Freeze-thaw treatment effects on the dynamic mechanical properties of articular cartilage.

BACKGROUND: As 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. METHODS: Both 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. RESULTS: Stiffness 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. CONCLUSIONS: Mechanical 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.

The possible influence of osmotic poration on cell membrane water permeability.

It has been hypothesized that pores in the plasma membrane form under conditions of rapid water efflux, allowing extracellular ice to grow into the cytoplasm under conditions of rapid freezing. When cells with intracellular ice are thawed slowly, the transmembrane ice crystal expands through recrystallization causing the cell to lyse. One of the implications of this hypothesis is that osmotic pores will provide an alternative route for water movement under conditions of osmotically induced flow. We show that the plasma membrane water permeability of a fibroblast cell changes as a function of the osmotic pressure gradient that is used to drive water movement. It is further shown that cell volume is more important than the magnitude of water flux in causing this departure from a uniform water permeability. We suggest that these data provide evidence of a transient route for water movement across cell membranes.

The salting-in hypothesis of post-hypertonic lysis.

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done. A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are "salted in" to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then "salted out" of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze-thaw injury in human red blood cells.

Osmoregulatory function of large vacuoles found in notochordal cells of the intervertebral disc running title: an osmoregulatory vacuole.

The nucleus pulposi of many species contain residual cells from the embryonic notochord, which exhibit a very unusual appearance (large vacuoles occupying approximately 80% of the cell volume, surrounded by an actin cytoskeleton). While the vacuoles have been qualitatively described, their composition and function has remained elusive. Given that these cells are believed to generate and experience significant osmotic pressures in both the notochord and intervertebral disc, we hypothesized that the vacuoles may serve as osmoregulatory organelles. Using both experimental and theoretical means, we demonstrated that the vacuoles contain a low-osmolality solution, generated via ion pumps on the vacuolar membrane. During hypotonic stress the vacuoles release their contents into the cytoplasm, diluting the cytoplasm and restoring the osmotic balance across the cell membrane. Thus the vacuoles function to regulate the cell volume and tonicity during rapid osmotic stress, protecting the cells from potentially damaging swelling pressures.

Retardation of ice growth in glass capillaries: measurement of the critical capillary radius.

An experiment was designed to compare the freezing of an aqueous solution in glass microcapillaries and in thin films. The velocity dependence of the ice front propagation in glass capillaries with radii of 87.5 microm-1.5 microm was observed. A critical capillary radius r(0), corresponding to certain thermal conditions, was obtained, below which the ice growth inside the capillaries was retarded. This critical capillary radius is further related to lambda(0), the smallest wavelength used in the Mullins-Sekerka criterion for the instability analysis of bulk solidifications [Mullins and Sekerka, J. Appl. Phys. 35, 444 (1964)]. It was found that for the present hypothesis, r(0)=lambda(0)/4 gives good predictions. The relation between the propagation velocity (or cooling rate) and the critical radius (or pore size) is summarized in a chart for applications in capillary-porous media, such as in the freezing of biological tissues.

A finite element model for ice ball evolution in a multi-probe cryosurgery.

The ice formation in a water body is examined for the computation of temperature field, phase change and a moving ice-water interface whose location is not known á priori. This is classically referred to as the Stefan problem [Rubinstein, L.I. (1971) The Stefan Problem (American Mathematical Society, Providence, Rhode Island 02904]. Based on the Duvaut [Duvaut, G. (1973) "Résolution d'un probléme Stefan" C.R. Acad Sci. Paris 276, 1461-1463] transformation, the governing equations for heat conduction are formulated within a variational principle that is readily amenable to a standard finite element solution without remeshing. Numerical simulation results pertaining to the freezing of tumour tissue in a multi-cryoprobe cryosurgery are presented. These results lend both quantitative and graphical support to the current empirical standards of "effective therapy" in view of refining clinical applications.

Measurement of freezing point depression of water in glass capillaries and the associated ice front shape.

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.

Osteoarthritis as an inevitable consequence of the structure of articular cartilage.

The hypothesis that osteoarthritis is an inevitable consequence of the structure of articular cartilage is proposed. Cartilage structure is viewed as an evolutionary solution to the optimization of articular cartilage function. A simplified model of the structural elements and constraints will be used to illustrate how the hypothesis leads to a mathematical solution and then a more realistic situation will be described, but not optimized rigorously. The goal for finding a solution will be to optimize the function of articular cartilage subject to the constraints of a biological tissue. In comparison, the structure of genuine articular cartilage will be discussed in the context of an optimal solution to the functional problem and, particularly, in relation to the disease of osteoarthritis.