Study On The Devitrification And Recrystallization In Nano-cryoprotectant Solutions

Vitrification is considered as the most feasible method for successful cryopreservation of cells, tissues and organs because of the ice-induced damages during freezing are totally avoided. Equally important as the induction of the vitreous state during cooling is to maintain a noncrystalline state during the subsequent warming, that is, to prevent devitrification and recrystallization during warming.Nanoparticles in solution offer unique electrical, mechanical and thermal properties due to their physical presence and interaction with the state of dispersion. The purpose of this paper is to study the devitrification and recrystallization behavior of two commonly used cryoprotectants namely glycerol(60%,w/w) and poly(ethylene glycol) PEG-600(50%,w/w) solutions with and without Hydroxyapatite(HA) nanoparticles, and to investigate the effect of nanoparticles on ice crystal characteristics of aqueous solutions during warming, as follows:1. Aqueous PEG-600 solution with 60 nm, 0.5%(w/w)HA nanoparticles was prepared by using ultrasonic oscillation method and the stability and uniformity of nanoparticle-containing suspensions were evaluated by using spectrophotometer. The results showed that the ultrasonic power and oscillation time are the main factors to influence stability of suspensions. When the power of ultrasound is 200 W and the oscillation time is 3 hours, the well-distributed and stable nano-fluid could be got to ensure that the agglomeration and sedimentation would not happen in 12 hours.2. The devitrification and recrystallization events of PEG-600(50%,w/w) solutions with HA nanoparticles of different particle sizes(20nm、40nm、60nm)and concentrations(0.1%、0.2%、0.4%、0.8%, w/w)were studied by cryomicroscope coupled with calorimetric analysis. Experimental results showed that the devitrification temperature of PEG-600 solution containing 40 nm 0.4% HA increased by 7℃. The temperature ranges of ice-crystal growth for PEG-600 solution containing 20 nm 0.4% and 40 nm 0.8% HA nanoparticles increased by 35% and decreased by 50% respectively. The morphology of ice crystal in PEG-600 solutions with HA nanoparticles changed remarkably.3. The freezing and thawing of glycerol solutions(40%,w/w) with HA nanoparticles of different particle sizes(20nm 、 40 nm 、 60nm) and concentration(0.5%,w/w) were observed under a cryomicroscope at different cooling and warming rates(5℃/min、10℃/min、15℃/min). Experimental results showed that the nucleation temperatures of glycerol solutions containing 60 nm HA decreased from-40℃ to-50℃ during the cooling, and the incipient melting temperatures of glycerol solutions containing 20 nm HA increased from-70℃ to-60℃ during the warming, with the speed-up of temperature change. The ice habits of glycerol solution containing 40 nm HA at the velocity of 10 ℃ /min is different remarkably. The glycerol solution containing 60 nm HA has no precipitation of solute molecules when comparing with the others.4. The effects of hydroxyapatite(HA) nanoparticles of different sizes(20nm, 40 nm, 60nm)and concentrations(0.1%, 0.5%, w/w)on the behaviors of devitrification and recrystallization of glycerol(60% w/w) and PEG-600(50% w/w) solutions during warming were studied by calorimetric analysis coupled with cryomicroscopy. Results showed that the presence of HA nanoparticles had little effect on the devitrification end temperatures, but affected the devitrification onset temperatures of glycerol and PEG-600 solutions remarkably. The investigation with the cryomicroscope observed that the ice morphologies of glycerol and PEG-600 solutions are dendritic and spheric respectively. The ice fraction of glycerol solution containing 60 nm 0.1% HA decreased to 2/5 of that of solution without nanoparticles at-45°C. The ice fractions of PEG-600 solutions increased significantly between-64?C and-54?C, and the ice fraction of PEG-600 solution without nanoparticles increased from 5% to 97% within the temperature range.5. The effects of hydroxyapatite(HA) nanoparticles on the thermo-physical properties and ice growth rate of glycerol(60% w/w) and PEG-600(50% w/w) solutions during warming were studied further by using differential scanning calorimeter(DSC) and cryomicroscopy. Experimental results showed that the glass transition temperatures and melting temperatures of glycerol and PEG-600 solutions did not change when adding nanoparticles. The devitrification and melting enthalpy changed remarkably in glycerol solutions, but changed little in PEG-600 solutions, with the introduction of nanoparticles. Ice growth rate has two maximum, there is only one maximum in glycerol solution containing 40 nm 0.5% HA, PEG-600 solutions with 60 nm HA, and PEG-600 solution without nanoparticles.These findings have significant implications for biomaterial cryopreservation, cryosurgery, and food manufacturing. The complexity of ice crystal growth kinetics in nanoparticle-containing dispersions remains to be poorly understood at the moment, further studies are needed to solve this problem. Results of the present study will be a basis regarding the application of nanoparticles in cryogenics.