Investigation Of Cryoprotective Agents For Vitrification Preservation Of Osteoblast

Bone grafts have been often used for reconstruction of bone defects caused by trauma, cyst, benign and malignant tumors, and congenital deformities. The problems of autografts limited by autograft quantity and donor site morbidity can be resolved by a promising alternative, tissue engineered bone. One major such obstacle is the issue of preservation and storage of living biomaterials to clinicians. Comparing with traditional slow-rate freezing, a new method of cryopreservation called vitrification, of which manipulation is simpler and less damage to cells and tissues on account of intracellular and extracellular ice-free during the freezing process. However, high concentration of cryoprotective agents (CPAs) necessary for vitrification can cause an osmotic shock and toxicity to cells. Up to now, many kinds of cells have been vitrified successfully , however , vitrification of osteoblasts and bone have not been reported in literature.The objective of this research is to find an appropriate CPA for the vitrification preservation of osteoblasts and bone tissues.The first step is to select vitrifying concentrations of three permeable CPAs: dimethyl sulfoxide (DMSO 45%w/w) , 2,3-butanediol (BD 32%w/w) and 1,2-propanediol (PD 45%w/w) , and verify their vitrifying properties with cryomicroscopic system and differential scanning calorimetry (DSC). Substituting sugars for a part of permeable CPAs is an alternative in order to reduce the overall toxicity of the cryopreservation solution, while retaining the required vitrifying properties. The results show that the vitrification concentration of BD in the present experiment is higher than that reported in literature and the mixtures combined with sugars retain the similar vitrification properties. Fast-rate freezing can prevent ice formation. Fracture temperature is lower than glass transition temperature and cells can be preserved between fracture temperature and glass transition temperature.Osteoblasts are exposed to vitrifying concentration of different CPAs at 0-4℃ for 5 min and after removal of the CPAs at -20℃, the effect of such solutions on cells are evaluation using a cell culture assay--MTT. The results show that there is measurable loss of cells after exposure to three permeable CPAs. Treatment with DMSO and sugars is less damaging, but PD and sugars is not significantly better.The numerical simulation is performed using the K-K model to predict the volume change for osteoblasts during CPA addition. The results show that the volume change increases with the increasing concentration of CPAs for one-step CPA addition. Comparing with one-step CPA addition, step-wise CPA addition can significantly decrease the volume change. Maximum volume excursion duringone-step PD addition is higher than that during one-step DMSO addition.