termijn vereisen. Daarom concluderen we dat het dagelijks kauwen van suikervrije

kauwgom zeker gezondheidsvoordelen voor de mond kan opleveren, maar dat de effecten

van toegevoegde ingrediënten moeilijk te bewijzen zijn naast de “basiseffecten” van

kauwen.

Het dagelijks gebruik van kauwgom met twee verschillende toegevoegde

ingrediënten; extract van de schors van de Magnolia officinalis plant (MBE) en

natriumhexametafosfaat (SHMP), werd geëvalueerd in hoofdstuk 3. Het kauwen van de

kauwgom werd geëvalueerd over een langere tijdsperiode waarbij gekeken is naar zowel

het totaal aantal bacteriën in de orale biofilm als de samenstelling van de biofilm. Hiertoe

werden er monsters genomen van de orale biofilm bij tien gezonde vrijwilligers die vier

SAMENVATTING

131

weken lang, drie maal daags, kauwgom kauwden met en zonder toegevoegde

ingrediënten. Vervolgens werd het totaal aantal, de levensvatbaarheid en de samenstelling

van de bacteriën in de biofilm bepaald. Er werd gevonden dat gedurende de vier weken

van kauwgomgebruik, voor zowel de kauwgom met als zonder toegevoegde ingrediënten

er geen significante daling was in zowel het totaal aantal bacteriën als hun

levensvatbaarheid in de biofilm. Desalniettemin werd er voor alle geteste kauwgoms, ook

zonder toegevoegde ingrediënten, wel een trend gevonden van toenemende diversiteit in

de bacteriële samenstelling van de biofilm. Hieruit werd geconcludeerd dat het dagelijks

kauwen van suikervrije kauwgom voor een langere tijd er voor kan zorgen dat de bacteriële

samenstelling van de orale biofilm verschuift in een meer diverse en daarmee gezondere

richting. Een effect van MBE en SHMP hierop kon niet worden geobserveerd.

De geadsorbeerde speeksellaag op tandoppervlakken is een belangrijke factor

voor de bevochtigbaarheid van het tandoppervlak en het mondgevoel. Met de opzet van de

studie uit hoofdstuk 3 als basis, ligt de focus van hoofdstuk 4 op de effecten van

kauwgom met en zonder MBE en SHMP op de bevochtigbaarheid van de tanden en de

perceptie van het mondgevoel onder vrijwilligers. Gedurende de vier weken dat vrijwilligers

kauwgom kauwden werd de perceptie van het mondgevoel bepaald middels een

vragenlijst en de intra-orale water randhoeken gemeten, beide zowel voor het kauwen als

tot en met 60 minuten na het kauwen. Er werd gevonden dat de perceptie van het

mondgevoel direct na het kauwen van kauwgom significant beter was dan voor het

kauwen, hetgeen tot 60 minuten lang kon duren. Tegelijkertijd daalden de intra-orale water

randhoeken ook significant direct na het kauwen, wat een teken is van een meer hydrofiel

tandoppervlak. Deze resultaten waren wederom ongeacht de toevoeging van MBE en

SHMP. Een positieve perceptie van het mondgevoel zou een stimulans voor mensen

kunnen zijn om kauwgom te kauwen en zodanig de speekselvloed te stimuleren en de

daarmee geassocieerde gezondheidsvoordelen te benutten.

In de volgende hoofdstukken beginnen we met het ontdekken van nieuwe

mogelijkheden om de orale gezondheidsvoordelen van kauwgom verder uit te breiden. Daarvoor gaan we in hoofdstuk 5 eerst terug naar de basis van de vorming van de orale

biofilm, waarbij er gekeken wordt naar de hechtingkrachten van bacteriën in de mondholte

in relatie tot de bacteriële samenstelling van het orale microbioom. Het orale microbioom

bestaat uit het planktonisch microbioom; de samenstelling van bacteriën in speeksel, en

het hechtende microbioom; de bacteriën die voorkomen in de biofilm die hechten aan

harde en zachte orale weefsels. De hypothese in dit hoofdstuk is dat mogelijke verschillen

in bacteriële samenstelling van het planktonische en hechtende microbioom gerelateerd

zijn aan de hechtingskrachten waarmee de verschillende bacteriën worden aangetrokken

DUTCH SUMMARY

132

tot het tandoppervlak. Hiertoe werd de relatieve aanwezigheid van zeven orale bacteriële

soorten tweemaal bepaald in speeksel en biofilm van tien gezonde vrijwilligers middels

denaturerende gradient gel elektroforese. Analyse liet een complete scheiding zien in de

bacteriële samenstelling van het planktonische microbioom ten opzichte van het hechtende

microbioom. Vervolgens werden de hechtingskrachten van bijbehorende bacteriële

stammen gemeten op, een met speeksel bedekt, oppervlak van tandglazuur middels

atomaire krachtmicroscopie. Bacteriële soorten die voornamelijk in het hechtende

microbioom werden gevonden hadden significant hogere hechtingskrachten richting

tandglazuur met een speeksellaag (-0.60 to -1.05 nN) dan soorten die voornamelijk

aanwezig waren in het planktonisch microbioom (-0.40 to -0.55 nN). Hieruit werd

geconcludeerd dat verschillen in de bacteriële samenstelling van het planktonische en

hechtende microbioom zouden kunnen worden toegeschreven aan de kleine verschillen in

hechtingskracht waarmee bacteriën worden aangetrokken tot tandglazuur met een

speeksellaag. Dit is een belangrijke stap in het begrijpen van de plaats- en materiaal-

afhankelijke verschillen in de bacteriële samenstelling van de orale biofilm.

In hoofdstuk 6 werd onderzocht of een stukje kauwgom gebruikt kan worden

voor het “vangen” van orale bacteriën om deze hiermee uit de mondholte te verwijderen.

Om dit te testen zijn er twee methoden ontwikkeld om het aantal bacteriën dat gevangen

wordt middels een stukje kauwgom te kwantificeren. In het geval van de eerste methode

werden bekende aantallen bacteriën in een stukje kauwgom “gekneed”, dit werd

vervolgens gevormd naar een standaard afmeting, gesonificeerd en uitgeplaat om het

aantal kolonievormende eenheden te bepalen. De hieruit voortkomende ijklijnen tonen het

aantal verkregen kolonievormende eenheden versus het aantal “ingeknede” bacteriën.

Voor de tweede methode werden de ijklijnen verkregen door wederom bekende aantallen

bacteriën in een stukje kauwgom te kneden en vervolgens de kauwgom op te lossen in

een mengsel van chloroform en (tris)-ethyleendiaminetetra-azijnzuur (TE)-buffer. De TE-

buffer werd geanalyseerd middels kwantitatieve polymerase kettingreactie (qPCR),

hetgeen ijklijnen voortbracht voor het totaal aantal bacteriën versus het aantal “ingeknede”

bacteriën. Vervolgens werden vijf vrijwilligers gevraagd om tot tien minuten lang kauwgom

te kauwen waarna het aantal kolonievormende eenheden en het totaal aantal bacteriën dat

gevangen was in het stukje gekauwde kauwgom werd bepaald middels de

bovengenoemde methodes. De qPCR methode, die zowel levende als dode bacteriën

betreft, toonde hogere aantallen gevangen bacteriën dan de kolonievormende eenheden

methode, die enkel levende bacteriën betreft. Het aantal gevangen bacteriën was

maximaal vlak na het starten van het kauwen waarna het aantal langzaam afnam

naarmate er langer werd gekauwd. Ongeveer 108 bacteriën per stukje gekauwde kauwgom

SAMENVATTING

133

werden gedetecteerd, afhankelijk van de gebruikte methode en de kauwgomsoort.

Daarentegen nam het aantal soorten bacteriën dat werd gevangen met een stukje

kauwgom toe naarmate er langer werd gekauwd. Gevangen bacteriën konden worden

gevisualiseerd in de gekauwde kauwgom middels een rasterelektronenmicroscoop.

Samenvattend kan gesteld worden dat door middel van nieuwe methodes het mogelijk is

gevangen bacteriën in een stukje gekauwde kauwgom te kwantificeren en kwalificeren.

Hiermee konden we bevestigen dat kauwgom daadwerkelijk gebruikt kan worden voor het

vangen van orale bacteriën en deze hiermee uit de mondholte te verwijderen.

In de preventieve tandheelkunde is er groeiende interesse voor een meer

doelgerichte aanpak van specifiek de ziekteverwekkende bacteriën in de mondholte. In

tegenstelling tot de huidige conventionele wijze van orale hygiëne, die voornamelijk gericht

is op het verwijderen van zo veel mogelijk bacteriën, ongeacht of deze een bijdrage

leveren aan gezondheid of ziekte. Het is bekend dat de hydrofobiciteit van het celoppervlak

van orale bacteriën veranderd kan worden middels componenten die in

mondverzorgingsproducten aanwezig zijn. Een meer hydrofoob celoppervlak stimuleert het

verwijderen van bacteriën uit de mondholte middels een hydrofobe ligand. In hoofdstuk 7 onderzochten we of de blootstelling van orale Gram-positieve en Gram-negatieve

bacteriën aan MBE de hydrofobiciteit dusdanig kon veranderen zodat de verwijdering van

bacteriën uit een waterige suspensie middels hechting aan hexadecaan vergemakkelijkt

werd. Elf orale bacteriestammen werden hiertoe in een waterige oplossing aan

verschillende concentraties MBE blootgesteld. Vervolgens werd de verwijdering van

bacteriën uit de waterfase door middel van hexadecaan gemeten met de kinetische

“microbiële hechting aan koolwaterstoffen” test. De blootstelling van bacteriën aan MBE in

een waterige oplossing resulteerde in een verandering van de hydrofobiciteit van Gram-

negatieve orale bacteriën volgens een dosis-respons relatie, wat een toename van de

verwijdering middels hexadecaan teweegbracht. Dit wekt de suggestie dat een combinatie

van MBE en een hydrofobe ligand toepassingen heeft in o.a. kauwgom om de prevalentie

van Gram-negatieve bacteriën in de mondholte en mogelijk de daarmee geassocieerde

ziekten te reduceren.

In hoofdstuk 8 worden de vindingen van de vorige drie hoofdstukken verder

uiteengezet richting applicaties in kauwgom. Hiertoe identificeren we twee benaderingen

voor de doelgerichte aanpak van specifiek de ziekteverwekkende bacteriën in de

mondholte. De eerste benadering is gebaseerd op het specifiek binden van ziekteverwekkende bacteriën aan kauwgom. Hoofdstuk 5 en 6 vormden hiervoor de

basis; bacteriële hechtingskrachten spelen een belangrijke rol in de mondholte en

bacteriën kunnen (niet specifiek) worden gevangen in een stukje kauwgom en daarmee

DUTCH SUMMARY

134

worden verwijderd uit de mondholte. Ondanks dat er meer experimenteel werk moet

worden verricht, konden we in een aantal proefprojecten aantonen dat bepaalde

eigenschappen van kauwgom de hechtingskracht van orale bacteriën aan kauwgom kan

beïnvloeden. Derhalve lijkt het plausibel dat kauwgom gebruikt kan worden voor het

doelgericht vangen van ziekteverwekkende bacteriën. De tweede benadering is een uitbreiding van hoofdstuk 7, waarin we mogelijkheden

voorstellen voor de toevoeging van ingrediënten aan kauwgom die specifiek gericht zijn op

de ziekteverwekkende bacteriën. Wederom is er meer onderzoek nodig, maar de

bevindingen van dit proefschrift laten zien dat beide benaderingen mogelijk zijn en het kan

het kader vormen voor de daadwerkelijke ontwikkeling van dit type kauwgom.

Acknowledgements

Dankwoord

ACKNOWLEDGEMENTS

136

You have reached the acknowledgements section of this thesis, which hopefully means

that you have read (at least some of) the contents of this thesis. A thesis that would have

been impossible to make without the people I have had the pleasure to encounter over the

course of the last four years, both inside and outside the scientific community. Here I would

like to express my gratitude to you.

Dear Henk, I am grateful for all the opportunities you have given me to advance on both a

scientific and a personal level. I admire your capability to manage a lot of projects at once

and be up to speed with all of them, always being able to come up with new creative

angles to advance a project. I really enjoyed our travels to Chicago and I yet have to find a

better place to eat steak than at the one you always recommend.

Dear Henny, thank you for all the advice and help you gave during my PhD. You are very

knowledgeable on the practical aspects of doing experiments and you have an amazing

eye for detail. I appreciate it that whenever I had a question I could just stop by and you

could always help me out. Together with Henk you form an excellent team, always

safeguarding progress and being passionate about science.

Dear Betsy and Marja, I am very sure that without your help with all the experimental work

this thesis would not have been possible. You showed me around in the lab, familiarized

me with various techniques and helped a lot with the gathering, analyzing and organizing

all the samples that were collected. Furthermore I would like to thank Jelly, for helping me

with the DGGE. Joop, for teaching me how to operate the AFM and Willy, for your

assistance with the qPCR work and the pipetting robot. Thank you Chris for providing me

with the opportunity to do some teaching and the guidance of a master student.

Dear Mike and Amarnath, thank you for the helpful discussions we had during visits and

via e-mail. I also would like to thank your colleagues Sophie, Taichi, Minmin and

Marcelo, who would often join the meetings and provide helpful insights. It was a pleasure

collaborating with you on this project.

Thank you, Ina and Wya for all the help with practical and financial affairs of any kind,

Gesinda, Minie and René, for happily helping with any question or problem on the lab.

Jelmer, Theo, Patrick, Roel, Ed, Danielle, Prashant, Corien, Hans, Tjarko, Marianne for actively or passively helping with daily affairs.

DANKWOORD

137

Thank you, Bastiaan for introducing me to the scientific world during my master project,

which eventually led to doing this PhD. Also I would like to thank my co-authors Edwin van den Heuvel, David Morando and Yun Chen for the collaboration on work in this thesis.

Thank you René, Willem, Brandon, Jiapeng (JP), Qihui (Jo) and Rebecca for the good

laughs around the lab. Minne, for all the work you did for this thesis during your internship

period. Over the four years many people have come and gone, I would like to thank Ferdi, Mark, Jesse, Akshay, Victor, Steven, Sara, Arina, Adhi, Helen, Lucja, Joana, Bart, Bu,

Brian, Mayeul, Romana, Sarthak, Anna, Song, Jenny, Niar, Marieke, Raquel, Simon,

Katya, Hilde, Vera and Marije for your help, work or non-work related conversations and

presence during my time at the department.

I am very grateful to all the volunteers that were willing to donate saliva, plaque and their

precious time to this research.

Dear reading committee, Prof. dr. J.M. ten Cate, Prof. dr. ir. W. Norde and Prof. dr. Y. Ren, thank you for all the time invested in reading and evaluating this thesis.

Together with Charlotte, Heleen, Tushar, Nicole, Ena, Dario and Mirjan I had the joy of

being in the GSMS PhD Development conference committee. Organizing a conference

was a great and new experience, in which I really enjoyed working with you guys. I think

we can be proud on the results and I hope we can keep on meeting each other.

Jan, Philipp, Ed, Deepak, Barbara, Agnieszka and Otto, thank you for the fun in the lab,

during coffee breaks and lunchtime, but even more outside the department; the parties

(and the day after), the football tournaments and the holiday in Greece.

Heren van AP, al vanaf het eerste jaar Life Science & Technology in 2005 is jullie

gezelschap goed voor mooie feestjes en schitterende momenten in binnen- en buitenland, bedankt hiervoor ! In het bijzonder Koen en Jan-Ytzen, die ik daarnaast ook met enige

regelmaat in het UMCG op de 8e en 9e verdieping kon ontmoeten voor een “bakkie”.

Team4mijl vrienden, de vele trainingsuren op de atletiekbaan, de wedstrijden en

trainingsstages door heel Europa, de onderlinge competitie en het toewerken naar topvorm

op het NK, kortom het samen bedrijven van topsport schept een geweldige band en was

naast het promoveren een belangrijk onderdeel van mijn leven. Gelukkig zie ik de meeste

van jullie, nu sport op een iets minder hoog plan staat, nog steeds zeer regelmatig, met als

ACKNOWLEDGEMENTS

138

één van de hoogtepunten de marathon van Rome. Ik heb nog nooit zo veel spierpijn

gehad, maar hopelijk kunnen we nog veel vaker dit soort grappen uithalen.

Tom en Sybren, het is een eer dat jullie mijn paranimfen willen zijn en jullie taken hierin

uiterst serieus nemen.

Robin, Tom, Jeroen en Paul, de tijd tussen ontmoetingen is meestal groot, maar voor

mijn gevoel maakt dit weinig uit en gaan we altijd verder waar we gebleven waren. Ik kijk

uit naar de volgende keer.

Liesbeth en Joost, jullie hebben altijd enorme belangstelling voor mijn werk en alle zaken

daarbuiten, waarvoor ik jullie zeer dankbaar ben.

Geert, Anja, Esther (en Kian) bedankt voor jullie interesse in mijn werk en ik waardeer het

om in jullie gezelschap te zijn.

Lieve Papa en Mama, bedankt voor jullie onvoorwaardelijke steun in alles wat ik doe. Jullie

staan altijd voor mij klaar en dankzij jullie heb ik dit mogen bereiken. Ik had mij geen betere

ouders kunnen wensen.

Liefste Evita, je bent mijn beste maatje en bij jou kan ik volledig mezelf zijn. Ik geniet van

elk moment samen en ik hoop dat ik dit nog lang mag blijven doen.

About the author

Curriculum Vitae

Curriculum Vitae

140

Stefan Wessel was born in Emmen, the Netherlands on May 3, 1987. After finishing

secondary school in 2005 at the “Hondsrug College” in Emmen he continued his education

at the University of Groningen and obtained a Bachelor of Science degree in Life Science

& Technology in 2009 and obtained a Master of Science degree in Biomedical Engineering

in 2011. The last internship of his Master’s degree was continued in a four year PhD

project at the department of Biomedical Engineering, part of the W.J. Kolff Institute at the

University Medical Center Groningen. This project resulted in the work presented this

thesis, two patents and presentations at various international conferences. In the four

years of doing a PhD project he participated in the organization of the Graduate School of

Medical Sciences PhD Development conference 2015 as a media director. Furthermore he

pursued a sport career in athletics, which resulted in a bronze medal at the national indoor

championships 1500m in 2013.

In 2015 he continued working at the University Medical Center Groningen as a post-

doctoral researcher.

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