Jiang Yanlin, Zhang Yi, Yang Jieliang, Luo Jingcong, Qin Tingwu
Regenerative Medicine Research Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 May;27(5):565-70.
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.
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 degreeC 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.
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) micr g/mg] was significantly lower than those in group A [(0.24 +/-0.12) micro g/mg] and group B [(0.16 +/-0.07) micro g/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).
Repeated freezing and thawing combining nuclease processing can effectively remove the component of cells, and simultaneously retain the original collagen fibrous structure, morphology, most of the extracellular matrix compositions, and mechanical properties of the bovine tendons.
探讨反复冻融联合核酸酶处理对牛肌腱脱细胞效果以及脱细胞肌腱的形态、结构、生化成分和力学性能的影响。
将48条1日龄新鲜牛跟腱随机分为3组（n = 16）：新鲜正常肌腱组（A组）、反复冻融5次组（液氮冷冻/37℃解冻，B组）、反复冻融联合核酸酶处理24小时组（C组）。每组中，2条肌腱用于扫描电子显微镜（SEM）观察，3条肌腱用于组织学和免疫组织化学观察，3条肌腱用于DNA含量检测，8条肌腱用于生物力学测试。
SEM观察显示，A组和B组胶原纤维完整、排列整齐且紧密堆积，无破坏；C组胶原纤维稍疏松，破坏较少。阿尔新蓝染色、天狼星红染色和免疫组织化学染色显示，C组脱细胞处理后大部分糖胺聚糖、Ⅰ型胶原、Ⅲ型胶原和纤连蛋白得以保留。HE和DAPI染色显示，A组和B组胶原纤维间细胞核清晰可见；而C组胶原纤维间细胞核几乎不可见，仅在内膜上有少量残留细胞核。DNA定量检测证实，C组DNA含量[（0.05±0.02）μg/mg]显著低于A组[（0.24±0.12）μg/mg]和B组[（0.16±0.07）μg/mg]（P < 0.05）。生物力学测试表明，3组的拉伸强度、断裂应变、刚度和弹性模量值不同，但差异无统计学意义（P > 0.05）。
反复冻融联合核酸酶处理可有效去除细胞成分，同时保留牛肌腱的原有胶原纤维结构、形态、大部分细胞外基质成分和力学性能。
