Experimental Study of Cryopreserved Allogenic Ear Cartilage

作者建立了一种长期保存的冷冻保存方法，并发表了大鼠、猪和猴同种异体皮肤、神经和血管移植的几篇报道。本文在前人实验的基础上，对兔耳软骨的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周龄雄性日本大耳白兔(2.0~2.5 kg)。在戊巴比妥钠静脉麻醉下，在耳根部切取一块带耳中血管的耳软骨。经耳中动脉注射甘油(1.4mol)进行冷冻保护。12只兔随机分为3组(冻存组、直接冷冻组和对照组)。超低温冻存组：取3×1 cm的耳软骨标本，分步冷冻至-70℃，在液氮储罐中-196℃保存数周以上。解冻过程包括快速分辨…至20℃，然后在4℃的生理盐水中浸泡1小时。直接冻结组：在没有任何冷冻保护的情况下，快速冷冻软骨标本，然后在-196℃下保存3周。利用光镜和电子显微镜进行了组织学研究。在软骨膜细胞、早熟软骨细胞和成熟期软骨细胞组成的三个领域进行活细胞计数，并与新鲜耳软骨作为对照组进行比较。实验二：将80个冷冻保存的兔耳软骨标本移植到不同兔的股筋膜上方作为异体移植。分别于术后1、2、4、6、8、12、16、20、21、28、31、32、46、52、55周取材进行光镜和电子显微镜观察。结果：实验1：超低温冻存组软骨细胞具有良好的细胞层次结构。实验2：组织学结果显示，冷冻异体移植组细胞浸润最少，与新鲜自体移植组相似，新鲜异体移植组细胞浸润明显。观察三组不同时期软骨细胞的形成情况。冷冻保存的同种异体软骨细胞数量下降非常缓慢，新鲜异体软骨细胞的数量下降相对较快。结论：作者的冷冻保存技术证明了该技术对于长期保存存活软骨细胞的通用性，尽管移植后的软骨细胞没有活跃的细胞渗透，但活细胞数量的下降明显缓慢。异体耳廓移植在临床上具有很大的应用潜力。较少

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