Experimental Study of Cryopreserved Allogenic Ear Cartilage

冷冻保存同种异体耳软骨的实验研究

• 批准号: 09671259
• 负责人: MIYAWAKI Takeshi
• 金额: $ 0.64万
• 依托单位: JIKEI UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671259/
• 关键词: cryopreservation; allograft; ear cartilage graft; cartilage preservation; 冷凍保存

The authors have established a cryopreservation method of long-term preservation nd published several reports on allogenic transfers of skin, nerves and vessels in rats, pigs and monkeys. Herein is reported a study of long-term storage and allogenic transplantation of the ear cartilage of rabbits which was based on previous ezperimental data.Materials and Methods : Experiment 1 ; Male 8-week-old Japanese white rabbits(2.0-2.5kg) were used. Under anesthesia by intravinous administration of pentobalbital sodium, a piece of ear cartilage with midauricular vessels was harvested at the base of an ear. Glycerol(1.4 mol)was injected through the midauricular artery for cryoprotection. Twelve rabbits were divided into three(cryopreservation, direct freezing and control)groups. In the cryopreservation group, 3×1 cm ear cartilage specimens were harvested, frozen stepwise to -70℃, and stored at -196℃ in a liquid nitrogen tank for more than weeks. The thawing process consisted of a rapid resolution … More to 20℃, followed by soaking in physiological saline at 4℃ for one hour. In the direct freezing group, each cartilage specimen was frozen very rapidly without any cryoprotection and then stored at -196℃ for three weeks. Histrogical study was conducted utilizing light and electron microscopy. Viable cells were counted in three fields consisted of perichondral cells and premature and cature chondrocytes and this was compared with fresh ear cartilages serving as a control group. Experiment 2 ; Eighty cryopreserved ear cartilage specimens were transplanted above the thigh fascia of different rabbits as allogenic transfers. At 1, 2, 4, 6, 8, 12, 16, 20, 21, 28, 31, 32, 46, 52, and 55 weeks after surgery, the grafted cartilage were harvested for histological examinations performed by light and electron microscopy. Viable cells were counted in the manner described in experiment 1 and compared with fresh auto and allografts.Results : Experiment 1 ; Chondrocytes in the cryopreserved group revealed a well-preserved cellular level structure. In constant, the freezing group demonstrated collapse and destruction of the cellular matrix.Experiment 2 ; Histological findings indicated cellular infiltration was minimal in the cryoallograft group, similar to the fresh autograft group, and marked in the fresh allograft group. Chondrocytegenesis was observed in three groups of different periods. There was a very slow decrease in the number of viable cells in the cryopreserved allograft group, and a relatively rapid decrease in the fresh allograft.Conclusion : The authors' cryopreservation technique proved its versatility for long-term storage of viable chondrocytes, and a significantly slow decrease in viable cell count was observed without active cellular infiltration despite alligenic transfers. It has great potential for allogenic transfer of ear catilage at clinics. Less

作者建立了一种长期保存的低温保存方法，并发表了大鼠、猪和猴皮肤、神经和血管的同种异体移植的报道。本文在前人实验基础上，对兔耳软骨长期保存和同种异体移植进行了研究，材料和方法：实验一：选用8周龄雄性日本白色家兔，体重2.0-2.5kg。在静脉注射戊巴比妥钠麻醉下，在耳根部切取一块带有耳中血管的耳软骨。通过耳中动脉注射甘油（1.4 mol）进行冷冻保护。将12只家兔分为3组（冷冻组、直接冷冻组和对照组）。冻存组取3× 1cm耳软骨标本，逐步冻存至-70℃，置于-196℃液氮罐中保存周以上。解冻过程包括快速分解 ...更多信息 20℃，然后在4℃的生理盐水中浸泡1小时。在直接冷冻组中，每个软骨标本在没有任何冷冻保护的情况下非常快速地冷冻，然后在-196℃下保存3周。利用光学和电子显微镜进行组织学研究。活细胞计数在三个领域组成的软骨膜细胞和未成熟和成熟的软骨细胞，并与新鲜的耳软骨作为对照组进行比较。实验二：将80例冷冻保存的兔耳软骨标本作为同种异体移植物移植于不同兔股筋膜上。分别于术后1、2、4、6、8、12、16、20、21、28、31、32、46、52、55周取移植软骨行光镜和电镜检查。活细胞计数在实验1中描述的方式，并与新鲜的自体和allografts.Results：实验1：冷冻保存组的软骨细胞显示了保存良好的细胞水平的结构。在常数，冷冻组表现出崩溃和破坏的细胞matrix.Experiment 2 ;组织学结果表明，细胞浸润是最小的cryoallograft组，类似于新鲜自体移植组，并在新鲜异体移植组显着。在三组不同时期均观察到软骨细胞发生。有一个非常缓慢的减少，在冷冻保存的同种异体移植物组中的活细胞的数量，并在新鲜allograft.Conclusion：作者的冷冻保存技术证明了其多功能性的长期存储的活软骨细胞，并观察到一个显着缓慢的减少，活细胞计数没有活跃的细胞浸润，尽管alligenic转让。同种异体耳廓移植在临床上有很大的应用潜力。少