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纸质出版:2010
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代震宇,李军,赵增辉,敖晓晓,蒋电明.纳米羟基磷灰石/聚氨基酸复合材料生物安全性评价[J].陆军军医大学学报(原第三军医大学学报),2010,32(21):2294-2299.
Dai Zhenyu,Li Jun,Zhao Zenghui,Ao Xiaoxiao,Jiang Dianming.Biological safety of nano-hydroxyapatite and poly-amino acid compound[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(21):2294-2299.
目的 初步评价新型骨修复材料纳米羟基磷灰石/聚氨基酸(nano-hydroxyapatite and poly-amino acid
n-HA/PAA)的生物安全性。 方法 按照ISO10993-1标准中规定以自制n-HA/PAA为实验组进行以下实验: ①体外细胞毒性实验:采用L929成纤维细胞,完全培养基为阴性对照,苯酚为阳性对照,通过细胞形态学、MTT试验以及流式细胞进行细胞毒性评价; ②全身急性毒性实验:以生理盐水为阴性对照组观察2组小鼠腹腔注射材料浸提液和生理盐水后72 h内有无中毒和死亡现象; ③致敏试验:白化豚鼠30只均分3组,以生理盐水为阴性对照组、二硝基氟苯丙酮溶液为阳性对照组观察各组动物致敏区有无红肿和红斑,并记分; ④溶血实验:生理盐水作为阴性对照组,灭菌蒸馏水为阳性对照组,分别加入抗凝兔血后计算溶血率; ⑤热源实验:新西兰大白兔3只,按10 ml/kg注射材料浸提液,测量正常体温与注射后连续3 h 内每小时体温的差值; ⑥遗传毒性实验:KM小鼠30均分3组,分别按100 g/kg间隔24 h注射材料浸提液、环磷酰胺及生理盐水2次,观察骨髓嗜多染红细胞及含微核小体的骨髓嗜多染红细胞,并计算微核率。 结果 体外细胞毒性实验观察材料对细胞形态与生长无明显影响,MTT检测实验组细胞毒性为1级,流式细胞仪检测材料不会引起细胞凋亡;全身急性毒性实验显示实验组动物无中毒及死亡;致敏试验显示致敏区无水肿及红斑;溶血实验表明材料溶血率为0.71%,符合小于5%的实验标准;热源实验显示实验动物无明显体温升高;遗传毒性实验显示材料微核试验阴性,无遗传毒性。 结论 n-HA/PAA复合材料具有良好的生物安全性。
Objective To evaluate the biological safety of nano-hydroxyapatite and poly-amino acid (n-HA/PAA) compound. Methods A n-HA/PAA compound was prepared according to the ISO10993-1 standards to carry out the following experiments. ① In vitro cytotoxicity test: L929 fibroblasts were divided into negative physiological saline group and positive phenol group. Morphologic change was observed and cytotoxicity was detected by MTT assay and flow cytometry. ②Systemic acute toxic test: intoxication and death were observed in mice injected with a solution of nHA/PAA compound extraction and physiological saline. ③Sensitization test: thirty mice were randomly divided into negative physiological saline group and positive dinitrofluorobenzene group. Edema and erythema were observed and scored. ④Hemolytic test: mice were divided into negative physiological saline group and positive phenol group. Hemolytic rate of n-HA/PAA compound was calculated after anticoagulant rabbit blood was added. ⑤Pyrogen test: 3 rabbits were injected with a solution of n-HA/PAA compound extract at the dose of 10 ml/kg. Difference in normal body temperature and temperature 3 h after injection of the solution was detected. ⑥Genetic toxicity test: 30 mice were randomly divided into three groups and injected with physiological saline
cyclophosphamide solution
and solution of n-HA/PAA compound extract
at the dose of 100 g/kg
respectively. Bone marrow polychromatic erythroblasts and polychromatic erythroblasts containing micronuclei were detected and the micronuclei rate was counted. Results In vitro cytotoxicity test showed that n-HA/PAA compound had no obvious effect on the morphology and growth of L929 fibroblasts. MTT assay revealed that the extract of n-HA/PAA compound could not inhibit the proliferation and intoxication of L929 fibroblasts. The apoptosis of L929 fibroblasts was not obvious. Systemic acute toxic test showed that n-HA/PAA compound could not cause intoxication and death of mice. Sensitization test demonstrated no edema and erythema. The hemolytic rate of n-HA/PAA compound was 0.71% (5%). Ppyrogen test showed no obvious temperature increase. Genetic toxicity test displayed no micronuclei and no genetic toxicity of n-HA/PA compound. Conclusion The biological safety of n-HA/PAA compound is rather good.
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