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

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.

取受精后第9天的鸡胚制备的离体股骨骨膜，用0.2M海藻糖、50%蛋黄、12%二甲亚砜（DNSO） （huecm AH25）冷冻保护剂进行低温保存。冷冻程序为室温至-7℃：-2.0℃/min, -7.0℃至-40℃：-1.0℃/min，转入液氮。冷冻骨膜包装在自来水（37℃）中解冻，在受精后第9天的鸡胚尿囊上培养。不加DNSO的骨膜低温保存不能成骨，低温保护中DMSO的最佳浓度为12%。添加蛋黄（50%）可提高成骨率，纯化蛋黄卵磷脂也有类似效果。电镜观察发现，解冻后表层细胞发生了变性，深层细胞保持了质膜和细胞器等细胞结构。带骨膜的股骨也以类似方式冷冻保存。培养10天后，解冻后的股骨被拉长，软骨组织增生。本研究结果表明，用DMSO作为冷冻保护剂，程序化慢速冷冻可以保存离体骨膜和带骨膜的股骨。接下来，我们将研究超高速冷冻（玻璃化）对分离骨膜和带骨膜的股骨的低温保存。

项目成果

Takahashi, M., Koyanagi, T., Takao, T., et. Al.: "The bone formation from the periosteum of chick embryonic femur cultured on egg chorioallantoic membrane."The Shikwa gakuho. 100. 1081-1089 (2000)

高桥，M.，小柳，T.，高尾，T.，等。

高橋正憲, 小柳貴裕, 高尾努, 田中洋一, 阿部伸一, 松坂賢一: "鶏卵漿尿膜上で培養された幼若骨膜からの骨形成過程の組織学的検討"歯科学報. 100. 1081-1089 (2000)

Masanori Takahashi、Takahiro Koyanagi、Tsutomu Takao、Yoichi Tanaka、Shinichi Abe、Kenichi Matsuzaka：“鸡蛋绒毛尿囊膜上培养的幼骨膜的骨形成过程的组织学研究”《牙科杂志》100。1081-1089（2000）。