胶原蛋白海绵的制备及安全性评价  

Fabrication and safety evaluation of collagen sponge

作　　者：刘超远 高田芳 黄伟 Liu Chaoyuan;Gao Tianfang;Huang Wei(Hangzhou Singclean Medical Products Co.,Ltd.,Hangzhou 310018,Zhejiang Province,China)

机构地区：[1]杭州协合医疗用品有限公司,浙江省杭州市310018

出　　处：《中国组织工程研究》2022年第22期3460-3466,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景:自动物体提取胶原蛋白并经冻干工艺得到胶原蛋白海绵,可赋予材料良好的临床止血性能,同时可实现在机体中的自身降解,但所提取的胶原蛋白也存在携带病毒及体内免疫原性风险。经过酸酶结合工艺处理去除胶原端肽、并灭活病毒,实现材料良好的生物安全性。目的:规模化生产制备胶原蛋白海绵,并对其进行免疫原性及病毒灭活有效性的整体生物安全性评价。方法:通过酸酶结合法从牛跟腱中提取胶原蛋白,经冷冻干燥工艺得到胶原蛋白止血海绵。将1,4片胶原蛋白海绵分别埋植于Wistar大鼠腹部皮下,埋植1,4,8,12周后对大鼠体征、血常规、T淋巴细胞增殖、免疫球蛋白含量等指标进行检测。取3批次的牛跟腱粉碎后中间体样品,分别浸泡于伪狂犬病毒、水泡性口炎病毒、猪细小病毒和猪脑心肌炎病毒液中,检验酸酶结合法处理的胶原蛋白海绵对病毒灭活的有效性。结果与结论:(1)于无菌车间规模化提取胶原蛋白,经冷冻干燥后得到胶原蛋白海绵,并完成内外包装和辐照灭菌的生产全过程;(2)埋植1,4片胶原蛋白海绵大鼠的生理体征、血常规、NK细胞杀伤活性、T淋巴细胞增殖状态及免疫球蛋白含量等指标均表现正常;(3)酸酶法工艺灭活的3批次牛跟腱粉碎后中间体样品均无可以检测到的病毒,可使病毒滴度下降4个logs以上;同时,对未能检测到的病毒滴度样品进行了敏感细胞接种盲传3代,结果未显示细胞病变;(4)免疫原性和病毒灭活检测表明,所制备的胶原蛋白止血海绵有着良好的生物安全性。BACKGROUND:Collagen could be extracted from the animal,and the collagen sponge could be fabricated by vacuum freeze-drying technology,which is used in clinical hemostasis.Besides,it would be degraded in vivo.However,there are some concerns about collagen biomaterials,owing to the potential risk of carrying viruses and immune rejection.The biological safety of collagen hemostatic sponge was achieved by removing the terminated peptide and inactivating the virus.OBJECTIVE:To investigate the immunogenicity and the validity of virus inactivation of collagen hemostatic sponge in commercial process.METHODS:The collagen was extracted from bovine tendons through acid-enzymatic hydrolysis.After the vacuum freeze-drying technology,the porous collagen hemostatic sponge was prepared.The immunogenicity was investigated by insert 1 or 4 pieces of collagen sponge in Wistar rats subcutaneously.The physical sign,blood routine examination,T lymphocyte proliferation,and immunoglobulin content were examined at 1,4,8 and 12 weeks.Pseudorabies virus,vesicular stomatitis virus,porcine parvovirus and porcine encephalomyocarditis virus were chose as indicator virus to investigate the validity of virus inactivation by three batches of crushed bovine tendons.RESULTS AND CONCLUSION:(1)The collagen sponge was extracted and fabricated by freeze-drying technology in clean workshop in commercial process.The irradiation sterilization was conducted after the whole process of packaging of the collagen sponge.(2)After inserting 1 or 4 pieces of collagen sponge,the physiological signs,routine blood tests,NK cell killing activity,T lymphocyte proliferation,and immunoglobulin content of the rats were found to be normal.(3)There was no detectable virus,which could reduce the titer of these four kinds of indicator virus by more than four logs,after the acid-enzymatic process of collagen sponge.In addition,sensitive cells inoculation and blind cultivation for three generations were performed on the virus titer samples that could not be detected,and the re

关 键 词：胶原蛋白 海绵 止血材料 端肽 免疫原性 冷冻干燥 酸酶结合法 病毒灭活 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]