Hendrich C, Geyer M, Scheddin D, Schütze N, Eulert J, Thull R
Orthopädische Universitätsklinik Würzburg, König-Ludwig-Haus.
Biomed Tech (Berl). 1996 Oct;41(10):278-83.
For in vitro testing of new biomaterials cultured fibroblasts are employed. In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested. Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated. In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described. Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts. For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed. The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin. Calcification was demonstrated using the v. Kossa stain. In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown. As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene. In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper. The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test. The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained. In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth. Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface.
对于新型生物材料的体外测试，会使用培养的成纤维细胞。在琼脂扩散试验中，细胞的存活与待测试材料的存在有关。关于生物材料生物学效应的进一步说明需要使用适应相关组织和所研究潜在问题的细胞培养物。在本研究中，描述了一种成骨细胞培养系统，利用该系统可按照相关标准要求测试与骨接触的植入物表面。在传统的琼脂扩散试验中使用了由钛、聚苯乙烯或铜制成的测试体，这些测试体也被成纤维细胞或人胎儿成骨细胞系覆盖。对于琼脂扩散试验，测试标准是用中性红染色后抑制区域的大小，而对于覆盖生长情况，则采用平均细胞直径和细胞数量。通过碱性磷酸酶免疫组织化学法或通过I型胶原蛋白和骨钙素免疫组织学法确定成骨细胞系的表型。使用冯·科萨染色法证明钙化情况。在成骨细胞的情况下，显示出从I型胶原蛋白和碱性磷酸酶阳性表型向骨钙素阳性表型的分化，再到钙沉积增加。与琼脂扩散试验一样，直接覆盖生长也显示钛和聚苯乙烯没有细胞毒性作用。相比之下，观察到铜具有细胞毒性作用，表现为细胞数量减少以及大小分布向左偏移。覆盖生长情况下各个测试的标准偏差比琼脂扩散试验的要小。因此，成骨细胞培养系统在所得结果的质量和准确性方面符合EN/DIN 30993-5的标准。除了排除直接细胞毒性外，该测试系统还提供了一种检查材料对细胞生长影响的新可能性。因此，它允许对每种材料表面上的成骨细胞增殖和分化进行可重复的检查。
