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

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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.