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Protocol no. 50
HEp-2 CYTOTOXICITY TEST FOR IMPLANT MATERIALS

44Two cytotoxicity tests are used in parallel to investigate the toxicity of implant materials used in medicine and dentistry.

CONTACT

Dr Miroslav Cervinka
Department of Biology
Medical Faculty of Charles University
Simkova 870 500 38 Hradec Kralove Czech Republic
Tel: Czech Republic- 49-23048 Fax: Czech Republic- 49-26595

RATIONALE

Exposing cultured cells to toxic chemicals may cause them to change their appearance, growth rate/pattern, or may result in an alteration in a specific chemical process. While an assay which detects one of these changes will allow some assessment of toxicity, a series of tests that detect different end points will complement each other in providing a more detailed toxicological profile of the test substance. The assay systems described in this protocol determine the changes in cell morphology and proliferation of HEp-2 cells that occur as a result of exposure to the test substance.

BASIC PROCEDURE

HEp-2 cells are plated out in 60mm petri dishes. To assay for changes in morphology as a result of contact cytotoxicity, cells are incubated for 24 hours, placed in a modified Rose chamber in the presence of the test material, and photographed at regular intervals over an 8 hour period. To assay for changes in cell proliferation, cells are exposed to various dilutions of the test compound and photographed at 0, 24, 48 and 72 hours. The changes in cell number in a defined area of the culture are recorded.

CRITICAL ASSESSMENT

This protocol enables the simultaneous investigation of the effects of a test substance on cell morphology and growth and incorporates the two common approaches to toxicity testing of implantation materials - direct contact of materials with cells and also the preparation of an eluate. Thus, these two assays provide complementary information on the toxicity of the test material. The epithelial cell line, HEp-2, derived from human carcinoma of the larynx, is well characterized, easy to handle, and is particularly suitable for studying changes in basal cell functions. Contact cytotoxicity is assessed by periodic observation of morphological changes occurring in cells that are in contact with the test material. The method is based on those of Haustveit et al (1984) and Rosenbluth et al.,(1965), modified so as to allow the material to be held in a fixed position in the dish (Puza & Novak 1971). The cell proliferation assay is based on the methods of Cur‚ & Bou‚ (1976) and Chen & Chen (1981). The number of cells within a defined area is recorded at intervals. The method has been modified by the use of a perforated self adhesive strip of paper to localise the desired area. Cells are exposed to an eluate of the test substance. One of the great advantages of this technique is that the original results in the form of photographs can be stored and used for further independent analysis if necessary. These tests also produce reliable results that have been used for several years for the testing of dental materials in compliance with the commission for New Dental Material of the Czech Ministry of Health and of most of the new materials used for stomatological care in Czechoslovakia. The results obtained using these techniques are in good agreement with the alternative method, that of implantation in vivo.

CHEMICALS TESTED

This method has been used for testing dental materials for the Commission for New Dental Materials of the Czech Ministry of Health. Materials tested include new filling polymers (Evicrol Esthetic Spofa, Czechoslovakia), new metal alloys (Kdynium, Kdyne, Czechoslovakia) and bioactive ceramic (BAS, Glass research institute Sklounion, Czechoslovakia).

TEST STATUS

The assay is in in house use.

REFERENCES

  1. Chen, T.R. 1977 In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res., 104, 255-262
  2. Chen, C. & Chen, S.C. 1981 Cell growth factor activity Exp. Cell Res., 136, 43-51 Cure, S. & Boue, A. 1976 Determination of the growth rate in human fibroblasts in cell culture. In: Methods in Cell Biology,XIV, (ed:Prescott, D.M.) pp 125-130
  3. Haustveit, G., Torheim, B., Fystro, D., Eidem, T. & Sandvik, M. 1984 Toxicity testing of medical device materials in human tissue cultures. Biomaterials, 5, 75-80
  4. Puza, V. & Novak, L. 1971 Zellkulturen als Mittel fur Toxizitatsteste zahnarztlicher Materialen. Schweiz. Mschr. Zahnheilk., 81, 75-84
  5. Rose, G.G. 1954 A separable and multipurpose tissue culture chamber. Tex. Rep. Biol. Med., 12, 1074-1083
  6. Rosenbluth, S.A., Weddington, G.R., Guess, W.L. & Autian, J. 1965 Tissue culture method for screening toxicity of plastic materials used in medical practice. J. Pharm. Sci., 54, 156-159

IP-50 © July 1992