dentistry journal

Review
Dental Biofilm and Laboratory Microbial Culture
Models for Cariology Research
Ollie Yiru Yu, Irene Shuping Zhao, May Lei Mei, Edward Chin-Man Lo and Chun-Hung Chu *
Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; yuyiru@hku.hk (O.Y.Y.);
irenezhao110@gmail.com (I.S.Z.); mei1123@hku.hk (M.L.M.); hrdplcm@hkucc.hku.hk (E.C.-M.L.)
* Correspondence: chchu@hku.hk; Tel.: +852-2859-0287

Received: 8 May 2017; Accepted: 15 June 2017; Published: 19 June 2017

Abstract: Dental caries form through a complex interaction over time among dental plaque,
fermentable carbohydrate, and host factors (including teeth and saliva). As a key factor, dental
plaque or biofilm substantially influence the characteristic of the carious lesions. Laboratory microbial
culture models are often used because they provide a controllable and constant environment for
cariology research. Moreover, they do not have ethical problems associated with clinical studies.
The design of the microbial culture model varies from simple to sophisticated according to the purpose
of the investigation. Each model is a compromise between the reality of the oral cavity and the
simplification of the model. Researchers, however, can still obtain meaningful and useful results from
the models they select. Laboratory microbial culture models can be categorized into a closed system
and an open system. Models in the closed system have a finite supply of nutrients, and are also simple
and cost-effective. Models in the open system enabled the supply of a fresh culture medium and the
removal of metabolites and spent culture liquid simultaneously. They provide better regulation of the
biofilm growth rate than the models in the closed system. This review paper gives an overview of the
dental plaque biofilm and laboratory microbial culture models used for cariology research.

Keywords: biofilm; dental plaque; demineralization; remineralization; caries; review

1. Introduction
Dental caries is the localized destruction of dental hard tissues by acidic byproducts from dental
plaque containing acid-producing bacteria. Cariology research allows the investigation of caries’
pathogenicity, testing the effects of new caries-prevention methods (i.e., some devices and drugs)
and developing new caries-preventing products. This review paper gives an overview of the dental
plaque biofilm and in vitro biofilm models used for cariology research. It aims to provide essential
and instructive information for researchers who seek to plan and design cariology research.

2. The Dental Plaque Biofilm

Dental plaque is an oral microbial biofilm that is found on exposed tooth surfaces in the mouth.
It has a large diversity of species and consists of densely packed bacteria embedded in a matrix of
organic polymers of bacterial and salivary origin. Dental plaque is the causal agent of dental caries in
the presence of sugar and time. In the oral cavity, the formation of dental plaque on the tooth surface
follows a similar sequence to that of biofilms in other natural ecosystems. A biofilm is formed by
bacteria sticking to each other and, often, adhering to a surface. The bacteria are embedded within
a self-produced matrix of extracellular polymeric substance. In dental biofilm, streptococcus mutans is
a major bacterium producing the extracellular polysaccharide matrix in dental biofilms. The bacterial
cells growing in a biofilm are physiologically distinct from planktonic cells which float or swim
in a liquid medium. Bacteria in the plaque biofilm can respond to many factors, such as cellular

Dent. J. 2017, 5, 21; doi:10.3390/dj5020021 www.mdpi.com/journal/dentistry

Dent. J. 2017, 5, 21 2 of 12

Dent. growing
cells J. 2017, 5, 21in a biofilm are physiologically distinct from planktonic cells which float or swim2 in of 12
a
liquid medium. Bacteria in the plaque biofilm can respond to many factors, such as cellular
recognition
recognition of of specific
specific or
ornon-specific
non-specificattachment
attachmentsites
sites
onon a surface
a surface andand nutritional
nutritional signals.
signals. Marsh
Marsh and
and Martin [1] divided the formation and growth of oral biofilm into five stages
Martin [1] divided the formation and growth of oral biofilm into five stages (Figure 1). (Figure 1).

Figure 1. Five stages of biofilm formation and growth (adapted from Stoodley et al., 2002 [2], with
Figure 1. Five stages of biofilm formation and growth (adapted from Stoodley et al., 2002 [2], with
permission from © 2002 Annual Reviews Directory. License number: 4131221128126).
permission from © 2002 Annual Reviews Directory. License number: 4131221128126).

Oral biofilms
Oral biofilms cancanformformon onalmost
almostany anysurface
surfacepresent
presentin inthe
theoral
oralcavity
cavityincluding
includingenamel,enamel,dentin,
dentin,
cementum, gingiva,
cementum, gingiva,oral oralmucosa,
mucosa, carious
carious lesion,
lesion,restoration,
restoration,dental implant,
dental and denture.
implant, and denture.DentalDental
plaque
will colonize
plaque rapidly, rapidly,
will colonize not only notthe coronal
only theenamel coronal surface
enamel butsurface
also the butexposed
also the root surface.root
exposed Thesurface.
growth
of microbiota
The growth ofon the exposed
microbiota on root surface proceeds
the exposed root surface more rapidly than
proceeds morethat on the
rapidly smooth
than that onenamel surface
the smooth
becausesurface
enamel of the irregular
because surface topography
of the irregular of the
surface exposed root
topography dentin
of the surface.
exposed rootThe organization
dentin surface. The and
structure of dental
organization plaque vary
and structure considerably
of dental plaqueaccording to the sitesaccording
vary considerably where plaque to the forms
sites[1]. The plaque
where growth
of microorganisms
forms [1]. The growth onofspecific oral niches
microorganisms onisspecific
affectedoralby niches
various is factors
affectedsuch as acidity
by various (pH)
factors of the
such as
environment,
acidity (pH) ofavailability
the environment,of nutrients, presence
availability of antimicrobial
of nutrients, presence agents, and host defense.
of antimicrobial agents, and host
Surface-bound microorganisms have a survival and/or selective advantage over their planktonic
defense.
phases [1]. Bacteria microorganisms
Surface-bound in dental plaque have have astronger
survivalresistance to antimicrobial
and/or selective advantageagents than planktonic
over their planktonic
bacteria.
phases [1].Bacterial
Bacteria in extracellular
dental plaque polysaccharides prevent the
have stronger resistance to perfusion
antimicrobial of antimicrobial agents to
agents than planktonic
bacterial targets; this acts as a barrier to protect the plaque bacteria
bacteria. Bacterial extracellular polysaccharides prevent the perfusion of antimicrobial agents to against certain environmental
threats such
bacterial as antibiotics,
targets; this acts as antibodies,
a barrier surfactant,
to protect bacteriophage,
the plaque bacteria and whiteagainstblood cellsenvironmental
certain [3]. Resistance
of biofilm
threats suchbacteria to antimicrobial
as antibiotics, antibodies,agents may also
surfactant, develop. As
bacteriophage, and a white
result,blood
the minimum inhibitory
cells [3]. Resistance
concentration of antimicrobial agents against bacteria in biofilm is
of biofilm bacteria to antimicrobial agents may also develop. As a result, the minimum inhibitory significantly higher (up to 1000-fold)
than that in liquid
concentration [1].
of antimicrobial agents against bacteria in biofilm is significantly higher (up to 1000-
fold) Though
than thatthere are many
in liquid [1]. bacteria associated with dental caries, a few groups of cariogenic bacteria
as streptococci,
suchThough there areactinomycetes,
many bacteriaand lactobacilli
associated withare found
dental to bea more
caries, closelyofassociated
few groups cariogenic than bacteriathe
others.
such as These groupsactinomycetes,
streptococci, of bacteria often anddominantly
lactobacilli are proliferate
found toinbe themore
dental biofilm
closely collected than
associated from thethe
carious lesions of teeth. Streptococcus is the predominant species in
others. These groups of bacteria often dominantly proliferate in the dental biofilm collected from the cariogenic microbe. It colonizes
clean tooth
carious lesionssurfaces
of teeth. at Streptococcus
an early stage, andpredominant
is the it also relates to root
species in caries.
cariogenic Themicrobe.
predominant coccal
It colonizes
isolated
clean toothfrom carious
surfaces at andentin
earlyin rootand
stage, caries it also S. mutans,
are relates S. sanguis,
to root caries. The and S. mitis [4]. coccal
predominant S. mutans and
isolated
S. sobrinus are difficult to distinguish. Hence, these two species are
from carious dentin in root caries are S. mutans, S. sanguis, and S. mitis [4]. S. mutans and S. sobrinus always lumped together and
regarded
are difficult mutans
as to streptococci.
distinguish. Hence, Mutans
these streptococci
two speciescan areadapt
always to lumped
acidic environments,
together and which regarded is the
as
key factor
mutans contributing
streptococci. Mutansto itsstreptococci
cariogeniccan adapt Actinomycetes
potential. is an initial which
to acidic environments, colonizer of human
is the root
key factor
surfaces. A. naeslundii
contributing and A.potential.
to its cariogenic viscosus can induce rootissurface
Actinomycetes caries
an initial [5]. Actinomycetes
colonizer of human root is often isolated
surfaces. A.
from subgingival
naeslundii and A. microflora
viscosus canand from root
induce plaque associated
surface carieswith
[5]. root caries [6] (they
Actinomycetes is oftenhave long surface
isolated from
appendages microflora
subgingival named fibrils, and or from
fimbriae).
plaque Theassociated
fibrils allow actinomycetes
with root caries to [6]
adhere
(they to have
the surface
long of tooth
surface
roots. Fibrils named
appendages also improvefibrils,the
orattachment
fimbriae). The of actinomycetes
fibrils allow to actinomycetes
other bacteriato in adhere
dental plaque. Lactobacilli
to the surface of
are aciduric
tooth bacteria,
roots. Fibrils also improveL.the
including acidophilus,
attachment L. rhamnosus, L. casei,toand
of actinomycetes L. oris
other [7]. Patients
bacteria in dental with caries
plaque.
have higher
Lactobacilli arecounts
aciduric of lactobacilli than those
bacteria, including with no caries.
L. acidophilus, Evaluating
L. rhamnosus, the amount
L. casei, and L. orisof Lactobacilli
[7]. Patients in
Dent. J. 2017, 5, 21 3 of 12

saliva is used as a caries-activity testing method in clinical assessment [8]. Lactobacilli is difficult to
grow and mature as a mono-species biofilm. However, it can be a predominate species in a substantial
biofilm in the presence of S. mutans [9]. A potential relationship was found among some species of
lactobacilli, streptococci, and actinomycetes in the root caries formation process [10].

3. Laboratory Microbial Culture Models

Laboratory microbial culture models simulate the oral environment for cariology study. Unlike
in vivo studies, they do not have problems relating to the uncontrollable fluctuating locus-specific of
the oral environment [11,12]. Two complementary microbiological approaches can be taken to generate
biofilm in microbial culture models. The first is the evolution of a plaque microcosm from natural
oral microflora. A microcosm is defined as “a laboratory subset of the natural system from which it
originates and from which it also evolves” [13]. Microcosm plaques are similar in composition, growth,
acidity (pH) behavior, biochemical properties, and (probably) in complexity to natural plaque.
The second approach is the construction of defined-species biofilm consortia with major plaque
species, or a mixture of different species of the acquired oral bacteria (such as the American Type
Culture Collection (ACTT) bacteria). Consortia are simpler than plaque microcosms; they have the
advantage of incorporating individual bacterial species. Even in a simple batch culture method, oral
multispecies consortia can develop complex biofilms on enamel and dentin that can induce carious
lesions similar to those in vivo. The designs of laboratory microbial culture models vary according
to the purpose of the laboratory studies. They can be classified as closed system and open system.
Each system is a compromise between the reality of the in vivo ecosystem and the simplification of
the system. However, a well-designed model and study allow researchers to obtain meaningful and
useful results [13].

3.1. The Closed System

Microbial culture models in the closed system have a finite supply of nutrients. The growth
rates of the biofilm are rapid at the beginning of the cultivation when there are ample nutrients.
However, this is uncommon in the natural growth of biofilm [14,15]. The growth conditions will change
considerably with consumption of the nutrients and the accumulation of metabolic products. Hence,
the physiological and biological properties of the biofilm are not comparable with the natural ones.
Researchers used closed system models because of their simplicity, high productivity, repeatability,
controllability of the experimental conditions, less contamination, and cost-effective properties.
The agar plate and microtiter biofilm models are two examples of the common microbial culture
models in closed system.

3.1.1. The Agar Plate

The agar plate is one of the simplest laboratory microbial culture models (Figure 2). The nutrient
supply is not continuous. Bacteria growth on the surface of the agar can only be supported until the
finite nutrient is exhausted. Thus, results of studies using this simplistic model should be interpreted
with caution. This situation is different from bacterial growth on a hard tissue surface, because
the biofilm consumes nutrients from the substrate. It resembles biofilms associated with soft tissue
infections or growing in an extracellular matrix. This model has been used to test the susceptibility of
oral biofilm to various antimicrobials, especially some light active chemicals [16,17]. The disc-diffusion
method is not an ideal way to predict the therapeutic effects of antimicrobial [18]. The effects of the
antibacterial agents can be misinterpreted because the cationic antibacterial agents may combine with
the anionic agar polysaccharide gel [19].
Dent. J. 2017, 5, 21 4 of 12
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Figure 2. Agar plate.

Figure 2. Agar plate.
Figure 2. Agar plate.
3.1.2. The Microtiter Biofilm Model
3.1.2. The Microtiter Biofilm Model
3.1.2.The
Themicrotiter
Microtiterbiofilm
Biofilmmodel
Modelis made of a multiple-well microtiter plate. A microtiter plate is
The
commonly microtiter biofilm
made of biofilm model
polystyrene, is made
it can ofbeaamanufactured
multiple-well in microtiter
a varietyplate. A microtiter plate is
The microtiter modelbutis made of multiple-well microtiter of materials.
plate. A microtiter
A microtiter plate is
commonly
plate made
is a flat of polystyrene,
plate but it
with multiple can be manufactured
“wells” (used as smallintest
a variety of materials. A microtiter plate
commonly made of polystyrene, but it can be manufactured in atubes).
varietyA ofstandard definition
materials. of a
A microtiter
is a flat plate
microtiter with
plate wasmultiple “wells”
developed by (used
the as
Society small
for test tubes).
Laboratory A standard
Automation definition
and of
Screening a microtiter
(SLAS) anda
plate is a flat plate with multiple “wells” (used as small test tubes). A standard definition of
plate was
published developed
by the by the Society for Laboratory Automation and Screening (SLAS) and published
microtiter plate wasAmerican
developedNational Standards
by the Society Institute Automation
for Laboratory (ANSI). Henceforth, the (SLAS)
and Screening microplate
and
by the American
standards National Standards Institute (ANSI). Henceforth, theof amicroplate standardsisare known
published are by known as ANSI/SLAS
the American Nationalstandards.
StandardsA configuration
Institute (ANSI). 96-well microtiter
Henceforth, shown
the microplate in
as ANSI/SLAS
Figure 3. Each standards. A configuration
well ofasa ANSI/SLAS
microplate typically of a 96-well microtiter is shown in Figure 3. Each well of
standards are known standards.holds several milliliters
A configuration of liquid.
of a 96-well The microplate
microtiter is shown in is
aregarded
microplateatypically holds in
several milliliters of liquid. Thethe
microplate
biofilm toisof regarded as a standard tool
Figure 3. asEachstandard
well of tool cariology
a microplate research,
typically allowing
holds several milliliters grow independently
liquid. in each
The microplate is
in cariology research, allowing the biofilm to grow independently in each well.
well.
regarded as a standard tool in cariology research, allowing the biofilm to grow independently in each
well.

Figure
Figure 3.
3. Configuration
Configuration of
of the
the 96-well microtiter.
96-well microtiter.
Figure 3. Configuration of the 96-well microtiter.
3.2. The
3.2. The Open System
Open System
3.2. The Open
openSystem
The open system can can be
be described
described as as aa continuous culture system.
system. It It enables
enables the
the supply
supply ofof aa fresh
The system continuous culture fresh
culture
Themedium
open and
system the
can removal
be of
describedmetabolites
as a and
continuous spent culture
culture liquid
culture medium and the removal of metabolites and spent culture liquid simultaneously.ofHence,
system. It simultaneously.
enables the supply Hence, the
a fresh
concentration
culture
the of
medium and
concentration bacteria
of bacteria and
the removal metabolic
and of products
metabolites
metabolic remains
and spent
products remainsconstant
culture [20]. Moreover,
liquid[20].
constant the
simultaneously.
Moreover, the biofilms
Hence, can
the
biofilms
stay in a stable
concentration of state
bacteria or keep
and in a
metabolicdynamic
products balance
remains [21]. Nevertheless,
constant [20]. the repeatability
Moreover,
can stay in a stable state or keep in a dynamic balance [21]. Nevertheless, the repeatability of the the of
biofilms the
can
experimental
stay in a stable
experimental result is low
state
result is low because
or because
keep in ofof the
a the heterogeneity
dynamic balanceof
heterogeneity of[21].
the biofilm
the biofilm in the
Nevertheless,
in the open
open system. Besides,
the system. Besides,
repeatability the
of the
possibility
experimental
possibility of contamination
result is low because
of contamination can be
can be ofhigh due
thedue
high to the
heterogeneity complexity of
of the biofilm
to the complexity the construction.
in the open system. Besides, the
of the construction.
The
possibilityopen
of system
contaminationsimulatescan the
be in
highvivo
due environment
to the better
complexity
The open system simulates the in vivo environment better than the than
of the the closed system.
system. It
construction.
closed It also
also allows
allows
better regulation
The open of
system the biofilm
simulates growth
the in rate
vivo and other
environment variables.
better Common
than the
better regulation of the biofilm growth rate and other variables. Common microbial culture models microbial
closed system. culture
It also models
allowsin
in the
better open system
regulation of include
the the
biofilm chemostat
growth model,
rate and the
otherflow cell
variables.biofilm
Common model,
the open system include the chemostat model, the flow cell biofilm model, the constant depth film the constant
microbial depth
culture film
models
fermenter
in the open
fermenter model,
system
model, the
theinclude
dripdrip flow
the
flow biofilm
chemostat
biofilm reactor,
model,
reactor, theflow
the the multiple
multiple cell Sorbarod
biofilm
Sorbarod model,
model, model,
thethe
and and
constantthedepth
multiple multiple
film
artificial
artificial
fermenter
mouth mouth model.
model, the drip flow biofilm reactor, the multiple Sorbarod model, and the multiple
model.
artificial mouth model.
Dent. J. 2017, 5, 21 5 of 12
Dent. J. 2017, 5, 21 5 of 12

3.2.1.
3.2.1. Chemostat
Chemostat is preferred
preferredfor forbiofilm
biofilmexperiments
experimentsbecause
because thethe continuous
continuous culture
culture of chemostat
of chemostat can
can provide homogeneity and a steady environment (Figure 4). The
provide homogeneity and a steady environment (Figure 4). The experimental parametersexperimental parameters can be
investigated
investigated independently
independently in the
in highly-controlled conditions
the highly-controlled [22]. Oral bacteria
conditions [22]. Oralgrowbacteria
planktonically
grow
in a conventional
planktonically in chemostat. A fresh
a conventional cultural medium
chemostat. is provided
A fresh cultural mediumat theissame rate asatthe
provided theculture waste
same rate as
liquid removal rate. Planktonic bacteria have the tendency to form biofilm at a solid-liquid
the culture waste liquid removal rate. Planktonic bacteria have the tendency to form biofilm at a solid- interface in
aliquid
chemostat. A substrate
interface such asAa substrate
in a chemostat. tooth slicesuch
can be
as suspended
a tooth sliceincanthebechemostat
suspended to provide a surface
in the chemostat
for bacteriala colonization
to provide and biofilm
surface for bacterial or dentaland
colonization plaque formation.
biofilm or dental Chemostat is generally
plaque formation. expensive
Chemostat is
and space-consuming in laboratory. Precaution is needed to prevent excessive
generally expensive and space-consuming in laboratory. Precaution is needed to prevent excessive bacteria growth in
chemostat, whichincan
bacteria growth block thewhich
chemostat, tubingcan[23].
block the tubing [23].

Figure 4. Schematic diagram of chemostat.

Figure 4. Schematic diagram of chemostat.

3.2.2. The Flow Cell Biofilm Model

3.2.2. The Flow Cell Biofilm Model
The flow cell biofilm model is used as perfusion chambers to observe the initial growth and
The flow cell biofilm model is used as perfusion chambers to observe the initial growth and
physiology of stationary bacterial cells [24]. The culture fluid passes through a tube and biofilms are
physiology of stationary bacterial cells [24]. The culture fluid passes through a tube and biofilms are
cultured in a flow reactor where the substratum is placed. Biofilms can grow on the surface of tooth
cultured in a flow reactor where the substratum is placed. Biofilms can grow on the surface of tooth
blocks [25], microscopy glass slides, or glass rods [26]. The flow cell biofilm model is shown in Figure
blocks [25], microscopy glass slides, or glass rods [26]. The flow cell biofilm model is shown in Figure 5.
5. Bacteria suspension stored in a chemostat (A) and bacteria-free medium (B) are stirred or pumped
Bacteria suspension stored in a chemostat (A) and bacteria-free medium (B) are stirred or pumped (D)
(D) to a mixed chamber (C) and go through the flow reactor (E) to create a flow. Therefore, the shear
to a mixed chamber (C) and go through the flow reactor (E) to create a flow. Therefore, the shear force
force will work on the microbe when the culture fluid passes through the surface of the biofilm. The
will work on the microbe when the culture fluid passes through the surface of the biofilm. The outside
outside chemostat in the flow cell biofilm model allows external biofilm growth, which means the
chemostat in the flow cell biofilm model allows external biofilm growth, which means the growth
growth condition can be controlled and the biofilm can grow for an extended period. Other
condition can be controlled and the biofilm can grow for an extended period. Other advantages are
advantages are flexibility of sample configuration, presence of fluid dynamics, plaque monitoring.
flexibility of sample configuration, presence of fluid dynamics, plaque monitoring. and the possibility
and the possibility of extra experimental treatments.
of extra experimental treatments.
The flow cell biofilm model simulates the in situ situation of undisturbed biofilm communities.
The flow cell biofilm model simulates the in situ situation of undisturbed biofilm communities.
The constant environment is provided with laminar flow [24]. The model has been adopted
The constant environment is provided with laminar flow [24]. The model has been adopted frequently
frequently in the evaluation of the effects of antimicrobial agents because it is convenient to make
in the evaluation of the effects of antimicrobial agents because it is convenient to make comparisons
comparisons of viability of microbes among different experimental groups [27]. In addition, the
of viability of microbes among different experimental groups [27]. In addition, the continuous flow
continuous flow system simulates the clearance of antimicrobial agents in the mouth. A limitation of
system simulates the clearance of antimicrobial agents in the mouth. A limitation of this device is that
this device is that the laminar fluid flows through the biofilm instead of across its surface. It mimics
the laminar fluid flows through the biofilm instead of across its surface. It mimics the flow of saliva
the flow of saliva on the surface of mucosal, but the pathways of saliva flowing on hard-surface
on the surface of mucosal, but the pathways of saliva flowing on hard-surface biofilms are different.
biofilms are different. Flow cell biofilm models are also expensive and space consuming.
Flow cell biofilm models are also expensive and space consuming.
 Dent. J. 2017, 5, 21 6 of 12
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Configuration
Figure5.5.Configuration
Figure of the
of the flowflow
cell cell biofilm
biofilm model
model (adapted
(adapted from from
HerlesHerles
et al., et al.,[28],
1994 1994with
[28],
with permission from © 1994 International & American Associations for Dental Research.
permission from © 1994 International & American Associations for Dental Research. License number: License
number: 4131180504654).
4131180504654).

3.2.3.The
3.2.3. TheConstant
ConstantDepth
DepthFilm
FilmFermenter
FermenterModel
Model
Themajor
The majorcomponents
componentsofofthe theconstant
constantdepth
depthfilmfilmfermenter
fermenter(CDFF)
(CDFF)model
modelareareplugs,
plugs,aarotating
rotating
stainless steel disk, and static scarper blades [23]. The plugs allow the growth of biofilm.
stainless steel disk, and static scarper blades [23]. The plugs allow the growth of biofilm. The rotating The rotating
stainlesssteel
stainless steeldisk
disk holds
holds thethe samples.
samples. The The
staticstatic
scarperscarper
blades blades
control control the of
the depth depth of the biofilm.
the biofilm. These
These components are put into a glass container where a fresh cultural medium
components are put into a glass container where a fresh cultural medium is provided and culture is provided and culture
wasteliquid
waste liquidisisremoved.
removed.The Theconfiguration
configurationof ofCDFF
CDFFisisshown
shownin inFigure
Figure6.6.
Thethickness
The thicknessofofbiofilms
biofilmsisiscontrolled
controlledtotoaapredetermined
predetermineddepth depthby bymechanically
mechanicallyremoving
removingthe the
excess biofilm. This simulates the tongue movement over the teeth. The thickness
excess biofilm. This simulates the tongue movement over the teeth. The thickness of biofilms can be of biofilms can be
200µm
200 [29,30]to
µm[29,30] tomimic
mimicdental
dentalplaques.
plaques.TheTheproperties
propertiesofofbiofilms
biofilmsthat
thatare
aredeveloped
developedare arerelatively
relatively
constant over time. The CDFF model supports restrained growth and produces
constant over time. The CDFF model supports restrained growth and produces a number of replicate a number of replicate
biofilms. Since the thickness of the biofilms is predetermined, subsampling and
biofilms. Since the thickness of the biofilms is predetermined, subsampling and effluent analysis are effluent analysis are
limited to
limited to some
someextentextent[31]. TheThe
[31]. model was used
model was to study
used toetiology of caries [32],
study etiology to assess
of caries antimicrobial
[32], to assess
effect on biofilm [33], and to investigate the structure of biofilm [34].
antimicrobial effect on biofilm [33], and to investigate the structure of biofilm [34].
Dent. J.J. 2017,
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Dent. J. 2017, 5, 21 7 of 12

Figure 6. Configuration of the constant depth film fermenter (adapted from Pratten et al., 2007 [35],
Figurewith
Figure Configuration
6. Configuration
6. of©the
of
permission from the constant
constant
2007 depthLibrary.
depth
Wiley Online film fermenter
film fermenter (adapted
(adapted
License number: from Pratten
from Pratten et
4131200175687). et al.,
al., 2007
2007 [35],
[35],
with permission
with permission from
from ©© 2007
2007 Wiley
Wiley Online
Online Library.
Library. License
License number:
number: 4131200175687).
4131200175687).
3.2.4. The Drip Flow Biofilm Reactor
3.2.4. The
The Drip
Drip Flow
Flow
The drip Biofilm
flow biofilmReactor
model is often used to grow and establish solid-liquid or solid-air interface
biofilms.
The The biofilm
drip flow
flow model usually
biofilm model iscontains fourto
often used
often used tochambers
grow and
grow andinestablish
an adjustable
establish inclinedor
solid-liquid
solid-liquid orfermenter. The
solid-air interface
solid-air interface
schematic diagram of drip-flow biofilm model is shown in Figure 7.
biofilms. The
Themodel
modelusually contains
usually containsfourfour
chambers in an adjustable
chambers inclined inclined
in an adjustable fermenter.fermenter.
The schematic
The
diagram ofdiagram
schematic drip-flowof biofilm
drip-flowmodel is shown
biofilm modelinisFigure
shown7.in Figure 7.

Figure 7. Schematic diagram of a drip flow biofilm reactor (adapted from McBain et al., 2009 [15],
with permission from © 2009 Elsevier. License number: 4130791265178).

The biofilms grow on angled tooth surfaces, which are continuously irrigated with small
Figure 7. Schematic
Schematic diagram
diagramofofa adrip
dripflow
flow biofilm
biofilm reactor
reactor (adapted
(adapted fromfrom McBain
McBain et2009
et al., al., 2009
[15], [15],
with
volumes of fresh medium from the inlet. The incline of the fermenter enables the medium to flow
with permission
permission from from
© 2009 © Elsevier.
2009 Elsevier.
LicenseLicense
number:number: 4130791265178).
4130791265178).
over the tooth surface with biofilm, providing a low-shear environment for the biofilm.
The model allows plaque to grow on the tooth surface and to stabilize for longer periods, which
The
The biofilmsgrow
biofilms growonon angled
angled toothtooth surfaces,
surfaces, which which are continuously
are continuously irrigated irrigated
with with
small small
volumes
enables relatively stable development of microbial communities [36]. However, as the medium flow
volumes
of fresh of fresh
onmedium
the surface medium
from
of the fromThe
thesubstrata
inlet. the inlet.
notThe
incline
might of
betheincline of the
fermenter
always fermenter
enables
consistent, aerialthe enables to
medium
heterogeneity the medium
flow
over over
the to tooth
the
surface flow
over the
surface tooth
of with surface
substratum
biofilm,may with biofilm,
exist [15].
providing providing
This model
a low-shear a low-shear environment
is commercially
environment for theavailable for the biofilm.
biofilm.(Biosurfaces Technologies
The model allows plaque to grow on the tooth surface
The model allows plaque to grow on the tooth surface and to researchers.
Corporation, Bozeman, MT, USA), and thus is commonly and
used to
by stabilize
stabilize for longer
forThis model
longer periods,
was used
periods, which
which
enablesto relatively
test stable
disinfection development
efficacy [37], to of microbial
investigate thecommunities
effect of [36].
powered
enables relatively stable development of microbial communities [36]. However, as the medium However,
tooth brushing as the
on medium
removal offlow
on thebiofilm
surface [38],
of and to comparemight
the substrata the antibacterial
not be alwayseffectsconsistent,
of anti-caries agents
aerial [36].
heterogeneity
flow on the surface of the substrata might not be always consistent, aerial heterogeneity over the over the surface
of substratum
surface may exist
of substratum may [15]. This This
exist [15]. model is commercially
model is commercially available
available(Biosurfaces
(Biosurfaces Technologies
Technologies
Corporation,
Corporation, Bozeman, MT, USA), and thus is commonly used by researchers. This
Bozeman, MT, USA), and thus is commonly used by researchers. This model
model was
was used
used
to
to test disinfection efficacy [37], to investigate the effect of powered tooth brushing on removal of
test disinfection efficacy [37], to investigate the effect of powered tooth brushing on removal of
biofilm [38], and to compare the antibacterial effects of anti-caries agents
biofilm [38], and to compare the antibacterial effects of anti-caries agents [36]. [36].
Dent. J. 2017, 5, 21 8 of 12
Dent. J. 2017, 5, 21 8 of 12

3.2.5. The Multiple Sorbarod Model

3.2.5. The Multiple Sorbarod Model
The multiple Sorbarod model uses a permeable Sorbarod membrane as the substratum. The
The multiple Sorbarod model uses a permeable Sorbarod membrane as the substratum. The fresh
fresh medium is supplied by continuous perfusion through the membrane. The exfoliated bacterial
medium is supplied by continuous perfusion through the membrane. The exfoliated bacterial cells and
cells and metabolic wastes will be removed with spent culture medium. The schematic diagram of
metabolic wastes will be removed with spent culture medium. The schematic diagram of the multiple
the multiple Sorbarod model is shown in Figure 8.
Sorbarod model is shown in Figure 8.
In this model, the flow rate of the medium can be controlled. Therefore, the growth rate of the
In this model, the flow rate of the medium can be controlled. Therefore, the growth rate of the
biofilm is controllable [15]. The multiple Sorbarod model was used to investigate the effect of oral
biofilm is controllable [15]. The multiple Sorbarod model was used to investigate the effect of oral
hygiene activities on anaerobic oral biofilms [39] and to assess the plaque-control effects of some
hygiene activities on anaerobic oral biofilms [39] and to assess the plaque-control effects of some specific
specific enzymes [40]. An advantage of this model is that the growth rate of the biofilm can be
enzymes [40]. An advantage of this model is that the growth rate of the biofilm can be controlled.
controlled. Another advantage is that the detached bacterial cells in the spent culture medium can be
Another advantage is that the detached bacterial cells in the spent culture medium can be studied to
studied to evaluate the biological effect of experimental treatment [36]. Since the model develops
evaluate the biological effect of experimental treatment [36]. Since the model develops heterogeneous
heterogeneous biofilm, it cannot be used in study design where homogeneity of the biofilm is
biofilm, it cannot be used in study design where homogeneity of the biofilm is important [15].
important [15].

Figure 8. A schematic diagram of a multiple Sorbarod device (adapted from McBain et al., 2005 [41],
Figure 8. A schematic diagram of a multiple Sorbarod device (adapted from McBain et al., 2005 [41],
with permission from © 2005 Wiley Online Library. License number: 4130790067572).
with permission from © 2005 Wiley Online Library. License number: 4130790067572).

3.2.6. The Multiple Artificial Mouth

3.2.6. The Multiple Artificial Mouth
The multiple artificial mouth (MAM) is a computer-controlled, multiple-station model. It has a
The multiple artificial mouth (MAM) is a computer-controlled, multiple-station model. It has
more complicated construction than the models discussed above.
a more complicated construction than the models discussed above.
A MAM can accurately simulate an in vivo environment using computer-controlled facilities
A MAM can accurately simulate an in vivo environment using computer-controlled facilities [42].
[42]. It has several microstations, which are relatively independent to one other (Figure 9). Different
It has several microstations, which are relatively independent to one other (Figure 9). Different
experimental conditions can be applied simultaneously in different microstations.
experimental conditions can be applied simultaneously in different microstations.
Environmental variables can be easily controlled in the MAM. This allows analysis of the biofilm
during its development, without contaminating other samples. Acidity can be monitored using a
Dent. J. 2017, 5, 21 9 of 12

pH electrode and a micro-reference electrode [12]. These well-controlled conditions improve the
standardization and flexibility of the MAM, and therefore enhance its ability to culture biofilms similar
to natural oral flora. Sissons et al. found that biofilms developed in this system exhibited metabolic
and pH behavior that resembled typical natural plaques [42]. The MAM has been adopted in different
studies, such as biodiversity of plaques [43], fluoride and phosphate assay [44], plaque calcium level
measurement [45], and the generation of consortia using major plaque species [46]. The biofilm
samples in this model were exposed to the same temperature and gas-phase fluctuation. The MAM
aims to mimic the oral environment. Therefore, saliva substitutes play an important role in the model.
Approximate laminar flows are applied to simulate the situations in the oral cavity, instead of turbulent
flow in chemostat.

Figure 9. Schematic diagram of a multiple artificial mouth (adapted from Sissons et al., 2000 [47], with
permission from © 2000 Springer. License number: 4130800878870).

4. Summary
Dental biofilm is an essential factor in the etiology of dental caries. Cariogenic bacteria streptococci,
actinomycetes, and lactobacilli are found to be more closely associated with dental caries. Laboratory
microbial culture models can provide a steady and controllable environment for cariology research.
Dent. J. 2017, 5, 21 10 of 12

The models play an important role in cariology research in investigating caries pathogenicity, testing
effects of new caries prevention methods, and developing new caries-preventing products. Each model
has its advantages and disadvantages from both experimental design and experiment cost. Table 1
shows a comparison of the discussed in vitro biofilm systems.

Table 1. Characteristics of common microbial culture models for cariology research.

Agar Drip
Parameter Microtiter Chemostat Flow Cell CDFF MSD MAM
Plate Flow
Hours to Hours to Hours to Hours to Days to Days to Days to Days to
Duration
days days days days weeks weeks weeks weeks
Planktonic
Controlled Controlled Controlled Controlled None None None None
phase
Growth
Via plank-
control by None Yes Yes Yes Yes Yes Yes
tonic phase
media
Fluid flow No No Turbulent Laminar Laminar Drop Laminar Drop
Shear force No No Yes Yes Yes No Yes No
Defined
No No Achievable Achievable Yes No No No
thickness
Timed Computer
No Manually Yes Pulse Yes Yes Yes
reagents control
Alternative
No Yes Yes Yes Yes Yes No Yes
substrate
Different
No No No No No Yes No Yes
conditions
Subsampling
Yes Yes Yes Yes Yes Yes Yes Yes
during growth
CDFF = Constant depth film fermenter; MSD = Multiple Sorbarod device; MAM = Multiple artificial mouth.

The designs of the biofilm models that are included vary from simple to sophisticated according
to the purposes of investigation. Agar plate and microtiter are microbial culture models in the closed
system that are low-cost and simple to manage. Microbial culture models in the open system are more
complex and the biofilms generated are closer to natural dental plaque. Selection of the type of model
used for a biofilm study depends on the growth conditions, requirements for the specific biofilm, and
purposes of the study.

Acknowledgments: This review is supported by HKU Seed Funding for Basic Research 201511159142.
Author Contributions: Ollie Yiru Yu did the literature search and prepared the first draft of this manuscript.
All authors contributed equally in order to finish this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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