Hayashi M, Tsuchiya H, Otoi T, Agung B, Yamamoto N, Tomita K
Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, Takara-Machi, Kanazawa, Japan.
Cryobiology. 2009 Aug;59(1):28-35. doi: 10.1016/j.cryobiol.2009.04.002. Epub 2009 Apr 9.
Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.
提高肉瘤患者的生存率使得肿瘤切除术后需要更具功能性和持久性的重建方法。冷冻骨关节移植物用于关节重建，但关节可能会发生骨关节炎变化。我们使用冷冻自体全兔膝关节移植模型来研究冷冻对关节组件的影响。将30个未经使用冷冻保护剂直接浸入液氮（L组）或生理盐水（C组）的兔膝关节重新植入。在4周、8周和12周后进行组织学观察。两组均有骨愈合。在L组中，尽管半月板和韧带恢复了细胞活性，但未观察到存活的软骨细胞，且软骨退变随时间进展。得出结论，软骨细胞的冷冻损伤导致了骨关节炎变化。然后我们测试了玻璃化方法是否能保护软骨免受冷冻损伤。将兔膝关节的全层关节软骨浸入有和没有玻璃化的液氮中。在培养24小时前后检查组织学、超微结构和软骨细胞活力。与新鲜软骨相比，玻璃化软骨细胞活力>85%。透射电子显微镜显示原始软骨细胞结构得以保留。我们的玻璃化方法对于保护软骨细胞免受冷冻损伤是有效的。由于用含有活软骨的骨关节移植物重建关节可避免骨关节炎变化，玻璃化方法可能有助于异体移植物活软骨的储存，或者在亚洲国家，用于含有肿瘤的冷冻自体移植物的重建。
