新型自组装肽纳米纤维支架材料的生物安全性评价

Biological safety of designer self-assembling peptide nanofiber hydrogel scaffolds

目的评价新型自组装肽纳米纤维水凝胶支架材料D-RADA16的生物相容性及安全性,为该材料的临床推广应用提供试验依据。方法根据GB/T 16886.1-2011/ISO 10993-1∶2009《医疗器械生物学评价标准》,选取体外细胞毒性试验、全身急性毒性试验、皮内刺激试验、溶血试验、热源试验、微核试验评价D-RADA16的生物相容性。结果细胞毒性试验中,材料共培养组相对增殖率均>90%,细胞毒性为1级,材料无细胞毒性。溶血试验结果显示,生物材料D-RADA16的溶血率为0.96%,符合<5%的国家标准。各组小鼠活动正常,7 d内无小鼠死亡,各组动物未见中毒症状或不良反应,7 d后各组小鼠体质量比较,差异无统计学意义(P>0.05)。7 d后处死动物,肝、脾、肾大体观察和苏木精-伊红染色法染色未见异常。热原试验显示,3只动物体温升高,最高0.5℃,升高总数为1.2℃,符合<1.4℃的国家标准。皮内刺激试验显示,兔各时间点注射肽溶液后原发性刺激指数均为0分。遗传毒性试验显示,各实验组与阴性对照组微核数比较,差异无统计学意义(P>0.05),各实验组与阳性对照组微核数比较,差异有统计学意义(P<0.05),材料未见明显致突变性。结论新型纳米生物支架材料D-RADA16无细胞毒性、溶血作用、急性毒性、致热原性、皮肤刺激性、遗传毒性,具有良好的生物安全性和生物相容性,为材料进一步的细胞生物学研究及临床应用提供理论依据。

Objective To evaluate the biological safety of designer self-assembling peptide （SAP） nanofiber hydrogel scaffolds D-RADA16, so as to provide experimental evidence for the clinical application of the material. Methods According to Biological Evaluation of Medical Devices （GB/T 16886.1-2011/ISO 10993-1： 2009）, physiological saline lixivium of the biomaterial or the hydrogel biomaterial was used for in vitro cytotoxicity test, hemolysis test, acute toxicity test, pyrogen test, intradermal irritation test, and micronucleus test to evaluate the biocompatibility of the material. Results In the cytotoxicity test, the relative growth rate of the experimental group was above 90%, and the toxicity of D-RADA16 was graded 1, indicating no cytotoxicity. The hemolysis rate of the lixivium was 0.96%, which was lower than the national standard （〈 5%）. The activity of mice was normal in the acute toxicity test. No animal died and no toxicity symptom or adverse effect was shown within 7 d. The increase of average daily weight showed no statistically significant difference between the experimental group and the control group after 7 d （P〉 0.05）. The mice were sacrificed after 7 d, and no abnormal changes were observed in general observation and HE staining of the liver, spleen or kidneys. The maximum increase of the body temperature in the experimental group was 0.5℃, and the total increase of body temperature of the three experimental animals was 1.2℃, which met the national standard in the pyrogen test （〈 1.4℃）. Intradermal irritation test showed that the primary irritation index was 0 at every defined time point. In the micronucleus test, the results showed that there was no statistically significant difference at micronucleus number between the experimental groups and the negative control group （P 〉 0.05）, and there were statistically significant differences between the experimental groups and the positive control group （P〈 0.01）. Therefore, potential mutagenicity was not observed in the micronucleus test. Conclusions Designer SAP hydro- gel scaffolds D-RADA16 does not have cytotoxicity, hemolytic effect, acute toxicity, pyrogenicity, skin irritation or genetic toxicity. It has good biological safety and biocompatibility, therefore exhibits a promising clinical application prospect and provides theoretical basis for further research of cell biology study and clinical application.