Cytotoxicity of resin composites as a function of interface area

The standardization protocols for biomaterial cytotoxicity testing require fine tuning for oral biomaterials to obtain international comparability as the basis for risk assessment. The principal aims were specifically to evaluate the effect of (i) relative interface area (ratio of specimen surface to cell layer surface) and (ii) volume of cell culture medium on cytotoxicity as a potential modification of ISO 10993-5.

ISO 10993-5 was followed with an interface area of 12.5%, as recommended, using primary human gingival fibroblasts and L-929 mouse fibroblasts. In another series of experiments (using L-929 cells) the interface area was varied between 12.5% and 0.71%. For each relative interface area, three conditions for affecting the cure of the resin composite were investigated by using three mould materials: white, transparent and black moulds. In addition, the volume of cell culture medium was varied. Composite specimens (Herculite XRV) were added to the cultures immediately after production or preincubation for 1, 2, 7 days or 6 weeks under cell culture conditions. Specimens were incubated with fibroblasts for 72 h and cell numbers determined by flow cytometry. Glass specimens resembling composite specimens in diameter and height were used as negative controls.

Cytotoxicity results with primary gingival fibroblasts were comparable to results with the cell line L-929. An effect from the color/material of the specimen moulds was found. Different ratios of specimen sizes to cell culture parameters (cell layer surface, volume of cell culture medium) produced different results. Three out of four differently designed specimens showed the same behavior in cell culture.

Cytotoxicity tests should be further standardized in line with existing standards with regard to specimen production protocols to ensure results are internationally comparable to validate these tests as tools for risk assessment.