OSTEOGENESIS FROM CRYOPRESERVED PERIOSTEUM- basic studies for tissue and organ cryopreservation

冷冻骨膜成骨——组织和器官冷冻保存的基础研究

• 批准号: 12671441
• 负责人: TAKAHASHI Masanori
• 金额: $ 1.92万
• 依托单位: TOKYO DENTAL COLLEGE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671441/
• 关键词: cryopreservation; periosteum; dimethyl sulfoxide; program freezing; electron microscopy; egg yolk; cryopreservation; periosteum; dimethyl sulfoxide; program freezing; electron microscopy; egg yolk

The isolated periosteum from Femur, which prepared from chick embryo of 9th day after fertilization, was cryopeserved with a cryoprotectant (0.2M treharose, 50% egg yolk, 12% dimethyl sulfoxide (DNSO) in huecm AH25). The following program of freezing was employed room temperaturo to -7℃ : -2.0s℃/min, -7.0℃ to -40℃ : -1.0℃/min, transfer in liquid nitrogen. The package of cryopresorved periosteum was thawed in tap water (37℃), and cultured on allantoic sac of chick embryo of 9th day after fertilization.The periosteum cryopreseved without DNSO gave no osteogenesis, and the optimum concentration of DMSO was found to be 12% DMSO in the cryoprotectat. Addition of egg yolk (50%) improved the rate of osteogenesis, and similar effect was observed by using purified egg yolk lecithin. Electron microscopic observation suggested that surface cell layer were denatured after thawing, whereas those of deep layers were kept their cell structures such as plasma membrane and organolas.The femur with periosteum was also cryopreserved in the similar manners. After 10 days culture, the thawed femur was elongated, and hypertrophic cartilaginous tissue was also observed.The present results suggested that the isolated periosteum as well as the femur with periosteum were able to preseved with DMSO as cryoprotectant and progarmmed slow rate freezing.We next would like to examine ultra rapid freezing (vitrification) for cryopreservation of the isolated periosteum as well as the femur with periosteum.

股骨的分离的骨膜是由受精后第9天的雏鸡胚胎制备的，在HUECM AH25中用冷冻凝集剂（0.2m的treharose，50％蛋黄，12％二甲基亚氧化物（DNSO））进行冷冻保存。以下冻结程序用室温为-7℃：-2.0S℃/min，-7.0℃至-40℃：-1.0℃/min，在液氮中转移。施肥后第9天的小鸡胚胎囊囊中培养了冷冻修复的骨膜包装。没有DNSO的骨膜冷冻定剂没有成骨，并且在冷冻保护物中发现最佳DMSO浓度为12％DMS​​O。蛋黄的添加（50％）提高了成骨的速率，并使用纯化的蛋黄卵磷脂观察到了相似的作用。电子显微镜观察表明，解冻后表面细胞层是变性的，而深层的细胞结构将其细胞结构（例如质膜和有机果）保持在类似的pryperem中。 After 10 days culture, the thawed femur was elongated, and hypertrophic cartilaginous tissue was also observed.The present results suggested that the isolated periosteum as well as the femur with periosteum were able to preseved with DMSO as cryopretectant and Progarmmed slow rate freezing.We next would like to examine ultra rapid freezing (vitrification) for cryopreservation of the isolated periosteum as well as the femur与骨膜。