Franz Alexander, König Franz, Skolka Astrid, Sperr Wolfgang, Bauer Peter, Lucas Trevor, Watts David C, Schedle Andreas
Bernhard Gottlieb University Clinic of Dentistry, Medical University of Vienna, Waehringerstrasse 25a, A-1090 Vienna, Austria.
Dent Mater. 2007 Nov;23(11):1438-46. doi: 10.1016/j.dental.2007.05.014. Epub 2007 Aug 3.
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.
生物材料细胞毒性测试的标准化方案需要针对口腔生物材料进行微调，以实现国际可比性，作为风险评估的基础。主要目的是具体评估（i）相对界面面积（标本表面与细胞层表面的比率）和（ii）细胞培养基体积对细胞毒性的影响，作为对ISO 10993 - 5的潜在修改。
按照ISO 10993 - 5进行操作，采用推荐的12.5%的界面面积，使用原代人牙龈成纤维细胞和L - 929小鼠成纤维细胞。在另一系列实验（使用L - 929细胞）中，界面面积在12.5%至0.71%之间变化。对于每个相对界面面积，通过使用三种模具材料（白色、透明和黑色模具）研究影响树脂复合材料固化的三种条件。此外，细胞培养基的体积也有所变化。复合材料标本（Herculite XRV）在生产后或在细胞培养条件下预孵育1、2、7天或6周后立即添加到培养物中。标本与成纤维细胞孵育72小时，并通过流式细胞术测定细胞数量。使用直径和高度与复合材料标本相似的玻璃标本作为阴性对照。
原代牙龈成纤维细胞的细胞毒性结果与细胞系L - 929的结果相当。发现标本模具的颜色/材料有影响。标本尺寸与细胞培养参数（细胞层表面、细胞培养基体积）的不同比例产生了不同的结果。四个不同设计的标本中有三个在细胞培养中表现出相同的行为。
细胞毒性测试应根据现有标准在标本生产方案方面进一步标准化，以确保结果具有国际可比性，从而验证这些测试作为风险评估工具的有效性。
