Title:  Methods for quantitating the efficacy of oral care products

United States Patent:  6,309,835

Assignee:  Koninkiijke Philips Electronics N.V. (Eindhoven, NL)

Filed:  May 27, 1999

Methods are included for quantitating the efficacy of oral care products at dislodging cells from biofilm test surfaces or inhibit or delay the accumulation of cells on a test surface. More specifically, the inventive methods measure the effectiveness of compositions and appliances in a test environment that models biofilm surface orientations that are encountered on a tooth surface, i.e. interproximal and subgingival tooth surfaces. The use of test surfaces that are removably attachable to a tooth prosthesis allow a variety of different quantitative methods to be applied to determine the amount of cells removed or deposited in a biofilm.

SUMMARY OF THE INVENTION

In one aspect, the present invention is a method for quantitating the efficacy of oral care products in the removal of dental plaque. The inventive plaque removal test method includes the following steps: 1) treating a test surface with saliva; 2) binding cells to the saliva treated test surface to form a biofilm on the test surface; 3) removing cells that are not bound to the biofilm test surface; 4) mounting the biofilm test surface in a tooth prosthesis to form a biofilm tooth prosthesis; 5) placing the biofilm tooth prosthesis into a typodont; 6) treating the biofilm tooth prosthesis with an oral care product; and 8) quantitatively determining the number of cells dislodged from the biofilm tooth prosthesis.

In yet another embodiment, the efficacy of an oral care product is determined using a method similar to that previously described except that rather than measuring the amount of cells removed from the biofilm tooth prosthesis, a quantitative method is used to determine the amount of cells that are retained in the biofilm and on the test surface after treatment of the biofilm tooth prosthesis with an oral care product.

In another aspect of the present invention a method for measuring the efficacy of an oral care product in preventing or inhibiting the formation of dental plaque is provided. The plaque inhibition test method includes the following steps: 1) treating a test surface with saliva; 2) treating the test surface with an oral care product; 3) incubating the test surface with cells under conditions conducive to the formation of a biofilm; 4) removing cells not bound to the biofilm or test surface; and 5) quantitatively determining the number of cells bound to the biofilm and test surface.

The inventive methods can be practiced using a wide variety of cell types. Many eukaryotic cell types are found in the mouth and can contribute to the formation of a biofilm. For example, an inflamed subgingival pocket surrounding a tooth contains cells mounting an immune response. In addition to bacterial and immune cells, the mouth also may contain fungal cells that contribute to biofilm formation or are present as an infective agent. The present invention provides methods for testing the efficacy of oral care products in removing or inhibiting the formation of in vitro biofilms on test surfaces that are composed of any type of eukaryotic and/or prokaryotic cells. A biofilm used in accordance with the present invention can be made using one cell type or many. More specifically, a biofilm can be formed from a combination of cells from different species, particularly cells of species selected from the large number of different bacterial species that are found in the mouth. In a preferred embodiment, the test surface is made from hydroxyapatite (HA). However, many other materials can be used as a test surface, including, but not limited to, bovine or other mammalian tooth sections, plastic, glass and metals, such as titanium, germanium and aluminum.

One aspect of the inventive methods is that the biofilm test surface can be removably positioned in a tooth prosthesis so as to more accurately model the different biofilm environments found on a natural tooth. For example, to model the removal of a cellular biofilm located in a subgingival location the biofilm test surface is mounted in a recess on the tooth prosthesis such that when the tooth prosthesis is placed into a typodont the biofilm test surface is below the artificial gum line. Similarly, to model the removal of bacteria located in an interproximal gap, the biofilm test surface is mounted on the tooth prosthesis such that the biofilm test surface is in an interproximal gap, preferably positioned so that the bristles of a tooth brush cannot make direct contact with the biofilm. In addition, a single typodont can be used simultaneously to test multiple biofilm test surfaces located on additional tooth prostheses.

A biofilm test chamber is also provided that is composed of a test chamber, which functions to retain fluid suspending the cells removed from a biofilm test surface; a typodont that is removably positioned in the test chamber and contains a plurality of tooth receiving holes; and one or more tooth prosthesis which are sized to fit snugly into the typodont tooth receiving holes. One or more of the tooth prosthesis also contain a recess sized to receive a biofilm test surface.

A number of different quantitative methods can be used to determine the number of cells either removed from a biofilm and test surface or deposited thereon. Such methods include the use of fluorescent dyes that bind specifically to cells and whose fluorescence characteristics allow live cells to be distinguished from dead cells. Further included are quantitative methods that rely upon the use of nucleic acid hybridization probes, antibody techniques, spectrophotometer readings of optical density, and other quantitative assays for cellular components such as enzymes or metabolic compounds.

Claim 1 of 35 Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for quantitating the efficacy of an oral care product in the removal of a biofilm, the method comprising:

(a) treating a test surface with saliva;

(b) binding cells to the test surface to form a biofilm test surface;

(c) removing cells not bound to the biofilm test surface;

(d) mounting the biofilm test surface in a tooth prosthesis to form a biofilm tooth prosthesis;

(e) placing the biofilm tooth prosthesis into a typodont;

(f) placing the typodont into a collection chamber;

(g) treating the biofilm tooth prosthesis with an oral care product; and

(h) quantitatively determining the amount of cells dislodged from the biofilm test surface, said quantitation of cells indicating the efficacy of said oral care product in biofilm removal.

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