Correlated parameter fit of arrhenius model for thermal denaturation of proteins and cells.

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy E a and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating E a, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher E a and A parameters were found at low end-temperature (50 °C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45-50 °C) vs. membrane dye assays (60-70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed.

Thermal injury prediction during cryoplasty through in vitro characterization of smooth muscle cell biophysics and viability.

Restenosis in peripheral arteries is a major health care problem in the United States. Typically, 30-40% of angioplasties result in restenosis and hence alternative treatment techniques are being actively investigated. Cryoplasty, a novel technique involving simultaneous stretching and freezing of the peripheral arteries (e.g., femoral, iliac, popliteal) using a cryogen-filled balloon catheter, has shown the potential to combat restenosis. However, evaluation of the thermal and biophysical mechanisms that affect cellular survival during cryoplasty is lacking. To achieve this, the thermal history in arteries was predicted for different balloon temperatures using a thermal model. Cellular biophysical responses (water transport (WT) and intracellular ice formation (IIF)) were then characterized, using in vitro model systems, based on the thermal model predictions. The thermal and biophysical effects on cell survival were eventually determined. For this study, smooth muscle cells (SMC) isolated from porcine femoral arteries were used in suspensions and attached in vitro systems (monolayer and fibrin gel). Results showed that for different balloon temperatures, the thermal model predicted cooling rates from 2200 to 5 degrees C/min in the artery. Biophysical parameters (WT & IIF) were higher for SMCs in attached systems as compared to suspensions. The "combined" fit WT parameters for SMCs in suspension (at 5, 10, and 25 degrees C/min) are L (pg) = 0.12 microm/(min atm) and E (Lp) = 24.1 kcal/mol. Individual WT parameters for SMCs in attached cell systems at higher cooling rates are approximately an order of magnitude higher compared to suspensions (e.g., at 130 degrees C/min, WT parameters in monolayer and fibrin TE systems are L (pg) = 18.6, 19.4 microm/(min atm) and E (Lp) = 112, 127 kcal/mol, respectively). Similarly, IIF parameters assessed at 130 degrees C/min are higher for SMCs in attached systems than suspensions (Omega 0 = 1.1, 354, 378 (x 10(8) (1/m(2) s)) and kappa(o) = 1.6, 1.8, 2.1 (x 10(9) K(5)) for suspensions, monolayer, and fibrin TE, respectively). One possible reason for the differences in IIF kinetics was verified to be the presence of gap junctions, which facilitate cell-cell connections through which ice can propagate. This is reflected by the change in the predicted IIF parameters when a gap junction inhibitor was added and tested in monolayer (Omega 0 (1/m(2) s)); kappa(o) = 2.1 x 10(9) K(5)). SMC viability was affected by the model system (lower viability in attached systems), the thermal conditions and the biophysics. For e.g., IIF is lethal to cells and SMC viability was verified to be the least in fibrin TE (most % IIF) and the most in suspensions (least % IIF) at all cooling rates. Using the results from the fibrin TE (suggested as the best in vitro system to mimic a restenosis environment), conservative estimates of injury regimes in the artery during cryoplasty is predicted. The results can be used to suggest future optimizations and modifications during cryoplasty and also to design future in vivo studies.

Water transport and IIF parameters for a connective tissue equivalent.

Understanding the biophysical processes that govern freezing injury of a tissue equivalent (TE) is an important step in characterizing and improving the cryopreservation of these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gels. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope allowing observation of the TEs cooled under controlled rates between 5 and 130 degrees C/min. Typically, freezing of cellular systems results in two biophysical processes that are both dependent on the cooling rate: dehydration and/or intracellular ice formation (IIF). Both these processes can potentially be destructive to cells. In this study, the biophysics of freezing cells in collagen and fibrin TEs have been quantified and compared to freezing cells in suspension. Experimental data were fitted in numerical models to extract parameters that governed water permeability, E(Lp) and L(pg), and intracellular ice nucleation, omega(o) and kappa(o). Results indicate that major differences exist between freezing HDFs in suspension and in a tissue equivalent. During freezing, 55% of the HDFs in suspension formed IIF as compared to 100% of HDFs forming IIF in collagen and fibrin TE at a cooling rate of 130 degrees C/min. Also, both the water permeability and the IIF parameters were determined to be higher for HDFs in TEs as compared to cell suspensions. Between the TEs, HDFs in fibrin TE exhibited higher values for the biophysical parameters as compared to HDFs in collagen TE. The observed biophysics seems to indicate that cell-cell and cell-matrix interactions play a major role in ice propagation in TEs.