Transport Phenomena For The Loading And Removing Processes Of CPA During Cryopreservation Of Bone Tissue

Cryopreservation is a technique by which cells and tissues can be long-term preserved. With the more rapid development of medical science and the more urgent demand of clinic treatment, the cryopreservation of engineered tissue and live organ becomes the most advanced and promising research field of biomedical engineering. The data shows that around the world, the number of bone transplant operation is the second large one only less than blood transfusion. It will facilitate the clinic therapy and lessen the patients' pain if the autograft and heterograft of bone used for large-area bone loss can be successfully cryopreserved. Therefore the cryopreservation of bone tissue has a promising future and an extensive practical value.At the beginning of the research of this thesis, osteoblasts and bone tissue is cultured in-vitro and the main techniques used in the experiment are cross-checked. By cross-checking the concentration of cryoprotective agent (CPA) and the viability of cells within bone tissue, we conclude that osmotic pressure technique and Typan blue stain method can precisely reflect two major parameters above.Meanwhile, the loading and removing process of cryopreservation are studied by choosing three different concentrations of two CPAs. Type and the concentration of CPA, operating temperature, loading and removing protocol and post-culture is set as parameters to check their effects on the characteristics of the whole process. Results show that the osmosis damage and the toxicity damage are two major factors for the loss of cells' viability, that continuous loading and removing is better than intermittent ones, that glycerol's osmosis ability is worse than DMSO, but its toxicity is less than DMSO and that the effect of temperature change on the glycerol's osmosis ability is much more than on DMSO's, but its effect on DMSO's toxicity is much more than on glycerol's.Finally, osteoblasts, the major cells in bone tissue, are freezed and thawed. By using three kinds of CPAs and changing the cooling rate and thawing rate, we conclude that for osteoblasts' cryopreservation, vitrification overmatches the crystallization. Non-vitrified CPA can partially vitrify, but can not show the superiority of vitrification, that with the increase of the cooling rate, vitrified CPA's ability to vitrify shows more greatly, that for two kinds of vitrified CPA studied in this experiment, VS1 is superior to 7mol/L DMSO in almost all aspects except the ability to overcome the devitrification, that the phenomena of the devitrification is closely related to the ice crystal appeared while freezing and the cryo-fragmentation and that the devitrification is blocked and finally prevented with the increasing of the thawing rate.