A Study on Reference Standard for Cytotoxicity Assay of Biomaterials

Abstract

The objective of the present study was to find a standard substance for use as a reference in the cytotoxicity assay of biomaterials, as an alternative to animal experiments in recent years. Eight kinds of rubber were made in a plate shape to keep their surface area at 1 cm² against 10 ml of extract volume. They were extracted by the following three extraction methods (a) dynamic extraction at 200 rpm gyration on alumina balls at 37°C for 24 h; (b) static extraction at 37°C for 24 h and (c) extraction by heating in an autoclave at 121°C for 60 min. At the end of each period each extract was examined for cell viability based on an evaluation by neutral red uptake. These methods were repeated up to seven times. Two kinds of chemicals were also tested. The extracts obtained were used to treat human gingival fibroblasts that have been cultured with DMEM supplemented with 5% fetal bovine serum into a 96 well tissue culture plate by 1 × 10⁵ cells/ml, in an incubator aerated with 5% CO₂, and 95% humidified air at 37°C for 48 h. The extracts of ethylene-propylene, butyl, nitrile rubbers, and two kinds of chemicals yielded strong cytotoxicity in all three kinds of extraction methods, while chloroprene, fluorine-contained, isoprene, India, and silicone rubbers showed little cytotoxicity. The results obtained by the three kinds of extraction methods revealed no differences in the order of cytotoxicity of the materials tested. Repetition of extraction did not change the degree or pattern of extraction in each extraction method compared to the single extraction. Therefore, a standard substance for reference should be chosen from the viewpoint of availability, handling, consistency, and safety. These findings suggested that several kinds of rubber may be candidate substances either for positive or negative controls in the cytotoxicity assay of biomaterials by the dynamic extraction method.