VITRIFICATION SOLUTIONS FOR THE CRYOPRESERVATION OF TISSUE-ENGINEERED BONE

摘要

Osteoblast (OB)-seeded hydroxyapatite (HA) scaffold cortical bone substitutes are being developed at Michigan State University. Preservation methods need to be developed to preserve such living products to ensure a steady supply for transplantation. Theoretically vitrification is an attractive method for the cryopreservation of tissue-engineered bone because it can eliminate the destructive effect of ice formation. However, relatively fast cooling and warming rates are required to avoid damage associated with ice crystallization and relatively high concentrations of cryoprotective agents (CPAs) are required to achieve a glassy (vitrified) state. These rapid rates of temperature change may not be possible as tissue-engineered structures become larger. In addition to cell damage, rapid rates may also cause destructive thermo mechanical damage to the scaffold itself. Slower rates can be used to achieve the vitrified state but this requires higher CPA concentrations, which are more toxic. As a means of studying the interactive determinants of an optimal vitrification process for osteoblasts, we have undertaken thermal analysis of a variety of vitrification solutions of interest using differential scanning calorimetry (DSC) to determine the critical cooling and warming rates. The toxicity dynamics and tendency for the scaffolds to be damaged mechanically by the vitrification process are also examined. Glycerol and dimethyl sulfoxide at a concentration of 40% were studied with and without an ice blocker. Two vitrification "cocktails" (VS55 and VEG) over a concentration range of 80% to 100% were studied with and without an ice blocker. On the basis of these studies 95% VEG with ice blocker was least toxic and yielded the highest recovery (～90%) for OBs vitrified in liquid suspension. Vitrification does not seem to be detrimental to the bending strength of high density (low porosity) HA scaffolds, but lower density HA scaffolds break more easily after vitrification in some instances.