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Protocol no.4
MODEL CAVITY METHOD

This method enables the in vitro cytotoxicity testing of dental restorative materials which may then be related to dental toxicity likely to occur in vivo.

CONTACT

INVITTOX
Russell&Burch House
96-98 North Serwood Street
Nottingham NG1 4EE, England
tel. (44)-115-9584 740 fax. (44)-115-950 3570

RATIONALE

The test system has been designed to mimic, as closely as possible, the contact which occurs between dental restorative and tooth pulp when cavities are filled in the clinical situation. A monolayer of cells is maintained in culture, the test material being in contact with the medium. A reduction in cell number, compared to control (non-exposed) cultures, indicates that the compound is cytotoxic and, hence, may cause damage to a tooth in the in vivo situation.

BASIC PROCEDURE

A hole is bored through the centre of a petri dish lid and a borosilicate glass cylinder, the lower aperture sealed with a microfilter, is attached to the underside. The resultant chamber, which represents the cavity, is packed with test material. The lid is placed over a culture dish containing a monolayer of either fibroblasts or macrophages (which represents the pulp), in such a way that the filter is just in contact with the medium. After a set exposure period the number of cells in the culture is determined and compared to that of control cultures. A reduction in cell number provides an indication of the cytotoxicity of the compound. If the reduction is 10% the compound is tested again in the presence of dentine. Artificial cavities are prepared as above with the addition of powdered dentine (obtained from non-carious human teeth) compacted into a 0.5mm layer at the base of the cylinder. (Or, alternatively, the filter can be replaced by an intact dentine slice.) The cytotoxicity of test compounds is then reassessed as before.

CRITICAL ASSESSMENT

This system provides a very reproducible, simple technique for the screening of large numbers of compounds. It is less time consuming and relatively inexpensive, especially in terms of animal usage, compared to in vivo tests. Comparison to the in vivo situation In vivo testing of dental restoratives is performed on the teeth of primates, dogs or ferrets. The wide variation in results, together with species differences, complicates any extrapolation to the situation in man. It has been suggested that this method, which attempts to mimic cavities in human teeth, may provide a more appropriate test system for comparing the relative toxicities of compounds, especially in view of its reproducibility. At the present time, however, the authors stress that the test cannot be categorically stated to be superior to in vivo tests. Comparison to other in vitro test systems This test has several advantages over other in vitro test systems as it conforms more accurately to the conditions which exist in the clinical situation. Certain in vitro systems produce inaccuracies, often due to the unrealistically large volume of material being tested producing toxic effects which would not be seen in vivo. In this test the ratio of volume and surface area of the test material, to the volume of the culture medium, is similar to that found in the clinical situation, thus producing more applicable results. Contact between the test material and underlying cells is through a permeable filter enabling the materials to be introduced into the system in their freshly mixed state when they are most irritant, as occurs in use. In conventional in vitro test systems the test chemical is often in direct contact with the target cells (representing the pulp cells of the tooth). In man, however, when a tooth is prepared for a filling it is usual for a certain amount of dentine to remain intact. This layer of dentine would, therefore, separate the material used to fill the cavity from the pulp of the tooth. This test system has been adapted so that, should a compound appear toxic, it can be retested in the same system, but one which incorporates a dentine component. The test compound must, therefore, penetrate the dentine before coming into contact with the medium bathing the pulp (i.e. macrophages or fibroblasts). The dentine component is prepared from non-carious human teeth (usually extracted wisdom teeth), which are readily available and again increase the applicability of the system for direct comparison to the clinical situation in man. Dentine can be prepared as a powder or in slices. In its powdered form the dentine provides an alternative to intact dentine as a chemically reactive substrate moderating toxicity and binding potentially toxic elements released from dental material. It is unsatisfactory, however, when the permeability characteristic of the dentine is important. In this instance a dental slice provides a better simulation of the clinical situation. The slices also possess an intact smear layer (a layer of cutting debri that serves as a natural cavity liner) which limits the access of certain toxic agents to the pulp. The choice between using slices or powdered dentine ultimately depends on the importance of standardisation against structural normality. However, if a material is to be tested according to the British Standard, dentine powder must be used. Choice of cell type The dental pulp is a specialised area of connective tissue composed of a peripheral layer of odontoblasts and a central mass of fibroblastic tissue. Although odontoblasts are the most likely to be initially affected by a toxic material they are highly specialised and difficult to grow in culture. Therefore, the cells of choice are either fibroblasts or macrophages. Mouse macrophages can be used as the target cells. The pulp beneath a carious dentine lesion may contain macrophages. Although much of this lesion is removed prior to placing a restoration the inflammation would still be present. In addition, trauma induced during cavity preparation prior to placing a restoration can result in the presence of inflammatory cells. Macrophages from two strains of mice have been compared (Meryon et al., 1985) and no difference in responsiveness was observed. The authors stress, however, that this does not guarantee that differences would not occur if other strains were used. On a routine basis the authors use fibroblasts rather than macrophages for several reasons, e.g. ease of handling and maintaining a cell line rather than having to obtain fresh cells, lack of animal involvement, etc. Pulp fibroblasts can be cultured as a primary diploid culture but this takes time, and the characteristics of the cells may vary from culture to culture. Fibroblasts are probably the most important cell type. It should be stressed that, as the two types of cell respond differently to certain toxic constituents of dental restoratives, results obtained using fibroblasts are not necessarily comparable to those obtained with macrophages.

TEST STATUS

Recently adopted as a British Standard. The Model Cavity system is now under consideration as an international testing method.

CHEMICALS TESTED

Dental restorative materials
ORGANISATIONS USING THE TEST
Dental materials have been tested from many different manufacturers and the author is in the process of liaising with others to test more materials.
Huntingdon Research Centre use the technique and have tested five materials in a collaborative study with the author funded by the DHSS (see Meryon et al, 1985).

REFERENCES

  1. Rabinovitch, M. & Destefano, M.J. (1975) In vitro, 11, 379-381. Tyas, M.J. (1977) A method for the in vitro toxicity testing of dental restorative materials. Journal of Dental Research, 56, 1285-1290.
  2. Meryon, S.D. & Browne, R.M. (1983) Evaluation of the cytotoxicity of four dental materials in vitro assessed by cell viability and enzyme cytochemistry. Journal of Oral Rehabilitation, 10, 363-372.
  3. Meryon, S.D. & Browne, R.M. (1983) Test methods for assessing the cytotoxicity of dental restorative materials using an in vitro model cavity system. In: Ceramics in Surgery (ed. Vincenzini, P., Elsevier Publishing Co., Amsterdam), 127-135.
  4. Meryon, S.D.; Stephens, P.G. & Browne, R.M. (1983) A comparison of in vitro cytotoxicity of two glass ionomer cements. Journal of Dental Research, 6, 769-773.
  5. Meryon, S.D. & Browne, R.M. (1984) In vitro cytotoxicity of a glass ionomer cement of a new generation. Cell Biochemistry and Function, 2, 43-48.
  6. Meryon, S.D.; Uphill, P.F.; Cordery, A.D. & Browne, R.M. (1985) A reproducibility study of the model cavity method for the in vitro toxicity testing of dental restorative materials. ATLA, 12, 215-223.
  7. Meryon, S.D. (1988) Model cavity method incorporating dentine. International Endodontic Journal, 21, 79-84.

IP-4 October 1989