Formation

of

Dental Plaque

Dr. Sandip Ladani

Post-Graduate

Department of Periodontics & Oral Implantology

Ahmedabad Dental College & Hospital

Guided by,

Dr. Mihir N. Shah Dr. Archita Kikani

Dr. Hiral Parikh Dr. Tejal Sheth

Introduction

Throughout life, all interface surfaces in the human body are exposed to colonisation by a

wide range of microorganisms. In general, microbiota live in harmony with the host. Constant

renewal of the epithelial surfaces by shedding prevents the accumulation of large masses of

microorganisms. In the oral cavity, teeth or prosthetic devices provide non-shedding surfaces

which allow extensive bacterial deposits to form. Such an accumulation of microbes on a hard

surface is commonly called “dental plaque” because of its yellowish colour reminiscent of the

mucosal plaques caused by syphilis. The accumulation and/or metabolic products of bacteria

have been associated with dental caries, gingivitis, periodontitis, peri-implant infections and

stomatitis.

From an ecological view point, the oral cavity and the oro-pharynx as a whole should be

considered as an “open growth system”. There is an uninterrupted ingestion and removal of

microorganisms and their nutrients. A dynamic equilibrium exists between the adhesion forces of

microorganisms and a variety of removal forces such as swallowing, friction by food intake, the

tongue and oral hygiene implements as well as the wash-out effect of the salivary and crevicular

fluid outflow. The latter is an inflammatory exudate that streams out of the periodontal. Most

organisms can only survive in the mouth when they adhere to non-shedding surfaces.

The formation of bacterial plaque is initiated by the adhesion of micro-organisms to the

tooth surface, and is the first step in the development of periodontal infections (Newman et al.

1978). Until now, no uniform theory has been developed to explain the fundamental mechanisms

of cell adhesion. Moreover, it would be impossible and erroneous to conclude that one single

mechanism dictates the adhesive tendency of microorganisms because the situation is too

complex (Quirynen and Bollen 1995).

The process of plaque formation can be divided into several phases:

1. Adsorption of Host and Bacterial Molecules to the tooth surface (Formation of pellicle)

2. Initial adhesion and attachment of bacteria

a) Transport to the surface

b) Initial / Reversible adhesion

c) Irreversible adhesion / Attachment

d) Colonization

e) Multiplication of the attached micro-organisms

3. Active detachment

1. Formation of pellicle

This conditioning film (the acquired pellicle) forms immediately following eruption or

cleaning and directly influences the pattern of initial microbial colonization (Marsh 2004).

Dental pellicles mediate many of the interactions that take place at intraoral surfaces. The term

pellicle is used to describe a thin, continuous membrane or cuticle, composed primarily of

salivary components deposited on a cleaned tooth surface (Al-Hashimi and Levine 1989). All

surfaces of the oral cavity, including all tissue surfaces (Bradway et al. 1989) as well as surfaces

of teeth-enamel (Al-Hashimi and Levine 1989), cementum (Fisher et al. 1987) and fixed, and

removable restorations (Edgerton and Levine 1992; Edgerton et al. 1996) are coated by dental

pellicle.

Pellicles contain salivary components, constituents

from gingival crevicular fluid, microbial, and cellular sources

(Scannapieco 1995). Enamel pellicle formation is driven by a

combination of physical forces (ionic, hydrophobic, hydrogen

loading and van der Waals) between molecules in saliva and

the tooth surface (Scannapieco et al. 1995). To study the

acquired enamel pellicle, it is convenient to examine the

freshly extracted teeth (Listgarten 1976) or by placing plastic

strips or epoxy crowns in the oral cavity as analogs to the tooth

(Brecx et al. 1981; Scannapieco 1995).

Early pellicle, formed within 2 h, contains both proteins and glycoproteins. Pellicle

contains components of salivary origin, like mucins (Kajisa et al. 1990; Fisher et al. 1987; Al-

Hashimi and Levine 1989), α-amylase (Al-Hashimi and Levine 1989; Scannapieco et al. 1995),

s-IgA (Al-Hashimi and Levine 1989; Orstavik and Kraus 1973), lysozyme (Orstavik and Kraus

1973), cystatins (Al-Hashimi and Levine 1989), proline-rich proteins (PRPs) (Bennick 1987), as

well as albumin originating from gingival crevicular fluid, (Al-Hashimi and Levine 1989; Kajisa

et al. 1990; Edgerton and Levine 1992), and bacterial products such as the glucosyltransferase of

Streptococcus mutans (Schilling and Bowen 1992).

The physical and chemical nature of the solid substratum significantly affects several

physicochemical surface properties of the pellicle, including its composition, packing density,

and its eonfiguration. Thus the characteristics of the underlying hard surface are transferred

through the pellicle layers and can still influence initial bacterial adhesion. Absolom et al. even

observed a clear relationship between the type of proteins adsorbed in the pellicle and the free

energy of the substratum surface. In an in vitro study, Busscher et al. observed that the

detachement of adhering bacteria might occur through a cohesive failure in the conditioning film

between bacteria and surface i.e. the pellicle.

2. Initial adhesion and attachment

(a) Transport of bacteria to the surface

Recent studies, applying the method of bacterial fingerprinting, have clearly proved that

periodontal pathogens are transmissible within members of a family (Zambon, 1996). The term

bacterial transmission between subjects should be used with caution. Indeed, transmission should

not be confused with contagion (the term contagious referring to the likelihood of a

microorganism being transmitted from an infected to an uninfected host and creating disease).

Intra-oral translocation of bacteria moving from one niche to another has also been proven

even if it has received less attention. This is surprising since transmission of these bacteria, from

one locus to another, can jeopardise the outcome of periodontal therapy. The introduction of two

phase type oral implants provided a favourable experimental setup to study intra-oral bacterial

translocation. Indeed, when the transmucosal part of the implant (the abutment) is inserted on top

of the endosseous part, a bacteriologically “virgin” surface is available. Since such implant

abutments can be replaced without any discomfort to the patient these “artificial” surfaces offer

an excellent model to study the build-up of a biofilm and the intra-oral translocation of bacteria

(Quirynen et al., 1996a). These abutments are also useful for the study of the influence of surface

characteristics (e.g. roughness, free energy) on initial supra-and subgingival colonisation

(Quirynen et al., 1994).

The first stage involves the initial transport of the bacterium to the tooth surface. Random

contracts may occur, for example, through Brownian motion (average displacement of 40

µm/hour), through sedimentation of microorganisms, through liquid flow (several orders of

magnitude faster than diffusion) of through active bacterial movement (chemotactic activity).

(b) Initial / Reversible adhesion

The second stage results in an initial, reversible adhesion of the bacterium, initiated by the

interaction between the bacterium and the surface, from a certain distance (50 nm), through long-

range and short-range forces, including van der Waals attractive forces and electrostatic

repulsive forces. Derjaguin, Landau, Verwey, Overbeek (DLVO) have postulated that, above a

separation distance of 1 nm, the summation of the previous two forces describes the total long-

range interaction. The total Gibbs energy GTOT is the total interaction energy.

GTOT = GA + GE

The result of this summation is a function of the separation distance between a negatively

charged particle and a negatively charged surface in a medium ionic strength suspension medium

GA = van der Waals attractive force

GE = electrostatic repulsive force

(e.g. saliva). For most bacteria, GTOT consist of a secondary minimum (where a reversible

binding takes place: 5-20 nm from the surface), a positive maximum (an energy barrier B) to

adhesion, and a steep primary minimum (Located at < 2nm away from the surface), where an

irreversible adhesion is established. For bacteria in the mouth, the secondary minimum does not

often reach large negative values, which means a “weak” reversible adhesion.

(c) Irreversible adhesion / Attachment

If a particle reaches the primary minimum (<1 nm from the surface), a group of short

range forces (e.g. hydrogen bonding, ion pair formation, steric interaction) dominates the

adhesive interaction and determines the strength of adhesion. This follows direct contact or

bridging true extracellular filamentous appendages (with length up to 10 nm).

Some of the adhesions that have been identified on subgingival species include fimbriae

(Cisar et al. 1984; Sandberg et al. 1988) and cell- associated proteins (Socransky and Haffajee

1992). Adhesions are often lectins which bind to saccharide receptors, but some adhesions are

thought to bind to proteinaceous receptors (Gibbons 1989). Receptors on tissue surfaces include

galactosyl residues, sialic acid residues (Murray et al. 1986), proline-rich proteins or statherin

and Type I and IV collagen (Socransky and Haffajee 1992). Oral bacteria generally possess more

than one type of adhesion on their cell surface and can participate in multiple interactions both

with host molecules and similar receptors on other bacteria (coadhesion) (Marsh 2004; Quirynen

and Bollen 1995).

Each Streptococcus and Actinomyces strain binds specific salivary molecules.

Streptococci (especially S. sanguis), the principal early colonizer, bind to acidic proline-rich-

proteins and other receptors in the pellicle, such as α-amylase and sialic acid. Actinomyces

species can also function as primary colonizers; for example, A.viscosus possesses fimbriae that

contain adhesins that specifically bind to proline-rich-proteins of the dental pellicle. Some

molecules from the pellicle (e.g. proline-rich-proteins) evidently undergo a conformational

change when they adsorb to the tooth surface so that new receptors become available. Indeed A.

viscosus recognizes cryptic segments of the proline-rich-proteins, which are only available in

adsorbed molecules. This provides a microorganism with a mechanism for efficiently attaching

to teeth and also offers a molecular explanation for their sharp tropisms. It is convenient to refer

to such hidden receptors for bacterial adhesins as cryptitopes (cryptic- hidden; topo-place).

(d) Colonization

This stage also involves specific interbacterial adhesion-receptor interactions (often

involving lectins) and leads to an increase in the diversity of the biofilm and to the formation of

unusual morphological structures, such as corn-cobs and rosettes (Marsh 2004; Kolenbrander

2000). Coaggregation (interactions between the suspended micro-organisms in a fluid phase)

between oral microbial pairs as well as its role in the sequential colonization of the tooth surface

has been studied extensively (Kolenbrander et al. 1994; Cisar et al. 1997). However, coadhesion

(interactions between suspended and already -adhering microorganism to a surface) may well be

equally important (Bos et al. 1996). Bacteria engage in a range of antagonistic and synergistic

biochemical interactions (Marsh and Bradshaw 1995). The efficiency of metabolic interactions

among bacteria in food chains may be enhanced if they are brought into close physical contact.

Likewise, the coadhesion of obligate anaerobic bacteria to oxygen-consuming species can ensure

their survival in overt aerobic oral environments (Marsh 2004).

The analysis of the coaggregation profiles of hundreds of subgingival isolates has

provided evidence that coaggregation might be important for subsequent plaque development.

Certain streptococci (for example, Streptococcus oralis), which bear receptors are coaggregation

partners of members of several genera. Early colonizing partners of receptor-bearing streptococci

include Streptococcus gordonii, Actinomyces naeslundii, Eikenella corrodens, Veillonella

atypica, Prevotella loescheii and Haemophilus parainfluenzae, as well as Capnocytophaga

ochracea. It is worth noting that these coaggregating partners of the initial colonizing

Streptococcus oralis, Streptococcus sanguinis and Streptococcus mitis are almost all gram-

negative, which correlates with the 40-year-old reports of a temporal shift from gram-positive to

gram-negative bacterial flora. The dominant species in initial dental plaque were Streptococcus

oralis that are receptor-bearing cells, indicating that receptor-bearing streptococci are an

abundant surface readily available for recognition by gram-negative bacteria expressing

complementary adhesions which recognize receptor polysaccharides. Possibly, receptor

polysaccharides on the early colonizing streptococci are a prerequisite for the shift from gram-

positive to gram-negative flora accompanying the shift from health to gingivitis (Kolenbrander et

al. 2006).

Special examples of coaggregations are the “corn cob” formation in which, for example,

streptococci adhere to filaments of Bacterionema matruchotii or Actinomyces species, and the

“test tube brush” composed of filamentous bacteria to which gram-negative rods adhere.

(e) Multiplication of the Attached Micro-Organisms

Cell division leads to confluent growth and, eventually, a three-dimensional spatially and

functionally organized, mixed-culture biofilm. Polymer production results in the formation of a

complex extracellular matrix made up of soluble and insoluble glucans, fructans and

heteropolymers. Such a matrix is a common feature of biofilms and makes a signifi cant

contribution to the known structural integrity and general resistance of biofilms; the matrix can

be biologically active and retain nutrients, water and key enzymes within the biofilm.

Endogenous substrates (derived from saliva or gingival crevicular fluid) are the main source of

nutrients for oral bacteria, but their catabolism requires the concerted and sequential action of

groups of microbes with complementary enzyme profiles, i.e., plaque functions as a true

microbial community (Marsh 2004).

3. Active Detachment

Once established, the resident plaque microfl ora remains relatively stable over time and is of

benefit to the host. The resident microflora of all sites plays a critical role in the normal

development of the physiology of the host and also reduces the chance of infection by acting as a

barrier to colonization by exogenous (and often pathogenic) species (“colonization resistance”).

Mechanisms contributing to colonization resistance include more effective competition for

nutrients and attachment sites, the production of inhibitory factors, and creation of unfavorable

growth conditions by the resident micro-flora. Thus, treatment should attempt to control rather

than eliminate the plaque microflora (Marsh 2004).

Test tube brush: (Listgarten MA,

Structure of the microbial flora

associated with periodontal health and

disease in man. A light and electron

microscopic study. J Periodontol 47:1-

18, 1976.)

De novo supraginigval plaque formation

Clinically, early undisturbed plaque on teeth follows an exponential growth curve when

measured planimetrically (Quirynen et al., 1989). During the first 24 h after perfect tooth

cleaning (including scaling and polishing), the increase in the amount of plaque is negligible

(<3% coverage of the vestibular tooth surface, which is clinically nearly undetectable). After 1 d

the term biofilm is fully deserved because organisation takes place within it. Microorganisms,

packed closely together, form a palisade while others start to develop pleomorphism. Each crack

is filled with one type of microorganisms. The thickness of the plaque increases slowly with

time, amounting to 20–30 μm after 3d. From day 2 to day 4, the plaque growth rate is much

faster but, from then on, there is a tendency for growth to slow down. After 96 h, on average

30% of the total tooth crown area will be covered with plaque. Although plaque does not

increase substantially with time after the fourth day, several reports have shown that its bacterial

composition will further change, with a shift towards a more anaerobic and a more Gram-

negative flora, including an influx of fusobacteria, filaments, spiral forms and spirochetes

(Theilade et al., 1966; Syed and Loesche, 1978).

Thin section of 24 h dental biofilm on

plastic film which is covered by

numerous layers of bacteria. Close to

the surface of the film a compact zone

of palisading organisms is seen

(arrows). In the external part of the

deposit (top right) an epithelial cell is

present. (Reproduced from Brecx et

al., 1983, with permission.) Bar=1 μm.

Thin section of 24 h dental biofilm on

plastic film containing numerous

bacteria comprising coci, rods and

filaments, one of which is branching.

(Reproduced from Brecx et al., 1983,

with permission.) Bar=1 μm.

In this ecologic shift within of the biofilm, there is a transition from the early aerobic

environment characterized by gram-positive facultative species to a highly oxygen-deprived

environment in which gram-negative anaerobic microorganisms predominate.

The slow start of the plaque growth curve can be explained partly by the fact that a single

colony of bacteria has to reach a certain size before it can be clinically detected. Brecx et al.

(1983) illustrated that the early increase in plaque mass originates largely from the proliferation

of bacteria already present, and only to a limited extent from new adhering species, an

observation which is also suggestive of an exponential curve. The increase in microbial

Clinical illustration of (a) (arrowheads) intra- and

(b) (arrows) intersubject variation in de novo

plaque formation. Plaque was visualized with an

erythrosin disclosing solution. Both photographs

represent undisturbed plaque formation in the upper

front region from two subjects with a healthy

periodontium after 4 d. In one subject (upper

picture, heavy plaque former), nearly 50% of the

tooth crown area was covered with plaque; in the

other (lower picture) the amount of biofilm was

relatively small. The formation of the biofilm

preferentially occurred both along the gingival

margin, from the interdental spaces, and from

surface irregularities, where bacteria are sheltered

against shear forces.

Thin section of 24 h dental biofilm on

plastic film. The microorganisms are

more or less arranged in colonies of

identical morphology. (Reproduced

from Brecx et al., 1983, with

permission.) Bar=1 μm.

generation time (1 h for initial plaque, 12 h for 3 day-old plaque), on the other hand, might

explain the leveling of the slope from day 4 onwards.

During the night, plaque growth rate is reduced by some 50% (Quirynen et al., 1989).

This was a surprising finding since it would be hypothesized that reduced plaque removal and

the decreased salivary flow (thus less antibacterial activity), at night would enhance plaque

growth. The fact that the supragingival plaque obtains its nutrients mainly from the saliva

(Carlsson, 1980) seems to be of more significance than the antibacterial activity of the latter.

De novo Subgingival Plaque formation

It is technically impossible to record the dynamics of subgingival plaque formation in an

established dentition for the simple reason that one cannot sterilize a periodontal pocket at

present. Some early studies, using culturing techniques, examined the changes within the

subgingival microbiota during the first week after mechanical debridement and reported only

partial reduction of about 3 logs (from 108 to 105), followed by a fast regrowth to almost

pretreatment levels (-0.5 log) within 7 days. The rapid recolonization was explained by several

factors. A critical review of the effectiveness of subgingival debridement, for example, revealed

that a high proportion of treated tooth surfaces (5% - 80%) still harbored plaque and calculus

after scaling. These remaining bacteria were considered the primary source for the subgingival

recolonization. Some pathogens penetrate the soft tissues

or the dentinal tubules and eventually escape

instrumentation.

Attempts to obtain realistic subgingival plaque

biofilms have been made by placing various insert

materials into the periodontal pockets of periodontitis

patients and then analyzing the bacterial components that

colonized the inserts (Takeuchi et al. 2004; Wecke et al.

2000). The noninvasive human model system, described

by Wecke et al. (2000) included a small rod surrounded

by a plastic membrane that is inserted into the periodon-

tal sulcus of a human volunteer. The carrier was fixed

supragingivally to the tooth surface by using cyanoacrylic

glue. The construction was guided by the assumption that

the carrier positioned in the pocket might be colonized

from both, the tooth and the soft tissue side. After 3 or 6

days of exposure, carriers were removed from the

periodontal pockets. Only those carriers that kept their

stable position during the exposure period can be used for

further investigations. After removal, the membrane is

embedded and minimally sectioned prior to examination by confocal microscopy or electron

microscopy. Fluorescence in situ hybridization has been used to stain these samples, but other

approaches, such as immunofluorescence, are also possible. Knowledge of sample orientation

allowed these investigators to conclude that spirochetes and gram-negative bacteria

predominated in deeper regions of the pocket, whereas streptococci were abundant in the shallow

regions.

Ecological differences in the supra- and subgingival environment which are of importance when

bacterial adhesion is considered.

Intraoral equilibrium between Cariogenic Species and Periopathogens

Several clinical studies followed the detection frequency and relative proportion of

cariogenic species after periodontal therapy (Quirynen et al. 1999). All suggest a relative

increase in the number as well as the detection frequency of S.mutans up to 8 months after

mechanical debridement. In a cross-sectional study, subgingival plaque samples from adult

periodontitis patients were tested for the presence and levels of mutans streptococci and putative

periodontal pathogens. Patients were divided into four groups based on the stage of their

periodontal treatment: 1) untreated, 2) after initial periodontal therapy, 3) maintenance phase

without periodontal surgery, and 4) after periodontal surgery. The prevalence of mutans

a. Enamel surface of tooth

b. Desquamating oral epithelium

c. Root cementum of dentin

d. Desquamating pocket epithelium

e. Swimming in GCF

f. Invasion of soft tissue

g. Invasion in hard tissue via dentine tubules

streptococci in the four groups was equivalent. The shift toward a more cariogenic flora observed

after initial therapy and after surgical periodontal therapy could be explained by:

1) Subgingival outgrowth by S. mutans occupying spots that become available after

periodontal therapy (e.g. increased number of free adhesion/receptor sites).

2) Creation of a new ecosystem in the subgingival area which is characterized by being

more anaerobic, and having a lower redox potential, lower pH and a protein concentrated

nutritional environment. This niche allows or facilitates the growth of S. mutants species,

and

3) “down growth” of S.mutans from supragingival area, where the species could survive in

the saliva.

Bacterial Colonization on Tooth Surfaces and Dental Materials

Differences in the amount of adherent plaque are observed in various materials (Siegrist

et al. 1991) and tissues (Nyvad and Fejerskov 1987; Carrassi et al. 1989). The pattern of

microbial colonization in vivo is determined by the surface structure of the tooth; on enamel

surfaces the first bacteria appeared in pits and surface irregularities followed by proliferation

along the perikymata, while on root surfaces bacterial colonization is characterized by a

haphazard distribution (Nyvad and Fejerskov 1987). It was also observed that within the initial

24-h period, root surfaces were more heavily colonized than were enamel surfaces (Nyvad and

Fejerskov 1987).

Different types of soft mucosal and hard dental surfaces may constitute various

prerequisites for bacterial colonization (Gibbons 1989). There are also more ecological

differences in the supra- and subgingival environment which are of importance when bacterial

adhesion is considered. Supragingivally, bacteria can adhere to the enamel surface or, to a lower

extent, to the desquamating oral epithelium. Subgingivally, more niches are available for

bacterial survival: adhesion to the root cementum, adhesion to the desquamating pocket

epithelium, swimming in the crevicular fluid, invasion in the soft tissue or invasion into the hard

tissue via the dentine tubules (Quirynen 1994).

The ultrastructural pattern of early plaque formation was studied on various dental

materials: amalgam, casting alloys, titanium, ceramics, glass polyalkenoate cement, composite

resins, unfilled resins, and bovine enamel (Hannig 1999). Because only less pronounced

variations could be detected in the ultrastructural appearance of the early plaque formed on the

different material surfaces, it was concluded that early plaque formation on solid surfaces is

influenced predominantly by the oral environment rather than by material-dependent parameters.

These findings may be ascribed to the presence of the pellicle layer, which apparently masks any

difference among materials, with regard to surface properties and biocompatibility.

Similar results were obtained by Leonhardt et al. (1995) who evaluated qualitative and

quantitative differences in bacterial colonization on titanium, hydroxyl-apatite, and amalgam

surfaces in vivo. No significant differences among the materials regarding colonization of

investigated bacteria were found during the study period. The different composition of materials

only slightly affects plaque colonization. The amount of the early plaque colonization seems to

be related more to the roughness degree than to material composition (Siegrist et al. 1991).

Materials used for dental restorations may also have antibacterial properties per se.

Several studies have shown that amalgam alloys have a bacteriostatic effect (Glassman and

Miller 1984). Titanium has been shown to inhibit plaque growth in vitro, particularly in the early

stages, probably due to the antimicrobial effect of metal ion release (Joshi and Eley 1988).

Nowzari et al. evaluated the amount of guided tissue regeneration membrane

contamination after treatment of mandibular bony defects in either a group of patients with a

healthy periodontium in the remaining dentition or a group of patients with multiple deep

pockets and numerous pathogens. The healthy group showed significantly less membrane

contamination both immediately after insertion as well as at removal after 6 weeks. The healthy

group also shoed significantly more clinical gain in attachment than the disease group (3.4 vs

1.4mm)

Macrostructural shielding and de novo Biofilm Growth

Early dental plaque formation on teeth follows a typical topographical pattern, with initial

growth along the gingival margin and from interdental spaces, areas protected against shear

forces, followed later by a further extension in the coronal direction (Mierau, 1984; Quirynen et

al., 1989). When the tooth surface demonstrates irregularities (Figure 7) biofilm formation does

not necessarily start from the gingival margin but from any groove, crack or pit. Scanning

electron microscopy studies (Lie, 1979; Lie and Gusberti, 1979; Nyvad and Fejerskov, 1987)

clearly revealed that the early colonisation of the surface starts from irregularities (Figure 5),

where bacteria escape shear forces allowing the time needed to change from reversible to

irreversible binding (Marshall et al., 1971; Quirynen and Bollen, 1995). By multiplication the

bacteria subsequently spread out from these areas as a relatively even monolayer. Surface

irregularities are also responsible for the so called “individualized” plaque growth pattern which

is reproduced in the absence of sufficient oral hygiene (Mierau and Singer, 1978; Mierau, 1984).

This phenomenon illustrates the importance of surface roughness in dental plaque growth.

Surface Micro-roughness

Besides the presence of pits or cracks, surface roughness (SR) as such affects the rate of

supragingival dental plaque formation. Rough intra-oral surfaces (crowns, implant abutments and

denture bases) accumulate and retain more plaque and calculus in terms of thickness, area, and

colony forming units (for review see Quirynen and Bollen, 1995). This plaque also reveals an

increased maturity of its bacterial component (characterized by an increased proportion of motile

organisms and spirochetes) and or a denser packing (Figures 3, 4). Smoothing of an intra-oral

surface decreases the rate of plaque formation. Below a certain surface smoothness (Ra<0.2 μm)

however, a further decrease in roughness does not result in an additional reduction in plaque

formation (Bollen et al., 1996; Quirynen et al., 1996b). Thus there seems to be a threshold SR

(Ra around 0.2 μm), above which bacterial adhesion will be facilitated (Bollen and Quirynen,

1998). The supragingival surface irregularities directly enhance initial bacterial adhesion but also

favour plaque growth indirectly by sheltering the attached microorganisms from oral cleaning.

Surface Free Energy

Glantz (1969) was the first to recognise in vivo the correlation between substratum

surface free energy (SFE) and the retaining capacity for intra-oral bacteria. Undisturbed

supragingival biofilm formation on test surfaces with different free energies, indicated a positive

correlation between substratum SFE and the weight of accumulated plaque (measured at days 1,

3, and 7).

Quirynen et al. (1989, 1990) studied the influence of SFE on undisturbed plaque growth

in man over a 9-day period. Small polymer films with different SFEs were glued on teeth and

allowed to accumulate bacteria. Hydrophobic surfaces (Teflon) harboured 10 times less plaque

than hydrophilic surfaces (enamel). Moreover, hydrophobic surfaces were found to be less able

to retain their plaque mass.

A microbiological examination of 3-day old plaque samples from the above mentioned

surfaces (Weerkamp et al., 1989) indicated that Teflon was preferably colonized by bacteria with

a low SFE whereas the opposite was observed for surfaces with a higher SFE (enamel).

Moreover, strains of S. sanguis I isolated from Teflon were found to be significantly more

hydrophobic than those isolated from higher energy surfaces (Weerkamp et al., 1989). This

suggests bacterial selection by, or adaptation to the surfaces, up to and even within the species

level.

The effect of substratum SFE on plaque maturation was investigated by comparing 3

month-old plaque from implant abutments with either a high (titanium) or a low (Teflon coating)

SFE. Low SFE substrata harboured a significantly less “mature” plaque both supraand

subgingivally, characterised by a higher proportion of cocci and a lower proportion of motile

organisms and spirochetes (Quirynen et al., 1994).

Interaction Between Surface Micro-roughness and SFE

The “relative” importance of the two parameters (SFE and roughness) on supragingival

plaque formation has been examined in vivo (Quirynen et al., 1990). Undisturbed plaque

formation was followed on polymer strips with low and medium SFE where one half was smooth

(Ra 0.1 μm) and the other half roughened (Ra>2.0 μm). After 3 d of undisturbed plaque

formation, as expected, significant inter-substratum differences were observed on the smooth

regions, while the rough regions of the strips all demonstrated an abundant bacterial

accumulation independent of the SFE. This indicates that, where bacterial adhesion is concerned,

SR overrules the influence of SFE.

The Impact of variables on Intra-Oral Biofilm Growth

The rate of plaque formation on teeth differs significantly between subjects (Figure 7).

There appear to be “heavy” (fast) and “light” (slow) plaque formers. Simonsson (1989) selected

one group of fast and one group of slow plaque formers from a group of 133 individuals. Both

groups were investigated for clinical, biochemical, biophysical and microbiological variables. In

comparative analyses only minor differences appeared between the groups, and no single

variable was considered as the only explanation to the great differences in the rate of dental

plaque formation. A multiple regression analysis explained 90% of the variation in initial plaque

formation by including the clinical wettability of the tooth surfaces, the saliva-induced

aggregation of oral bacteria and the relative salivary flow conditions. The saliva from slow

plaque formers reduced the colloidal stability of bacterial suspensions, such as for Streptococcus

sanguis (Simonsson and Glantz, 1988).

In a study by Zee et al. (1997) de novo plaque formation was followed on small enamel

blocks that were bonded onto the teeth of slow and heavy plaque formers. After a 1-day fast,

plaque formers showed more plaque with a more complex supragingival structure. However,

from day 3 to 14 there were no discernible differences between the groups, except for a more

prominent inter-microbial matrix in the rapid growth group. Moreover, rapid plaque formers

showed higher proportions of Gramnegative rods (35% vs 17%) after 2 weeks (Zee et al., 1996).

Variation within the Oral Cavity

Within the dental arch, large differences in plaque growth rate can be detected. In

general, plaque grows more quickly in the lower jaw, the vestibular tooth surfaces and the

interdental spaces (Lang et al., 1973; Quirynen, 1986; Furuichi et al., 1992).

Impact of Gingival Inflammation

Several studies clearly indicate that early in vivo plaque formation is more rapid on tooth

surfaces facing inflamed gingival margins than on those adjacent to healthy gingiva (Saxton,

1973; Hillam and Hull, 1977; Brecx et al., 1980; Quirynen et al., 1991; Ramberg et al., 1994;

1995). These studies suggest that the increase in crevicular fluid production plays a keyrole in

enhanced plaque formation, Probably, some substance(s) from this exudate (e.g. minerals,

proteins or carbohydrates) favour both the initial adhesion and/or the growth of the early

colonising bacteria (Hillam and Hull, 1977).

Impact of Patients’ Age

A subject‟s age does not influence de novo plaque formation, either in terms of the

amount produced or the composition of the plaque, except during the initial hours of biofilm

accumulation where less bacteria are present on the teeth of elderly individuals (Brecx et al.,

1985). In a study no differences could be detected in de novo plaque formation between a group

of young (20–25 years of age) and older (65–80 years of age) subjects who abolished mechanical

tooth cleaning measures for 21 d (Fransson et al., 1996). These observations largely confirm data

by Holm-Pedersen et al. (1975) and Winkel et al. (1987). The developed plaque in the older

patient group resulted, however, in a more severe gingival inflammation, which seems to indicate

an increased susceptibility to gingivitis with ageing.

Inter-subject Variation

Besides differences in surface characteristics and/or in the degree of gingival

inflammation inter-subject variation in plaque formation might also be explained by diet

(Ainamo et al., 1979), chewing fibrous food (Arnim, 1963), smoking (Sheiham, 1971), the

presence of copper amalgam (Hyyppä and Paunio, 1977), tongue and palate brushing (Jacobson

et al., 1973), the colloid stability of bacteria in the saliva (Simonsson, 1989), anti-microbial

factors present in the saliva (Adamson and Carlsson, 1982), the chemical composition of the

pellicle (Simonsson et al., 1987) and the retention depth of the dento-gingival area (Simonsson et

al., 1987). Rinsing with glucose or sucrose however, was found to have no detectable effect on

biofilm growth during the first hours of plaque accumulation (Brecx et al., 1981).

Conclusions

Intra-oral biofilm formation on a clean tooth/prosthetic surface follows an exponential

growth curve. Large intra- and inter-individual differences appear. Surface characteristics (SR

and to a lesser degree SFE) are responsible for the majority of dental plaque growth variability.

The similarity with the observations made in other environments (e.g. larynx or cardiovascular

prostheses, submarine surfaces, pipelines, etc.) is striking. Since the study of bacterial growth in

the oral cavity does not involve elaborate or invasive techniques, it deserves more attention in the

context of the study and control of biofilms.

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