脱细胞、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响  

Physical and chemical properties of porcine dermis matrix affected by process integration for decellularization,viral inactivation and sterilization

作　　者：黄森丽 孙海港 孙文全 Huang Senli;Sun Haigang;Sun Wenquan(Ruijian Gaoke Biotechnology Co.,Ltd.,Beijing 102200,China;School of Health Science and Engineering,Shanghai University for Science and Technology,Shanghai 200093,China)

机构地区：[1]北京瑞健高科生物科技有限公司,北京市102200 [2]上海理工大学健康科学与工程学院,上海市200093

出　　处：《中国组织工程研究》2023年第21期3349-3355,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景:脱细胞、去抗原、病毒灭活与终端灭菌工艺的组合优化是制备符合临床要求组织再生材料的关键技术。目的:系统比较分析两种脱细胞、去抗原、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响。方法:取新鲜猪皮,分两组工艺制备猪真皮基质:①方法A:采用1%Triton X-100、DNase和RNase、1%磷酸三丁脂进行脱细胞处理,1%过氧乙酸+25%乙醇病毒灭活,伽马射线辐照终端灭菌;②方法B:采用中性蛋白酶、0.5%Triton X-100和DNase进行脱细胞处理,α-半乳糖苷酶去除抗原,0.1%过氧乙酸病毒灭活,伽马射线辐照终端灭菌。对两种方法制备的猪真皮基质进行表征。结果与结论:①A组胶原蛋白含量低于B组,弹性蛋白、糖含量高于B组,材料硬度值高于B组;A组基质材料的悬垂性较差,B组基质材料的柔韧性较好;②A组脱细胞处理不影响基质材料的热稳定性,B组脱细胞略降低基质材料的热稳定性;伽马射线灭菌后,A组基质材料的热稳定性大幅度下降,B组基质材料的热稳定性下降很小;③与B组相比,A组基质材料的拉伸强度和弹性较小;体外酶降解实验显示,A组基质材料对胶原酶降解具有很强的抗性,对胰蛋白酶敏感、易降解,B组基质材料则相反;④扫描电镜显示,伽马射线灭菌后,A组基质材料基本看不到胶原三维结构,胶原纤维排列致密,胶原纤维之间存在非纤维结构性凝胶样物质;B组基质材料胶原纤维结构清晰,胶原纤维之间不存在非纤维结构性胶样物质。苏木精-伊红和三色染色显示,伽马射线灭菌后,A组基质材料可见少量细胞核,胶原纤维排列紧密,材料致密;B组基质材料无细胞核,保留了较好的胶原纤维三维空间结构;⑤结果表明,不同组合的制备工艺对脱细胞基质材料的理化性能影响极大,方法B在有效脱细胞和去抗原的同时较完整地保留了天然组织结构,方法A对材料的破坏性较大,制备的�BACKGROUND:Process integration of decellularization,antigen removal,viral inactivation and sterilization is essential for producing regenerative biomaterials that meet clinical requirements.OBJECTIVE:To systematically compare the effects of two combined processes of decellularization,antigen removal,virus inactivation and sterilization on the physicochemical properties of porcine dermal matrix.METHODS:Fresh porcine dermis was obtained and processed by two processes into acellular matrices.(1)Method A:1%Triton X-100,DNase and RNase,and 1%tributyl phosphate were applied for decellularization,1%peracetic acid and 25%ethanol for viral inactivation,and gamma-irradiation for terminal sterilization.(2)Method B:Dispase Ⅱ,0.5%Triton X-100 and DNase were used for decellularization,α-galactosidase for the antigen removal,0.1%peracetic acid for viral inactivation and gamma-irradiation terminal sterilization.Porcine dermal matrices prepared by two methods were characterized.RESULTS AND CONCLUSION:(1)The collagen content of group A was lower than that of group B.The contents of elastin and sugar of group A were higher than those of group B.The material hardness value of group A was higher than that of group B.The drapability of the matrix material in group A was poor and the flexibility of the matrix material in group B was better.(2)The decellularization of group A did not affect the thermal stability of the matrix material.In group B,decellularization slightly reduced the thermal stability of the matrix material.After gamma ray sterilization,the thermal stability of the matrix material in group A decreased greatly and the thermal stability of the matrix material in group B decreased very little.(3)Compared with group B,the tensile strength and elasticity of the matrix material in group A were smaller.In vitro enzymatic degradation experiments showed that the matrix materials in group A had strong resistance to collagenase degradation,were sensitive to trypsin and were easily degraded;the matrix materials in group B were t

关 键 词：猪真皮 组织再生支架 生物材料 细胞外基质 脱细胞 病毒灭活 伽马辐照 灭菌 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R-331