脱细胞小牛肌腱组织形态学和生物力学特性研究  

HISTOMORPHOLOGICAL AND BIOMECHANICAL CHARACTERISTICS OF DECELLULARIZED BOVINE TENDONS

作　　者：江燕林[1] 张忆[1] 杨洁亮[2] 罗静聪[1,2] 秦廷武[1,2] 

机构地区：[1]四川大学华西医院再生医学研究中心,成都610041 [2]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2013年第5期565-570,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划(863)资助项目(2012AA020502);教育部留学回国人员科研启动基金资助项目(2010609)~~

摘　　要：目的研究反复冻融结合核酸酶处理小牛肌腱脱细胞效果以及对肌腱组织形态、结构、生化成分和力学特性的影响。方法取新鲜1日龄西门塔尔小牛跟腱48根,随机分为3组(n=16):A组为正常对照组,B组采用液氮冷冻/37℃复温反复冻融5次,C组在反复冻融基础上结合核酸酶处理24 h。每组取2根跟腱用于扫描电镜观察,3根用于、组织学及免疫组织化学染色观察,3根用于DNA残留量检测,8根用于生物力学检测。结果扫描电镜观察示A、B组胶原纤维排列致密有序,形态完整,未见断裂;C组胶原纤维排列松散,几乎无断裂。阿利新蓝、天狼星红染色及免疫组织化学染色示C组糖胺聚糖、Ⅰ型胶原、Ⅲ型胶原、纤维连接蛋白大部分被保留;HE染色和DAPI染色示A、B组胶原纤维间可见较多完整的细胞核,而C组胶原纤维之间未见完整细胞核,但腱内膜上仍有部分细胞核残留。A、B、C组的DNA残留量分别为(0.24±0.12)、(0.16±0.07)、(0.05±0.02)μg/mg,C组显著低于A、B组(P<0.05);A、B组间差异无统计学意义(P>0.05)。生物力学检测显示,A、B、C组间拉伸强度、断裂应变、刚度和弹性模量差异均无统计学意义(P>0.05)。结论反复冻融结合核酸酶处理小牛肌腱,可有效去除细胞成分,同时基本保留肌腱原始胶原纤维结构、形态、大部分细胞外基质成分和力学特性。Objective To investigate the effect of repeated freezing and thawing combining nuclease treatment on the decellularization of bovine tendons, and the morphology, structure, biochemical compositions, and mechanical properties of the decellularized tendons. Methods A total of 48 fresh 1-day-old bovine Achilles tendons were randomly divided into 3 groups （n=16）： fresh normal tendons （group A）, repeated freezing and thawing for 5 times （liquid nitrogen refrigeration/37℃ thawing, group B）, and repeated freezing and thawing combining nuclease processing for 24 hours （group C）. In each group, 2 tendons were used for scanning electron microscope （SEM）, 3 tendons for histological and immunohistochemical observations, 3 tendons for DNA content detection, and 8 tendons for biomechanical testing. Results SEM observation indicated the intact, aligned, and densely packed collagen fibers with no disruption in groups A and B, and the slightly loose collagen fibers with little disruption in group C. The alcian blue staining, sirius red staining, and immunohistochemical staining showed that the most of glycosaminoglycan, collagen type I, collagen type III, and fibronectin in group C were retained after decellularization treatment. HE and DAPI staining showed that the cell nuclei between the collagen fibers were clearly visible in groups A and B; however, the cell nuclei between collagen fibers almost were invisible with a few residual nuclei on the endotendineum in group C. DNA quantitative detection confirmed that DNA content in group C [（0.05 ± 0.02） μg/mg] was significantly lower than those in group A [（0.24 ± 0.12） μg/mg] and group B [（0.16 ± 0.07） μg/mg] （P 〈 0.05）. Biomechanical testing showed that the values of tensile strength, failure strain, stiffness, and elastic modulus were different among 3 groups, but no significant difference was found （P 〉 0.05）. Conclusion Repeated freezing and thawing combining nuclease processing can effectively remove the component of cells

关 键 词：脱细胞肌腱 形态学 组织学 生物力学 小牛 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]