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Review Dental Biofilm and 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; [email protected] (O.Y.Y.); [email protected] (I.S.Z.); [email protected] (M.L.M.); [email protected] (E.C.-M.L.) * Correspondence: [email protected]; 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 , fermentable , and host factors (including teeth and ). 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 , 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 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 . 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 surfaces in the mouth. It has a large diversity of species and consists of densely packed bacteria embedded in a matrix of organic of bacterial and salivary origin. Dental plaque is the causal agent of dental caries in the presence of 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, mutans is a major bacterium producing the extracellular 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 cellsDent. growing J. 2017, 5, 21 in a biofilm are physiologically distinct from planktonic cells which float or swim2 in of 12a liquid medium. Bacteria in the plaque biofilm can respond to many factors, such as cellular recognitionrecognition of specificspecific oror non-specificnon-specific attachment attachment sites sites on on a surfacea surface and and nutritional nutritional signals. signals. Marsh Marsh and andMartin Martin [1] divided[1] divided the formationthe formation and and growth growth of oral of oral biofilm biofilm into into five stagesfive stages (Figure (Figure1). 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).

OralOral biofilms can can form form on on almost almost any any surface surface pres presentent in in the the oral oral cavity cavity including including enamel, enamel, , dentin, ,cementum, gingiva,gingiva, oraloral mucosa, mucosa, carious carious lesion, lesion, restoration, restoration, dental , implant, and denture. and denture. Dental Dental plaque plaquewill colonize will colonize rapidly, rapidly, not only not the coronalonly the enamel coronal surface enamel but surface also the but exposed also the root exposed surface. root The surface. growth Theof growth of on microbiota the exposed on rootthe exposed surface proceeds root surf moreace proceeds rapidly thanmore that rapidly on the than smooth that on enamel the smooth surface enamelbecause surface of the irregularbecause surfaceof the irregular topography surface of the topography exposed root of the dentin exposed surface. root The dentin organization surface. The and organizationstructure of dental and structure plaque vary of denta considerablyl plaque according vary considerably to the sites acco whererding plaque to the forms sites [ 1where]. The plaque growth formsof [1]. The growth on of specific microorganisms oral niches on is specific affected oral by niches various is factorsaffected such by various as acidity factors (pH) such of theas acidityenvironment, (pH) of availability the environment, of nutrients, availability presence of nutr of antimicrobialients, presence agents, of and host defense. agents, and host defense.Surface-bound microorganisms have a survival and/or selective advantage over their planktonic phasesSurface-bound [1]. Bacteria microorganisms in dental plaque have have a stronger survival resistance and/or selective to antimicrobial advantage agents over their than planktonic planktonic phasesbacteria. [1]. BacterialBacteria in extracellular dental plaque have stronger resistance prevent the to perfusionantimicrobial of antimicrobialagents than planktonic agents to bacteria.bacterial Bacterial targets; thisextracellular acts as a barrierpolysaccharides to protect prevent the plaque the bacteria perfusion against of antimicrobial certain environmental agents to bacterialthreats such targets; as , this acts as , a barrier surfactant,to protect ,the plaque bacteria and whiteagainst blood certain cells environmental [3]. Resistance threatsof biofilm such bacteria as antibiotics, to antimicrobial antibodies, agents surfactant, may alsobacteriophage, develop. As and a white result, blood the minimum cells [3]. Resistance inhibitory ofconcentration biofilm bacteria of antimicrobial to antimicrobial agents agents against may bacteria also indevelop. biofilm As is significantly a result, the higher minimum (up to inhibitory 1000-fold) concentrationthan that in liquid of antimicrobial [1]. agents against bacteria in biofilm is significantly higher (up to 1000- fold) Thoughthan that there in liquid are many [1]. bacteria associated with dental caries, a few groups of cariogenic bacteria suchThough as streptococci there are, actinomycetes, many bacteriaand associatedlactobacilli withare dent foundal caries, to be a morefew groups closely of associated cariogenic thanbacteria the suchothers. as Thesestreptococci groups, actinomycetes, of bacteria often and dominantlylactobacilli are proliferate found to in be the more dental closely biofilm associated collected than from the the others.carious These lesions groups of teeth. of bacteriaStreptococcus often dominantlyis the predominant proliferate species in the in dental cariogenic biofilm microbe. collected It colonizesfrom the cariousclean tooth lesions surfaces of teeth. at Streptococcus an early stage, is the and predominant it also relates species to root in caries.cariogenic The microbe. predominant It colonizes coccal cleanisolated tooth from surfaces carious at an dentin early instage, root and caries it also are relatesS. mutans to root, S. sanguiscaries. The, and predominantS. mitis [4]. coccalS. mutans isolatedand fromS. sobrinus cariousare dentin difficult in root todistinguish. caries are S. Hence,mutans, theseS. sanguis, two speciesand S. mitis are always[4]. S. mutans lumped and together S. sobrinus and areregarded difficult as tomutans distinguish. streptococci Hence,. Mutans these streptococcitwo speciescan are adapt always to lumped acidic environments, together and whichregarded is theas mutanskey factor streptococci contributing. Mutans to its streptococci cariogenic can potential. adapt Actinomycetesto acidic environments,is an initial which colonizer is the of human key factor root contributingsurfaces. A. naeslundiito its cariogenicand A. potential. viscosus can Actinomycetes induce root is surface an initial caries colonizer [5]. Actinomycetes of human rootis often surfaces. isolated A. naeslundiifrom subgingival and A. microfloraviscosus can and induce from root plaque surface associated caries with [5]. rootActinomycetes caries [6] (theyis often have isolated long surface from subgingivalappendages microflora named fibrils, and or from fimbriae). plaque The associated fibrils allow withactinomycetes root cariesto [6] adhere (they to have the surface long ofsurface tooth appendagesroots. Fibrils named also improve fibrils, the or attachmentfimbriae). The of actinomycetes fibrils allowto actinomycetes other bacteria to in adhere dental plaque.to the surfaceLactobacilli of toothare aciduric roots. Fibrils bacteria, also including improveL. the acidophilus attachment, L. rhamnosus of actinomycetes, L. casei to, and otherL. orisbacteria[7]. Patients in dental with plaque. caries Lactobacillihave higher are counts aciduric of lactobacillibacteria, includingthan those L. acidophilus with no caries., L. rhamnosus Evaluating, L. casei the amount, and L. oris of Lactobacilli [7]. Patientsin

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 plate and microtiter biofilm models are two examples of the common microbial culture models in closed system.

3.1.1. The The agar plate is one of the simplest laboratory microbial culture models (Figure2). The 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 or growing in an . This model has been used to the susceptibility of oral biofilm to various , 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 Dent.Dent. J. 20172017,, 55,, 2121 44 ofof 1212

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 The microtiter Microtiter biofilm Biofilm model Model is made of a multiple-well microtiter plate. A microtiter plate is The microtiter biofilm model is made of a multiple-well microtiter plate. A microtiter plate is commonlyThe microtiter made of biofilmpolystyrene, model but is madeit can ofbe amanufa multiple-wellctured in microtiter a variety plate. of materials. A microtiter A microtiter plate is commonly made of polystyrene, but it can be manufactured in a variety of materials. A microtiter plate platecommonly is a flat made plate of polystyrene,with multiple but “wells” it can be(used manufa as smallctured test in atubes). variety A ofstandard materials. definition A microtiter of a is a flat plate with multiple “wells” (used as small test tubes). A standard definition of a microtiter microtiterplate is a plateflat plate was developedwith multiple by the “wells” Society (used for Laboratory as small test Automation tubes). A and standard Screening definition (SLAS) ofand a plate was developed by the Society for Laboratory Automation and Screening (SLAS) and published publishedmicrotiter plateby the was American developed National by the Society Standards for Laboratory Institute Automation(ANSI). Henceforth, and Screening the (SLAS) and by the American National Standards Institute (ANSI). Henceforth, the microplate standards are known standardspublished areby knownthe American as ANSI/SLAS National standards. Standards A conf Instituteiguration (ANSI). of a 96-well Henceforth, microtiter the ismicroplate shown in as ANSI/SLAS standards. A configuration of a 96-well microtiter is shown in Figure3. Each well of Figurestandards 3. Each are known well of as a ANSI/SLASmicroplate typicallystandards. hold A confs severaliguration milliliters of a 96-well of liquid. microtiter The microplate is shown inis a microplate typically holds several milliliters of liquid. The microplate is regarded as a standard tool regardedFigure 3. asEach a standard well of toola microplate in cariology typically research, hold allowings several the milliliters biofilm to ofgrow liquid. independently The microplate in each 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 3. ConfigurationConfiguration of the 96-well microtiter. Figure 3. Configuration of the 96-well microtiter. 3.2. The Open System 3.2. The Open System The open system can be described as a continuous culture system. It enables the supply of a fresh cultureThe medium open system and and the thecan removal removalbe described of of metabolites metabolites as a continuous and and spent culture spent culture culture system. liquid liquid It enables simultaneously. simultaneously. the supply Hence, of Hence,a fresh the theconcentrationculture concentration medium of andbacteria of bacteriathe removaland andmetabolic of metabolic metabolites products products and remains spent remains constantculture constant liquid [20]. [ 20Moreover,simultaneously.]. Moreover, the biofilms the Hence, biofilms canthe canstayconcentration stayin a in stable a stable of bacteriastate state or orandkeep keep metabolic in ina adynamic dynamic products balance balance remains [21]. [21 constant]. Nevertheless, Nevertheless, [20]. Moreover, the the repeatability repeatability the biofilms of can thethe experimentalstay in a stable result state isis low or becausekeep in ofa thedynamic heterogeneityheterogene balanceity of of[21]. the biofilmbiofilmNevertheless, inin the open the system.repeatability Besides, of thethe possibilityexperimental of contaminationresult is low because cancan bebe of highhigh the duedueheterogene toto thethe complexitycomplexityity of the biofilm ofof thethe construction.inconstruction. the open system. Besides, the possibilityThe open of contamination system simulates can thebe highin vivo due environmentenvironmen to the complexityt better ofthan the the construction. closed system. It also allows betterThe regulationregulation open system ofof thethe simulates biofilmbiofilm growth growth the in ratevivo rate and environmenand other other variables. variables.t better Commonthan Common the closed microbial microbial system. culture culture It also models models allows in theinbetter the open openregulation system system includeof include the biofilm the the chemostat growth rate model, model, and the otherthe flow flow variables. cell biofilm biofilm Common model, model, microbial thethe constantconstant culture depthdepth models filmfilm fermenterin the open model,model, system the theinclude drip drip flow theflow biofilmchemostat biofilm reactor, model,reactor, the the multiplethe flow multiple cell Sorbarod biofilm Sorbarod model, model, model, and the the constant and multiple the depth multiple artificial film mouthartificialfermenter model. mouth model, model. the drip flow biofilm reactor, the multiple Sorbarod model, and the multiple artificial mouth model.

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3.2.1. Chemostat Chemostat is preferred preferred for for biofil biofilmm experiments experiments because because the the continuous continuous culture culture of chemostat of chemostat can canprovide provide homogeneity homogeneity and and a steady a steady environment environment (F (Figureigure 4).4). The The experimental experimental parameters cancan bebe investigatedinvestigated independentlyindependently in thein highly-controlledthe highly-controlled conditions conditions [22]. Oral bacteria[22]. Oral grow bacteria planktonically grow inplanktonically a conventional in chemostat.a conventional A fresh chemostat. cultural mediumA fresh cultural is provided medium at the is same provided rate as at the the culture same rate waste as liquidthe culture removal waste rate. liquid Planktonic removal bacteria rate. Planktonic have the tendencybacteria have to form the biofilmtendency at to a solid-liquid form biofilm interface at a solid- in aliquid chemostat. interface Asubstrate in a chemostat. such as A a substrate tooth slice such can beas suspendeda tooth slice in can the be chemostat suspended to providein the chemostat a surface forto provide bacterial a colonizationsurface for bacterial and biofilm colonization or dental and plaque biofilm formation. or dental Chemostat plaque formation. is generally Chemostat expensive is andgenerally space-consuming expensive and in space-consuming laboratory. Precaution in labora is neededtory. Precaution to prevent is excessiveneeded to bacteria prevent growth excessive in chemostat,bacteria growth which in can chemostat, block the which tubing can [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 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], 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 Figure5. 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.

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FigureFigure 5. 5. ConfigurationConfiguration of the of theflow flow cell cellbiofilm biofilm model model (adapted (adapted from fromHerles Herles et al., et1994 al., [28], 1994 with [28 ], permissionwith permission from © from 1994 ©International1994 International & American & American Associations Associations for Dental for Research. Dental Research. License number: License number: 4131180504654). 4131180504654).

3.2.3.3.2.3. The The Constant Constant Depth Depth Film Film Fermenter Fermenter Model Model TheThe major major components components of of the the constant constant depth depth film film fermenter fermenter (CDFF) (CDFF) model model are are plugs, plugs, a arotating rotating stainlessstainless steel steel disk, disk, and and st staticatic scarper scarper blades blades [23]. [23]. The The plugs plugs allow allow the the growth growth of of biofilm. biofilm. The The rotating rotating stainlessstainless steel steel disk disk holds holds the the samples. samples. The Thestatic static scarper scarper blades blades control control the depth the of depth the biofilm. of the biofilm.These componentsThese components are put are into put a intoglass a glasscontainer container where where a fresh a fresh cultural cultural medium medium is isprovided provided and and culture culture wastewaste liquid liquid is is removed. removed. The The configuration configuration of of CDFF CDFF is is shown shown in in Figure Figure 6.6. TheThe thickness thickness of of biofilms biofilms is is controlled controlled to to a apr predeterminededetermined depth depth by by mechanically mechanically removing removing the the excessexcess biofilm. biofilm. This This simulates simulates the the tongue tongue movement movement over over the the teeth. teeth. The The thickness thickness of of biofilms biofilms can can be be 200200 µmµm [29,30] [29,30 ]to to mimic mimic dental dental plaques. plaques. The The proper propertiesties of of biofilms biofilms that that are are developed developed are are relatively relatively constantconstant over over time. time. The The CDFF CDFF model model supports supports restra restrainedined growth growth and and produces produces a anumber number of of replicate replicate biofilms.biofilms. Since Since the the thickness thickness of of th thee biofilms biofilms is is predetermined, predetermined, subsam subsamplingpling and and effluent effluent analysis analysis are are limitedlimited to somesome extent extent [31 [31].]. The The model model was usedwas toused study to etiologystudy etiology of caries [of32 ],caries to assess [32], antimicrobial to assess antimicrobialeffect on biofilm effect [33 on], andbiofilm to investigate [33], and to the investigate structure the of biofilm structure [34 of]. biofilm [34].

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Figure 6. Configuration of the constant depth film fermenter (adapted from Pratten et al., 2007 [35], Figurewith 6. ConfigurationConfiguration permission from of of © the the 2007 constant constant Wiley On depth depthline Library. film film ferm fermenter Licenseenter number: (adapted (adapted 4131200175687). from from Pratten Pratten et et al., al., 2007 2007 [35], [35], with permission from © 2007 Wiley OnlineOnline Library. LicenseLicense number:number: 4131200175687).4131200175687). 3.2.4. The Drip Flow Biofilm Reactor 3.2.4. The The Drip DripThe drip Flow Flow flow Biofilm Biofilm biofilm Reactor model is often used to grow and establish solid-liquid or solid-air interface Thebiofilms. drip flow flowThe biofilm biofilmmodel usuallymodel is contains often often used used four to tochambers grow grow and and in establish establishan adjustable solid-liquid solid-liquid inclined or or fermenter. solid-air solid-air interface interfaceThe biofilms.biofilms.schematic TheThe model modeldiagram usually usually of drip-flow contains contains biofilm four four chambersmodel chambers is shown in an in adjustablein Figure an adjustable 7. inclined inclined fermenter. fermenter. The schematic The schematicdiagram of diagram drip-flow of biofilmdrip-flow model biofilm is shown model in is Figureshown7 .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 diagramdiagramof of a a drip drip flow flow biofilm biofilm reactor reactor (adapted (adapted from from McBain McBain et al., et 2009al., 2009 [15], [15], with volumes of fresh medium from the inlet. The incline of the fermenter enables the medium to flow withpermission permission from from © 2009 © Elsevier.2009 Elsevier. License License 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 biofilms grow on angled tooth surfaces, which are continuously irrigated with small Theenables biofilms relatively grow stable on angled development tooth surfaces, of microbia whichl communities are continuously [36]. However, irrigated as the with medium small volumesflow volumesof freshon medium theof freshsurface frommedium of the the substrata inlet. from The the might incline inlet. not The ofbe the always inc fermenterline consistent, of the enables fermenter aerial the heterogeneity medium enables tothe over flow medium the over surface the to toothflow oversurface theof with toothsubstratum biofilm, surface may providing with exist biofilm, [15]. a low-shear Thisprovidin model environmentg ais low-shear commercially for environment the available biofilm. for(Biosurfaces the biofilm. Technologies TheCorporation, model allows Bozeman, plaque MT, to USA), grow and on thusthe tooth is commonly su surfacerface used and byto researchers.stabilize for This longer longer model periods, periods, was used which enablesto relativelytest relatively disinfection stable stable efficacy development development [37], to investigateof ofmicrobia microbial thl ecommunities effect communities of powered [36]. [ 36tooth However,]. However,brushing as theon as removalmedium the medium of flow biofilm [38], and to compare the antibacterial effects of anti-caries agents [36]. onflow the on surface the surface of the ofsubstrata the substrata might mightnot be notalways bealways consistent, consistent, aerial heterogeneity aerial heterogeneity over the over surface the ofsurface substratum of substratum may exist may [15]. exist [This15]. Thismodel model is commercially is commercially available available (Biosurfaces (Biosurfaces Technologies Technologies Corporation, Bozeman, MT, USA), and thus is commonly used by researchers. This model was used Corporation, Bozeman, MT, USA), and thus is commonly used by researchers. This model was used toto test disinfection efficacy efficacy [37], [37], to investigate th thee effect of powered on removal of biofilmbiofilm [38], [38], and to compare the antibacterialantibacterial effects of anti-caries agentsagents [[36].36].

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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 Figure8. 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 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 [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 (Figure9). 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. Table1 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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