BackgroundAs a relatively non-regenerative tissue, articular cartilage has been targeted for cryopreservation as a method of mitigating a lack of donor tissue availability for transplant surgeries. In addition, subzero storage of articular cartilage has long been used in biomedical studies using various storage temperatures. The current investigation studies the potential for freeze-thaw to affect the mechanical properties of articular cartilage through direct comparison of various subzero storage temperatures.MethodsBoth subzero storage temperature as well as freezing rate were compared using control samples (4°C) and samples stored at either -20°C or -80°C as well as samples first snap frozen in liquid nitrogen (-196°C) prior to storage at -80°C. All samples were thawed at 37.5°C to testing temperature (22°C). Complex stiffness and hysteresis characterized load resistance and damping properties using a non-destructive, low force magnitude, dynamic indentation protocol spanning a broad loading rate range to identify the dynamic viscoelastic properties of cartilage.ResultsStiffness levels remained unchanged with exposure to the various subzero temperatures. Hysteresis increased in samples snap frozen at -196°C and stored at -80°C, though remained unchanged with exposure to the other storage temperatures.ConclusionsMechanical changes shown are likely due to ice lens creation, where frost heave effects may have caused collagen damage. That storage to -20°C and -80°C did not alter the mechanical properties of articular cartilage shows that when combined with a rapid thawing protocol to 37.5°C, the tissue may successfully be stored at subzero temperatures.

A hypothesis of the nature of intracellular ice formation is proposed in which the osmotically driven water efflux that occurs in cells during freezing (caused by the increased osmotic pressure of the extracellular solution in the presence of ice) is viewed as the agent responsible for producing a rupture of the plasma membrane, thus allowing extracellular ice to propagate into the cytoplasm. This hypothesis is developed into a mathematical framework and the forces that are present during freezing are compared to the forces which are required to rupture membranes in circumstances unrelated to low temperatures. The theory is then applied to systems which have been previously studied to test implications of the theory on the nature of intracellular ice formation. The pressure that develops during freezing due to water flux is found to be sufficient to cause a rupture of the plasma membrane and the theory gives an accurate description of the phenomenology of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.

Variations of the Kelvin equation [W. Thomson, Philos. Mag. 42, 448 (1871)] to describe the freezing point depression of water in capillaries exist in the literature. The differing equations, coupled with the uncertainty in input parameters, lead to various predictions. The difference between the predictions may become substantial when the capillary size decreases much below micron dimensions. An experiment was designed to investigate the predicted values using a customized directional solidification stage. The capillary freezing point depression for glass tubes with radii of 87 microm-3 microm was successfully measured. The image of the ice-water interface at equilibrium was also digitally captured and analyzed to examine the contact angle and the interface shape as well. Both are important for examining the hemispherical interface assumption that was exclusively used in the theoretical derivations. Finally, an equilibrium analysis of the thermodynamic system leads to a theoretical discussion of the problem. The effect of the temperature gradient on the interface shape is addressed, and an engineering criterion for the critical temperature gradient above which the effect must be considered for the interface shape calculation is derived.

A time dependent three-dimensional finite difference model of iceball formation about multiple cryoprobes has been developed and compared to experimental data. Realistic three-dimensional probe geometry is specified and the number of cryoprobes, the cryoprobe cooling rates, and the locations of the probes are arbitrary inputs by the user. The simulation accounts for observed longitudinal thermal gradients along the cryoprobe tips. Thermal histories for several points around commercially available cryoprobes have been predicted within experimental error for one, three, and five probe configurations. The simulation can be used to generate isotherms within the iceball at arbitrary times. Volumes enclosed by the iceball and any isotherms may also be computed to give the ablative ratio, a measure of the iceball's killing efficiency. This ratio was calculated as the volume enclosed by a critical isotherm divided by the total volume of the iceball for assumed critical temperatures of -20 and -40 degrees C. The ablative ratio for a single probe is a continuously decreasing function of time but when multiple probe configurations are used the ablative ratio increases to a maximum and then essentially plateaus. Maximum values of 0.44 and 0.55 were observed for three and five probe configurations, respectively, with an assumed critical temperature of -20 degrees C. Assuming a critical temperature of -40 degrees C, maximum ablative ratios of 0.21 and 0.3 for three and five probe configurations, respectively, were observed.