超临界二氧化碳萃取用于异种脱细胞骨修复材料的制备  

Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique

作　　者：郝峰 泮凯锋 黄柳云 陈叙宏 尉海坤 陈献花 张建锋[2] HAO Feng;PAN Kaifeng;HUANG Liuyun;CHEN Xuhong;WEI Haikun;CHEN Xianhua;ZHANG Jianfeng(Zhejiang Decellmatrix Biotech Co.,Ltd.,Hangzhou 310018,China;Department of Orthopedic Surgery,Sir Run Run Shaw Hospital,Zhejiang University School of Medicine,Hangzhou 310016,China;Zhejiang Institute of Medical Device Testing,Hangzhou 310018,China)

机构地区：[1]浙江狄赛生物科技有限公司,浙江杭州310018 [2]浙江大学医学院附属邵逸夫医院骨科,浙江杭州310016 [3]浙江省医疗器械检验研究院,浙江杭州310018

出　　处：《浙江大学学报(医学版)》2024年第6期772-778,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省“尖兵”“领雁”研发攻关计划(2022C03089)。

摘　　要：目的:评价采用超临界二氧化碳脱细胞后动物源性骨修复材料的免疫原性和成骨能力。方法:将猪股骨前处理后随机分成两组,分别使用常规方法(常规对照组)和超临界二氧化碳(实验组)进行脱细胞处理,并以同种脱钙骨基质作为阳性对照。通过酶联免疫吸附试验和荧光染色法检测半乳糖-α-1,3-半乳糖(α-Gal)抗原清除率和DNA残留量。9只6周龄SPF级雄性裸鼠随机分成实验组、常规对照组和阳性对照组,在裸鼠股二头肌肌间隙中植入不同骨组织,术后4周取材,分别用苏木精-伊红(HE)染色和免疫组织化学检测植入部位诱导成骨能力及成骨相关蛋白表达。结果:实验组和常规对照组α-Gal抗原清除率分别为(99.09±0.26)%和(30.18±2.02)%,差异有统计学意义(t=58.67,P<0.01)。实验组、常规对照组和阳性对照组的DNA残留量分别为(13.49±0.07)、(15.20±0.21)和(14.70±0.17)ng/mg,其中实验组DNA残留量低于常规对照组(t=-13.41,P<0.01)和阳性对照组(t=-11.30,P<0.01)。HE染色结果显示,实验组术后4周植骨部位有多个骨生发中心伴成骨活跃、骨髓丰富;常规对照组和阳性对照组仅有少量骨生发中心,无明显的成骨细胞。免疫组织化学结果提示,实验组成骨相关蛋白碱性磷酸酶、Runt相关转录因子2、Ⅰ型胶原蛋白和骨钙蛋白的表达量较常规对照组和阳性对照组均有增加的趋势。结论:经超临界二氧化碳脱细胞处理的骨组织比临床上常用的同种脱钙骨基质和常规方法处理的骨修复材料具有更低的免疫原性和更好的诱导成骨能力。Objective:To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide.Methods:Porcine femurs were randomly divided into two groups after preliminary treatment,and decellularized with conventional method(control group)or supercritical carbon dioxide(experimental group).Allogenic demineralized bone matrix was used as positive control.Clearance rate of galactose-α-1,3-galactose(α-Gal)antigen was determined by enzyme-linked immunosorbent assay and residual DNA was detected by a fluorescence method.Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental,control and positive control groups.Samples were implanted over biceps femoris muscle of athymic nude mice.The explants were collected 4 weeks post implantation.Hematoxylin and eosin(HE)staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants.Results:The clearance rates ofα-Gal antigen in the experimental group and the control group were(99.09±0.26)%and(30.18±2.02)%,respectively(t=58.67,P<0.01).The residual DNA of the experimental,control and positive control groups were(13.49±0.07),(15.20±0.21)and(14.70±0.17)ng/mg.The residual DNA in the experimental group was significantly lower than that in the control group(t=-13.41,P<0.01)and positive control group(t=-11.30,P<0.01).HE staining results showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation,but only a small number of bone formation centers were observed in the control and positive control groups,with no obvious osteoblasts present.Immunohistochemistry results indicated that the expressions of alkaline phosphatase,Runt-related transcription factor 2,collagen typeⅠand osteocalcin in the experimental group showed an increasing trend compared with those in the control and positive control groups.Conclusion:Comp

关 键 词：骨修复 异种移植 脱细胞支架 超临界二氧化碳萃取 小鼠 

分 类 号：R683[医药卫生—骨科学] R318.08[医药卫生—外科学]