Response of Murine Osteoblasts and Porous Hydroxyapatite Scaffolds to Two-Step, Slow Freezing and Vitrification Processes

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Abstract
The emergence of tissue engineering has created the need for cryopreservation processes to store tissue-engineered products. This paper describes the response of murine osteoblasts (OBs) to slow freezing and vitrification processes as a first step towards the rational design of optimal cryopreservation of OB-seeded hydroxyapatite (HA) scaffold cortical bone substitutes. The results show that exposure time has little effect on the survival of cells for low cryoprotective agent (CPA) concentrations [5% or 10% (vol/vol)] for dimethyl sulfoxide (DMSO) and 1,2-propanediol. Survival decreased significantly as the concentration of these CPAs was increased to 20% and 30%, especially for 1,2-propanediol. For a "two-step" freezing process, high survival (approximately 85-90%) was obtained with 5% and 10% DMSO and 1,2-propanediol solutions. Survival was significantly lower (approximately 10-60%) for higher concentration solutions. For vitrification, 40% DMSO or glycerol solutions produced high survival (78% and 79%, respectively). Lower CPA concentrations (30%) resulted in devitrification during warming, whereas higher concentrations (50% or 60%) were more toxic. High survival (approximately 83%) was also obtained using the vitrification solution VS55. Preliminary results reveal that low density HA scaffolds are damaged when cryopreserved by either two-step freezing or vitrification.