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University of Groningen
Oral health benefits of chewing gumWessel, Stefan
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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.
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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/tri- phosphates in sugar-free gums (37) (Table 1).
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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
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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
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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).
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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)
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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
CHEWING GUM: FROM CANDY TO NUTRACEUTICAL
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.
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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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3
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
EFFECTS OF CHEWING GUM ON ORAL BIOFILM VIABILITY AND COMPOSITION
35
3
(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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3
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
EFFECTS OF CHEWING GUM ON ORAL BIOFILM VIABILITY AND COMPOSITION
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
EFFECTS OF CHEWING GUM ON ORAL BIOFILM VIABILITY AND COMPOSITION
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.
Authors were employed by their own organizations. HJB is also director-owner of a
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.
EFFECTS OF CHEWING GUM ON ORAL BIOFILM VIABILITY AND COMPOSITION
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.
3. Zarco MF, Vess TJ, Ginsburg GS. The oral microbiome in health and disease and the potential impact on personalized dental medicine. Oral Dis. 2012; 18(2):109–20.
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.
11. Mickenautsch S, Leal SC, Yengopal V, Bezerra AC, Cruvinel V. Sugar-free chewing gum and dental caries: a systematic review. J Appl Oral Sci. 2007; 15(2):83–8.
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.
16. Campus G, Cagetti MG, Cocco F, Sale S, Sacco G, Strohmenger L, et al. Effect of a sugar-free chewing gum containing magnolia bark extract on different variables related to caries and gingivitis: a randomized controlled intervention trial. Caries Res. 2011; 45(4):393–9.
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.
19. Walters P. Benefits of sodium hexametaphosphate-containing chewing gum for extrinsic stain inhibition. J Dent Hyg. 2004; 78(4):1–9.
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.
Materials and methods
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
Materials and methods
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.
EFFECTS OF CHEWING GUM ON TOOTH SURFACE WETTABILITY AND MOUTHFEEL
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
EFFECTS OF CHEWING GUM ON TOOTH SURFACE WETTABILITY AND MOUTHFEEL
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.
EFFECTS OF CHEWING GUM ON TOOTH SURFACE WETTABILITY AND MOUTHFEEL
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.
Authors were employed by their own organizations. HJB is also director-owner of a
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.
EFFECTS OF CHEWING GUM ON TOOTH SURFACE WETTABILITY AND MOUTHFEEL
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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.
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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,
Anje M. Slomp, Henk J. Busscher and Henny C. van der Mei
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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Materials and methods
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.
ADHESION FORCES AND COMPOSITION OF PLANKTONIC AND ADHERING MICROBIOMES
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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.
ADHESION FORCES AND COMPOSITION OF PLANKTONIC AND ADHERING MICROBIOMES
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5
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.
ADHESION FORCES AND COMPOSITION OF PLANKTONIC AND ADHERING MICROBIOMES
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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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References
1. Aas J, Paster B, Stokes L. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005; 43(11):5721–32.
2. Keijser B, Zaura E. Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res. 2008; 87(11):1016–20.
3. Marsh PD. Dental plaque as a biofilm and a microbial community - implications for health and disease. BMC Oral Health. 2006; 6:S14.
4. Zaura E, Keijser BJF, Huse SM, Crielaard W. Defining the healthy “core microbiome” of oral microbial communities. BMC microbiol. 2009; 9:259.
5. Kreth J, Merritt J, Qi F. Bacterial and host interactions of oral streptococci. DNA Cell Biol. 2009; 28(8):397–403.
6. Hojo K, Nagaoka S, Ohshima T, Maeda N. Bacterial interactions in dental biofilm development. J Dent Res. 2009; 88(11):982–90.
7. Kolenbrander PE, Palmer RJ, Periasamy S, Jakubovics NS. Oral multispecies biofilm development and the key role of cell-cell distance. Nat Rev Microbiol. Nature Publishing Group; 2010; 8(7):471–80.
8. Liu Y, Black MA, Caron L, Camesano TA. Role of cranberry juice on molecular-scale surface characteristics and adhesion behavior of Escherichia coli. Biotechnol Bioeng. 2006; 93(2):297–305.
9. Syed SA, Loesche WJ. Survival of human dental plaque flora in various transport media. Appl Microbiol. 1972; 24(4):638–44.
10. Signoretto C, Bianchi F, Burlacchini G, Sivieri F, Spratt D, Canepari P. Drinking habits are associated with changes in
the dental plaque microbial community. J Clin Microbiol. 2010; 48(2):347–56.
11. Mei L, Busscher HJ, Van der Mei HC, Chen Y, De Vries J, Ren Y. Oral bacterial adhesion forces to biomaterial surfaces constituting the bracket-adhesive-enamel junction in orthodontic treatment. Eur J Oral Sci. 2009; 117(4):419–26.
12. Yongsunthon R, Fowler VG, Lower BH, Vellano FP, Alexander E, Reller LB, et al. Correlation between fundamental binding forces and clinical prognosis of Staphylococcus aureus infections of medical implants. Langmuir. 2007; 23:2289–92.
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.
70
Appendices
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
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
APPENDICES
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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.
APPENDICES
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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
1. Ferreira AVB, Glass NL. PCR from fungal spores after microwave treatment. Fungal Genet Newsl. 1996; 43:25–6.
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).
Materials and methods
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
QUANTIFICATION AND QUALIFICATION OF BACTERIA TRAPPED IN CHEWED GUM
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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
QUANTIFICATION AND QUALIFICATION OF BACTERIA TRAPPED IN CHEWED GUM
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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.
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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.
7. 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.
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.
11. Edgar M, Dawes C. Saliva and oral health. 3rd ed. London: BDJ Books; 2004. 146 p.
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.
14. Mickenautsch S, Leal SC, Yengopal V, Bezerra AC, Cruvinel V. Sugar-free chewing gum and dental caries: a systematic review. J Appl Oral Sci. 2007; 15(2):83–8.
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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.
16. Imfeld T. Chlorhexidine-containing chewing gum. Schweiz Monatsschr Zahnmed. 2006; 116:476–83.
17. 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.
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.
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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.
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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.
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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
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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).
Materials and methods
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.
MBE INCREASES ADHESION OF ORAL GRAM-NEGATIVE BACTERIA TO A HYDROPHOBIC LIGAND
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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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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
MBE INCREASES ADHESION OF ORAL GRAM-NEGATIVE BACTERIA TO A HYDROPHOBIC LIGAND
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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.
* *
*
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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*
*
MBE INCREASES ADHESION OF ORAL GRAM-NEGATIVE BACTERIA TO A HYDROPHOBIC LIGAND
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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.
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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.
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Periodontol. 1967; 36:177–87. 13. Goldberg S, Konis Y, Rosenberg M.
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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.
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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.
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Grenier D. Subinhibitory concentrations of triclosan promote Streptococcus mutans biofilm formation and adherence to oral epithelial cells. PLoS One. 2014; 9(2):e89059.
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General discussion
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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
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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,
CHAPTER 8
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
CHAPTER 8
120
8
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.
4. Yamanaka A, Saeki Y, Seki T, Kato T, Okuda K. Adsorption of oral bacteria to porous type calcium carbonate. Bull Tokyo Dent Coll. 2000; 41(3):123–6.
5. Wacker Chemie. Info sheet Vinnapas ® Polyvinylacetate.
6. Imfeld T. Chlorhexidine-containing chewing gum. Schweiz Monatsschr Zahnmed. 2006; 116:476–83.
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
121
8
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.