Bacterial Arrangement on the Teeth

Dental caries and periodontal disease are closely associated with the oral biofilm known as
dental plaque. Much is known about the makeup of this biofilm, especially at its early stage
of development (i.e. up to 12 hrs following tooth cleaning). The primary initial colonizers
are streptococci, and actinomyces. These bacteria coaggregate frequently with one another by
a mechanism that involves binding of protein adhesins on the surface of one cell to
complementary cell surface receptor polysaccharides (RPS) on streptococci.

Coaggregation profiles are known for a wide range of oral actinomyces and streptococci.
However, despite the potential significance of these interactions in biofilm formation,
little is known about their occurrence within oral biofilms in situ.

Objective

The objective of this study is to determine and compare the influence of coaggregation on the
spatial organization of bacteria in supragingival dental plaque formed under conditions of
normal salivary flow (Arm 1 of the protocol) or increased salivary flow (Arm 2 of the
protocol).

Study Population

The study population will consist of up to twenty-five (25) healthy adult subjects 18 years
of age and older. All subjects will participate in Arm 1 of the protocol. In addition, at
least three volunteers from Arm 1 will participate in Arm 2 of the protocol.

Study Design

This will be an observational, longitudinal protocol to determine the spatial organization of
bacteria found within dental plaque of normal healthy subjects. Enrolled subjects will have a
standard oral examination (including oral cancer screening) performed in the NIDCR clinic by
protocol-listed dental clinicians. A mandibular stent will be custom fabricated based on
impressions taken during the exam. Enamel chips cut from previously extracted third molars
will be sterilized by ethylene oxide then inserted into the stent on each side over the molar
area. We currently have enough extracted third molars in storage to accommodate tooth chips
for the entire protocol if four subjects complete the study as projected. If additional chips
are needed, the NIDCR is currently in the process of preparing a protocol that will provide
extracted teeth, if needed, for this and other protocols. In Arm 1 of the study, a
custom-fitted stent will be worn in the mouth of each subject, and chips removed for analysis
after 4 or 8 hours of wearing. The bacterial biofilm on the chips will be stained with
fluorescent antibodies against specific microbial adhesins or receptors, after which the
spatial organization of labeled cells within the biofilm will be documented by laser confocal
microscopy. Chips may be used for bacterial isolation and subjects will be asked to provide
saliva samples, and site-specific plaque samples (curette scrapings, gum swabs) for molecular
taxonomic analysis. Decisions on which subjects will have these samples taken will be based
on the reactivity of available antibodies with bacterial communities. Subjects from Arm 1 of
the study who are chosen to participate in Arm 2, will chew a gum base for 10 minutes to
increase salivary flow while wearing their stent. The stent from these individuals will be
removed after 4 hours of additional wear and analyzed as above. Prior to participation in Arm
2 of the protocol, subjects will be advised that the experiments are supported by a CRADA
agreement. Through this agreement, only coded research information will be exchanged. The
samples will remain with the NIH research team. Individuals may participate in the first arm
only, or in both arms of the study, as indicated in their consent document.

Outcome Measures

Outcome measures for this protocol will be the qualitative and quantitative description of
the oral bacterial community during development of initial plaque.

- INCLUSION CRITERIA:

- Healthy adult subjects (as determined from self-reported medical history, evaluation
by study dentist and nursing assessment, per policy).

- 18 years of age or older

- Good oral health as determined by screening oral examination with:

- no history of oral cancer

- no active periodontal disease.

EXCLUSION CRITERIA:

- History of any medical conditions affecting salivary flow, such as
Sj(SqrRoot)(Delta)gren s syndrome

- History of any immunosuppressant therapy, such as glucocorticoids (within the last
year), auto-immune or immune diseases such as ulcerative colitis or systemic lupus
erythematosis, or current steroid therapy, other than topical, within the last 30 days

- History of cardiac, kidney, liver and lung disorders

- Use of tobacco within the last year

- Use of antibiotics within the preceding four months

- Use of medications thought to affect salivary flow such as head/neck radiation
therapy, diuretics, or nitrates. Medications will be checked against product
information for effect and reviewed by the protocol dentist.

- Clinically visible active dental caries.

- Active periodontal disease as defined as no more than 4 sites in the entire mouth with
greater than or equal to 5mm probing depths.

- History of severe allergic reactions (Anaphylaxis) to several allergins and specific
allergies to or other substances found in the stent or impression material (ie:
Plasticized Methacrylate Polymer, methyl methacrylate monomer, alkyl dimethacrylate,
benzophenone, Toluidine and Hydroquinone), waxes containing Pine Rosin, and to the
list of ingredients for the impression material.