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.
GA = van der Waals attractive force
GTOT = GA + GE
GE = electrostatic repulsive force
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
(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.
 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.)

(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).
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.
 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.

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.

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
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.

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

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
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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