CPA Loading Process For Engineering Bone Cryopreservation

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 clinictreatment, the cryopreservation of engineering tissues and live organs becomes the mostpriority and promising research field of biomedical engineering. The statistical data show thataround the world, the number of bone transplant operation is the second largest one only lessthan blood transfusion. It will facilitate the clinic therapy and lessen the patients' pain if theengineering bone can be successfully cryopresered. Therefore the cryopreservation ofengineering bone has a promising future and an extensive practical value.Firstly, cells from cranium of Sprague-Dawley (SD) rats were used for primary andpassaged cultures. The biological morphology and properties were assayed, and the culturedcells were purified and determined to be osteoblasts without pollution of other cells. Theosteoblasts could provide sufficient seed cells for the further researches and ensure theaccuracy and repetition of the experiments.Micro-photography technique was employed to measure of the cell volume excursion.Osmotically inactive volume of SD rat osteoblasts was determined to be 0.261V₀. And thenosteoblast membrane permeability parameters for K-K model of DMSO was determined: L_p=1.8×10^-13m³Â·N^-1·s^-1,ω=8.5×10^-11mol·N^-1·s^-1,σ=1.5.Using the K-K model's permeability parameters in the present paper, the process ofDMSO addition to osteoblast can be simulated and optimized. The results indicate that forhigh concentration CPA, stepwise addition and continuous-gradient addition are much betterthan one-step addition due to the smaller injuries caused by both the volume change and theosomosity oscillation during the cryopreservative agent loading. Four-step addition is goodamong other stepwise additions. For continuous-gradient addition, linear and S-mould loadingprotocols were preferred.Finally, the FLUENT software was used to study the velocity and force distributionswith in the scaffold of simulated engineering bone. A optimal perfusion mode at the middle ofperfusion chamber with small mechanical damage to cells in the scaffold is presented.