反复冻融处理同种异体移植兔跟腱组织学及生物力学变化

Effects of repetitive freeze-thawing on histology and biomechanics of rabbit Achilles tendon following allograft

背景:近年来反复冻融处理技术作为一种同种异体肌腱的保存处理方法,主要原理是反复利用0～-60℃腱细胞最易损伤的冷冻温度,使得肌腱内细胞无活性,从而降低免疫原性。目的:拟观察反复冻融处理对同种异体移植兔跟腱组织学和生物力学的影响。设计、时间及地点:组织形态学及生物力学水平的对照动物实验,于2007-10/2008-02在北京大学运动医学研究所实验室完成。材料:选用17只成年健康新西兰大白兔。方法:无菌条件下取兔跟腱并带0.6cm×0.4cm跟骨骨块,按照组织库程序行60Co照射68min,照射剂量为25kGy。分为3组,-80℃冷冻,室温融解,反复冻融次数分别是1,3和10次,每组又抽取7条肌腱作生物力学测定,4条作组织学观察。主要观察指标:取肌腱行苏木精-伊红染色、胶原VanGieson染色和透射电镜扫描,观察跟腱组织形态学改变,并进行跟腱拉伸实验以观察生物力学变化。结果:组织学观察发现,随着反复冻融次数的增加,腱束和胶原纤维排列变得混乱直至断裂,细胞崩解,并且由于冰晶形成造成明显的腱束间裂隙。生物力学方面,与冻融1次组比较,冻融3,10次组的最大载荷、最大载荷能量及最大应力均显著降低(P<0.05),其他指标如弹性模量、能量密度也有下降但不明显。结论:反复冻融处理肌腱是一种有创的处理方法,正如组织学表现的可以引起细胞崩解、腱纤维紊乱甚至断裂等,最终会导致其生物力学水平的下降。

BACKGROUND： Recently, repetitive freeze-thawing treatment of tendon is used to deactivate cells and reduce immunogenicity. OBJECTIVE： To observe the effects of repetitive freeze-thawing treatment before allograft tendon transplantation on histology and biomechanics of rabbit Achilles tendon. DESIGN, TIME AND SEI-FING： Controlled animal experiment of histology and biomechanics was performed at the laboratory of Institute of Sports Medicine, Peking University from October 2007 to February 2008. MATERIALS： A total of 17 adult and healthy New Zealand White rabbits were selected. METHODS： Rabbit Achilles tendons were harvested with calcaneus bone block, 0.6 cm × 0.4 cm, and exposed to ^60Co irradiation for 68 minutes at 25 kGy. The tendons were divided into three groups, stored at -80 ℃, and thawed for 1, 3 and 10 times. In addition, seven tendons were selected from each group for biomechanical determination, and 4 tendons for histological observation. MAIN OUTCOME MEASURES： HE staining, Van Gieson staining and transmission electron microscopy were used to observe tendon histological alterations. Tension test was performed to observe the biomechanical changes. RESULTS： Histological observation showed that with increasing freeze-thawing, the ranks of tendon bundles and collagen fibrils become disordered until ruptured; cell disrupted, and apparent tendon bundle gaps were caused by ice crystal formation. Biomechanics results showed that the values of maximum load, maximum load energy, and maximum stress were significantly decreased with increasing freeze-thawing times （P 〈 0.05）, while elastic modulus and energy density were slightly decreased. CONCLUSION： Repetitive freeze-thawing has apparent histological and biomechanical effects on rabbit Achilles tendon before allograft tendon transplantation, such as cell disruption, collagen fibrils disorder, ultimately biomechanical reduction.