The Materials and Methods

The Materials and Methods section should provide a detailed description of the materials and procedures used to conduct the investigation in order to assure the possibility of replication for future studies.3 The items described should include: subjects of the study (i.e., patients, extracted teeth, experimental animals); their characteristics (i.e., mean age, sex ratio, etc.); the method for obtaining the sample; sample size; the procedures used to assign subjects to treatment groups; data collection instruments; the statistical tests used; and, in the case of human subjects, methods used to protect their rights. This generally includes obtaining permission to do the study from an appropriate institutional review board (IRB). A justification for the sample size, often based on a power analysis, may also be included.

Below is an example of the Materials and Methods section:

The Clinical Effect of Dentifrices Containing Stabilized Stannous Fluoride on Plaque Formation and Gingivitis - A Six-Month Study with Ad libitum Brushing4

Materials and Methods

Study Design
Six-hundred and twenty healthy adult volunteers from the greater Indianapolis metropolitan area were accepted as subjects for this six-month, double-blind, placebo controlled, parallel group, single center clinical trial. To participate in the study, subjects had to have at least 16 natural teeth, including 4 molars, and five gingival bleeding sites at the initial examination. Subjects were excluded if they had rampant caries, advanced periodontal disease, chronic dental neglect which required prompt treatment or serious medical conditions as determined by the investigator. Subjects were also excluded if they had taken antibiotics within seven days of the start of the study. Pregnant women were not entered into the study.

After baseline examinations to determine initial levels of gingivitis, plaque accumulation, extrinsic dental stain, and oral soft tissue status, subjects were separated by gender and stratified with respect to their baseline gingivitis scores. Within strata, subjects were assigned to treatment groups by random permutations of four. Following the initial examinations, all subjects received intraoral photographs and had an alginate impression of the mandibular and maxillary arches taken. The impressions were used to produce models for the construction of stain masking tooth covers. Following this procedure, all subjects received a thorough oral prophylaxis and a supply of their assigned test dentifrice. Each subject was provided with six 4.6 ounce plain white tubes containing one of the following dentifrices: (1) 0.243% sodium fluoride dentifrice (control), (2) 0.454% SnF2 dentifrice stabilized with 1.5% SnCI2 and 2.08% sodium gluconate (lowGluc stabilized stannous fluoride), (3) 0.454% SnF2 stabilized with 1.5% SnCI and 4.16% sodium gluconate (highGluc stabilized stannous fluoride), and (4) experimental dentifrice. The dentifrices were packaged in uniquely labeled identical white tubes to ensure that neither the examiners nor subjects knew the identity of the dentifrices throughout the course of the trial.

Dentifrice use instructions were given to each subject verbally and in writing at the time of product distribution. These instructions were for the subject to use their assigned dentifrice according to their own individual habits. Subjects were also instructed to use only their assigned dentifrice and no other toothpastes or mouth rinses. Subjects did not receive a new toothbrush at the start of the study. Rather they were told to use their existing toothbrushes and to replace them from commercial sources as they normally would.

Subjects returned for examinations after three and six months of dentifrice use. At these examinations, gingival inflammation and gingival bleeding, plaque accumulation, extrinsic dental stain, and oral soft tissue status were recorded. Supragingival plaque was sampled at baseline and after three and six months. At the six-month examination, tooth covers, previously fabricated for each subject and covering the tooth to the cervical margin, were employed to evaluate the potential for examiner bias resulting from stannous fluoride staining. To construct the tooth covers, dark gray plastic sheets were vacuformed onto full-arch models and then carefully trimmed around the cervical areas of the teeth. This produced a "cover" which obscured the teeth from view, but still permitted access for conducting gingivitis examinations. In order to determine the effort expended by the dental hygienists to remove stain at the end of the study, questionnaires were completed by the hygienist at the conclusion of each subject's prophylaxis. The data collected on the questionnaires included the time it took to scale and polish each subject as well as the severity and ease of removal of each subject's accumulated stain. A complete schedule of study visits and corresponding clinical measurements is shown in Figure 1.

Figure 1. Schedule of Study Visits and Clinical Examinations
3 Months 3 Months Exit Study
Screening Baseline Exam 3-Month Exam 6-Month Exam
Plaque Sampling Plaque Sampling Plaque Sampling
Oral Soft Tissue Oral Soft Tissue Oral Soft Tissue
Stain Stain Stain
Plaque Plaque Plaque
Gingivitis Gingivitis Gingivitis
Prophylaxis
Product Distribution

Conduct of Clinical Examinations
Oral examinations were conducted under dental clinic conditions employing good oral illumination, compressed air, mouth mirrors, and periodontal probes.

Oral Soft Tissue Health
To monitor oral soft tissue health, comprehensive visual-tactile examinations of the oral mucosa were conducted to detect pathoses that could possibly be related to dentifrice use. Structures examined included the gingival, hard and soft palate, oropharynx, buccal mucosa, tongue, floor of the mouth, labial mucosa, and lips.

Gingivitis and Gingival Bleeding
Gingivitis was measured using the Gingival Index developed by Loe and Silness.14 This index measured the qualitative changes in the gingival tissue adjacent to the mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and mesiolingual surfaces of all natural teeth, excluding third molars, for a total of 168 potential sites. After drying the teeth and gingival areas with a stream of air, a score was assigned to each site according to the following criteria:

0= Normal gingiva (no inflammation)
1= Mild inflammation evidenced by a slight change in color, slight edema, but no bleeding on probing
2= Moderate inflammation evidenced by redness, edema, glazing and bleeding on probing
3= Severe inflammation evidenced by marked redness and edema. Ulceration with a tendency to spontaneous bleeding.

For gingivitis, results are reported as the mean GI score for each surface averaged across all teeth evaluated. For bleeding, results are reported as the number of sites bleeding on probing or bleeding spontaneously (grades 2 or 3).

Plaque
Plaque was measured using the P1I Index described by Silness and Loe.15 After drying the teeth and gingival areas with a stream of air, undisclosed plaque was measured on the mesiobuccal, buccal, distobuccal, mesiolingual, lingual, and mesiolingual surfaces of all teeth (except third molars) for a total of 168 potential sites (6 sites per tooth). A score was assigned to each surface according to the following criteria:

0= No plaque in the cervical area
1= A film of plaque adhering to the free gingival margin or the adjacent cervical area of the tooth. The plaque is not visible to the naked eye and is only recognized by running a probe across the surface.
2= Moderate accumulation of plaque on the gingival margin and/or adjacent tooth surface, which can be seen by the naked eye
3= Abundance of plaque on the gingival margin and adjacent tooth surface, filling the gingival sulcus.

An average plaque score was obtained for each subject at each examination by summing the scores and dividing by the number of sites graded for that subject.

Dental Stain
Extrinsic dental stain was scored on the facial and lingual surfaces of all natural teeth (excluding third molars) resulting in a total of 56 possible sites. The stain was graded by first assigning an intensity score (0-3 scale) for a surface and then estimating the area covered on that surface to the nearest five percent.16 The intensity score was multiplied by the area covered for each site graded, summed for each subject, and divided by the number of sites graded for that subject.

Statistical Analysis
Covariance analyses17 were performed on the plaque, gingivitis, gingival bleeding, and dental stain scores using the baseline score as covariate. All statements of significance were based on alpha = 0.05, two-tailed test, and all percent reductions were calculated versus the sodium fluoride control. Significance testing among the four treatment groups was performed using Student-Newman-Keuls multiple comparison techniques as described by Snedecor.18