骨组织工程经多聚赖氨酸修饰的脱钙骨基质富集材料的细胞毒性研究

目的：评价多聚赖氨酸修饰的脱钙骨基质富集材料的细胞毒性，为该材料应用于临床提供实验依据。方法：参照GB／T16886．5—2003-ISO 10993—5：1999标准，将不同浓度的材料浸提液分别与人间充质干细胞（Mscs），成骨细胞（OS）体外复合培养。倒置相差显微镜行细胞形态学观察；MTT比色法测定1、3、5、7d的MSCs增殖活性；金氏比色法检测OS碱性磷酸酶活性。结果：不同时间点、不同浓度材料浸提液培养的MSCs均良好增殖，毒性0～1级；5d后浸提液培养组细胞增殖率优于阴性对照组（P〈0．05），随材料浸提液浓度增加而增加；浸提液培养组OS碱性磷酸酶活性与阴性对照组相比差异无显著性（P〉0．05）。结论：经多聚赖氨酸修饰的脱钙骨基质富集支架材料无细胞毒性，实验浓度范围内利于MSCs增殖，不影响OS的成骨活性。

Cytotoxicity Tests of Decalcified Bone Matrix Decorated with Poly-L-Lysine for Bone Tissue Engineer

Objective： To evaluate the cytotoxicity of decalcified bone matrix decorated with poly-L-lysine in order to provide experimental proof for its clinical application. Methods： According to the criteria for evaluation of cellular trials recommended in GB/T16886.5--2003--ISO 10993--5：1999, the mesenchymal stem cells （MSCs） and osteoblast cells （OS） were cocultured with different doses of extracts of decalcified bone matrix decorated with poly-L-lysine in vitro. Cell morphology were observed by invert phase contrast microscope, MSCs proliferation and relative multiplication rate were calculated with MTT assay at 1, 3, 5 and 7 days. The activity of alkaline phosphatase （ALP） in OS was tested at the same time. Results： The growth and proliferation of MSCs were normal in different doses of extracts at different time point, levels of toxicity ranged from 0 to 1 grade. Relative multiplication rate of MSCs co-cultured with extracts was significantly higher than that without extracts after 5 days （P~0.05） and went higher as the extract dose denser. There was no obvious difference in ALP activity between OS co-cultured with and without extracts. Conclusion： The decalcified bone matrix decorated with poly-L-lysine showed no cytotoxicity and within experimental density, it could promote the proliferation of MSCs without affecting osteogenesis activity of OS.