Biomedical subjects

John C Bischof

Publications and source records attributed to John C Bischof.

At least 19 recordsLinked to original sources

PubMed2006

Effects of freezing and cryopreservation on the mechanical properties of arteries.

Cryoplasty, a freezing therapy, is being used for the treatment of restenosis in peripheral arteries. In addition, cryo-preserved arteries are increasingly used in vascular grafts. While studies are being performed to establish the efficacy of such treatments, very little is known about the postcryosurgical or postcryo-preservation changes in mechanical properties of the arteries. Few studies have examined the effect of freezing in the absence of cryoprotective agents (CPAs), and the several studies done in the presence of CPAs have given mixed results. To examine this issue further, we froze pig femoral arteries in a controlled rate freezer, using an aluminum probe, both in the presence at (-80 degrees C to 1 degrees C/min) and absence (at -20 degrees C for 2 or 5 mins) of CPA and Fetal bovine serum (FBS). Following freezing, artery samples were subjected to uniaxial tensile testing. The weights of the tissue were measured before and after freezing. Our results suggest that freezing does have an effect on stress-strain properties, particularly in the low stress region corresponding to physiological conditions. The mechanisms of this change in mechanical properties may include the loss of smooth muscle cell viability, damage to extra cellular matrix (ECM), bulk redistribution of water, or changes in alignment caused by ice crystal growth. In the case of samples frozen in the absence of CPA or FBS, the results indicated a drastic reduction in weight of the tissue suggesting the importance of bulk water redistribution as one underlying mechanism. To further examine potential mechanisms, we subjected cryopreserved vessels to the same uniaxial tests. The extent of changes in mechanical properties and bulk water redistribution was greatly attenuated; reinforcing that water movement might play a role in the changes observed with freezing.

Adaptation, Physiological↗

PubMed2006

Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-alpha delivery.

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-alpha is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol-coated colloidal gold nanoparticles (PT-cAu-TNF-alpha) with incorporated TNF-alpha payload (several hundred TNF-alpha molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-alpha. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 microg/kg PT-cAu-TNF-alpha alone or followed by local heating at 42.5 degrees C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-alpha alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-alpha or PT-cAu-TNF-alpha. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-alpha alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage-mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-alpha-coated gold nanoparticles when given i.v. at the proper dosage and timing.

Animals↗

PubMed2005

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.

Biological Transport↗

PubMed2005

A quantitative analysis on latent heat of an aqueous binary mixture.

The latent heat during phase change of water-NaCl binary mixture was measured using a differential scanning calorimeter, and the magnitude for two distinct phase change events, water/ice and eutectic phase change, were analyzed considering the phase change characteristics of a binary mixture. During the analysis, the latent heat associated with each event was calculated by normalizing the amount of each endothermic peak with only the amount of sample participating in each event estimated from the lever rule for the phase diagram. The resulting latent heat of each phase change measured is 303.7 +/- 2.5 J/g for water/ice phase change, and 233.0 +/- 1.6 J/g for eutectic phase change, respectively regardless of the initial concentration of mixture. Although the latent heats of water/ice phase change in water-NaCl mixtures are closely correlated, further study is warranted to investigate the reason for smaller latent heat of water/ice phase change than that in pure water (335 J/g). The analysis using the lever rule was extended to estimate the latent heat of dihydrate as 115 J/g with the measured eutectic and water/ice latent heat values. This new analysis based on the lever rule will be useful to estimate the latent heat of water-NaCl mixtures at various concentrations, and may become a framework for more general analysis of latent heat of various biological solutions.

Calorimetry, Differential Scanning↗

PubMed2005

Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice.

Activation of vascular endothelium plays an essential role in vasoocclusion in sickle cell disease. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to inhibit endothelial cell activation and hypoxia-induced vasoocclusion. Transgenic sickle mice, expressing human alpha-, beta(S)-, and beta(S-Antilles)-globins, had an activated vascular endothelium in their liver, lungs, and skin, as exhibited by increased activation of NF-kappaB compared with normal mice. NF-kappaB activation increased further in the liver and skin after sickle mice were exposed to hypoxia. Sickle mice had decreases in red blood cell (RBC) velocities and developed vasoocclusions in subcutaneous venules in response to hypoxia. Dexamethasone pretreatment prevented decreases in RBC velocities and inhibited vasoocclusions and leukocyte-endothelium interactions in venules after hypoxia. Dexamethasone treatment inhibited NF-kappaB, VCAM-1, and ICAM-1 expression in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation. VCAM-1 or ICAM-1 blockade with monoclonal antibodies mimicked dexamethasone by inhibiting vasoocclusion and leukocyte adhesion in sickle mice, demonstrating that endothelial cell activation and VCAM-1 and ICAM-1 expression are necessary for hypoxia-induced vasoocclusion in sickle mice. VCAM-1, ICAM-1, and vasoocclusion increased significantly 3 days after dexamethasone discontinuation, possibly explaining rebounds in vasoocclusive crises observed after withdrawal of glucocorticosteroids in sickle patients. We conclude that anti-inflammatory treatments that inhibit endothelial cell activation and adhesion molecule expression can inhibit vasoocclusion in sickle cell disease. Rebounds in vasoocclusive crises after dexamethasone withdrawal are caused by rebounds in endothelial cell activation.

Anemia, Sickle Cell↗

PubMed2005

The kinetics of thermal injury in human renal carcinoma cells.

In this study, the thermal injury behavior of both suspended and attached SN12 human renal carcinoma cells (RCC) under thermal therapy conditions (i.e., heating cells to elevated temperature for seconds to minutes) was investigated using a non-isothermal method. This non-isothermal method entailed heating the cells using a programmable heating stage from room temperature at 130 degrees C min(-1) to various peak temperatures from 45 to 70 degrees C, held for 0-10 min, and then cooling down to room temperature at 65 degrees C min(-1). It was found that the suspended SN12 cells are more heat susceptible than attached ones. The non-isothermal portions (i.e., the heat-up and cool-down portions) of the thermal histories were found to be able to cause significant injury (> 10%) in both suspended and attached SN12 cells when the peak temperature is above 60 degrees C. Therefore, a non-isothermal method, which accounts for both the isothermal and non-isothermal portions of the thermal histories, was used to extract the kinetic parameters (i.e., the activation energy and frequency factor) in the Arrhenius injury model for SN12 cells. Furthermore, these results suggest that this non-isothermal method can be used to extract kinetic parameters from in vivo heating studies using minimally invasive surgical probes, where it is very difficult to get a thermal history in tissue with a dominant isothermal portion.

Carcinoma, Renal Cell↗

PubMed2005

A cryoinjury model using engineered tissue equivalents for cryosurgical applications.

Cryosurgery is emerging as a promising treatment modality for various cancers, but there are still challenges to be addressed to improve its efficacy. Two primary challenges are determining thermal injury thresholds for various types of cell/tissue, and understanding of the mechanisms of freezing induced cell/tissue injury within a cryolesion. To address these challenges, various model systems ranging from cell suspensions to three-dimensional in vivo tissues have been developed and used. However, these models are either oversimplifications of in vivo tissues or difficult to control and extract precise experimental conditions from. Therefore, a more readily controllable model system with tissue-like characteristics is needed. In this study, a cryoinjury model was developed using tissue engineering technology, and the capabilities of the model were demonstrated. Engineered tissue equivalents (TEs) were constructed by seeding and culturing cells in a type I collagen matrix. Two different cell lines were used in this study, AT-1 rat prostate tumor cells and LNCaP human prostate cancer cells. The constructed TEs underwent a freeze/thaw cycle imitating in vivo cryosurgery. Thermal conditions within TEs during freeze/thaw cycles were characterized, and the responses of TEs to these thermal conditions including freezing induced cellular injury and extracellular matrix damage were investigated at three different time points. The results illustrate the feasibility to establish thermal thresholds of cryoinjury for different cell/tissue types using the presently developed model, and its potential capabilities to study cell death mechanisms, cell proliferation or migration, and extracellular matrix structural damage after a freeze/thaw cycle.

Animals↗

PubMed2005

In vitro model systems for evaluation of smooth muscle cell response to cryoplasty.

Restenosis is a major health care problem, with approximately 40% of angioplasties resulting in restenosis. Mechanisms related to elastic recoil, cell proliferation, and extracellular matrix (ECM) synthesis are implicated. In vivo studies have demonstrated the potential for cryotherapy to combat the process of restenosis, but the mechanisms whereby freezing and/or cooling can reduce or eliminate smooth muscle cell (SMC) proliferation and ECM synthesis are not well known. While in vivo testing is ultimately necessary, in vitro models can provide important information on thermal parameters and mechanisms of injury. However, it is important to carefully choose the model system for in vitro work on cryoinjury characterization to adequately reflect the clinical situation. In this study, we examined the differences in response to cryoinjury by SMCs from different species (rat, pig, and human) and in different cellular environments (suspension vs. tissue equivalent). Tissue equivalents, composed of cells embedded in collagen or fibrin gel, provide a 3-D tissue-like environment, while allowing for controlled composition. As reported here, all SMCs showed similar trends, but rat cells appeared less sensitive to cooling at faster cooling rates in suspension, while human SMCs were less sensitive to temperatures just above freezing when embedded in collagen. In addition, the SMCs were less sensitive in suspension than they were in collagen. Cells in suspension exhibited 70% viability at -11 degrees C, whereas cells in the tissue equivalent model showed only 30% survival. Future studies will aim to more adequately represent the conditions in restenosis by providing inflammatory and proliferative cues to the cells.

Angioplasty↗

PubMed2005

Polynitroxyl albumin inhibits inflammation and vasoocclusion in transgenic sickle mice.

Individuals with sickle-cell disease (SCD) and transgenic sickle mice expressing human betaS globin exhibit enhanced reactive oxygen species (ROS) production, vascular inflammation, and episodic vasoocclusion. We hypothesize that reduction of ROS will reduce endothelial-cell activation and adhesion-molecule expression, thereby inhibiting vasoocclusion. To test this hypothesis, we measured endothelial-cell activation, adhesion-molecule expression, and vasoocclusion in sickle mice after administering i.v. polynitroxyl albumin (PNA), a superoxide dismutase and catalase mimetic. Untreated sickle mice, compared with normal mice, showed increased activation of nuclear factor-kappaB (NF-kappaB), an oxidant-sensitive transcription factor, in their lungs, livers, and skin. NF-kappaB activation was increased further in the livers and skin of sickle but not normal mice after hypoxia-reoxygenation. IV administration of PNA inhibited NF-kappaB activation by 60% (P < .01) in the lungs and by 33% (P < .05) in the livers of sickle mice after hypoxia-reoxygenation. PNA also reduced the expression of vascular cell-adhesion molecule-1 (VCAM-1) by 57% in lung (P < .05) and by 33% in liver (P < .05) and reduced the expression of intercellular-adhesion molecule-1 (ICAM-1) by 40% in lung (P < .05) and by 53% in liver (P < .05). PNA inhibited a hypoxia-reoxygenation-induced increase in leukocyte rolling (P < .01) and adhesion (P < .05) in venules of the dorsal skin. Most importantly, PNA completely inhibited hypoxia-reoxygenation-induced vasoocclusion (P < .001). Control albumin had no effect on NF-kappaB, VCAM-1, ICAM-1, rolling, adhesion, or vasoocclusion. We speculate that therapies to reduce oxidative stress will inhibit inflammation and vasoocclusion in SCD.

Albumins↗

PubMed2005

Analysis of thermal stress in cryosurgery of kidneys.

In this study, the thermal stress distribution in cryosurgery of kidney was investigated using a multiphysics finite element model developed in ANSYS (V8.1). The thermal portion of the model was verified using experimental data and the mechanics portion of the model (elastic) was verified using classic analytical solutions. Temperature dependent thermal and mechanical properties were used in the model. Moreover, the model accounts for thermal expansion due to both thermal expansion in single phase and volumetric expansion associated with phase change of tissue water to ice. For a clinical cylindrical cryoprobe inserted into the renal cortex from the top-middle renal capsule, it was found that the thermal stress distributions along the radial position are very different at different depths from the top renal capsule. The thermal stress is much higher at both ends than in the middle of the cryoprobe surface. It was found that there might be more tissue next to the top renal capsule than other region undergoing microcrack formation or plastic deformation. Furthermore, it was found that macrocrack formation is more likely to occur in tissue adjacent to the cryoprobe surface (especially on the sharp point tip) and during the thawing phase of cryosurgery. It was further found that the volumetric expansion associated with phase change induced much higher thermal stress than thermal expansion in a single phase and might therefore be the main cause of the frequently observed crack formation shortly after initiation of thawing in cryosurgery. Because the thermal stress adjacent to the cryoprobe is much higher than the yield stress of frozen renal tissue, a plastic stress model is required for better modeling of the thermal stress distribution in cryosurgery of kidney in future. However the computational effort will then be drastically increased due to the strong nonlinear nature of the plastic model and more experimental studies are indispensable for better understanding of the mechanical behavior of frozen tissue in cryosurgery.

Animals↗

PubMed2005

Thermal stability of proteins.

Protein stability is critical to the outcome of nearly all thermally mediated applications to biomaterials such as thermal therapies (including cryosurgery), burn injury, and biopreservation. As such, it is imperative to understand as much as possible about how a protein loses stability and to what extent we can control this through the thermal environment as well as through chemical or mechanical modification of the protein environment. This review presents an overview of protein stability in terms of denaturation due to temperature alteration (predominantly high and some low) and its modification by use of chemical additives, pH modification as well as modification of the mechanical environment (stress) of the proteins such as collagen. These modifiers are able to change the kinetics of protein denaturation during heating. While pH can affect the activation energy (or activation enthalpy) and the frequency factor (or activation entropy) of the denaturation kinetics, many other chemical and mechanical modifiers only affect the frequency factor (activation entropy). Often, the modification affecting activation entropy appears to be linked to the hydration of the protein. While the heat-induced denaturation of proteins is reasonably well understood, the heat denaturation of structural proteins (e.g., collagen) within whole tissues remains an area of active research. In addition, while some literature exists on protein denaturation during cold temperatures, relatively little is known about the kinetics of protein denaturation during both freezing and drying. Further understanding of this kinetics will have an important impact on applications ranging from preservation of biomaterials and pharmaceutics to cryosurgery. Interestingly, both freezing and drying involve drastic shifts in the hydration of the proteins. It is clear that understanding protein hydration at the molecular, cellular, and tissue level will be important to the future of this evolving area.

Animals↗

PubMed2004

Microvascular blood flow and stasis in transgenic sickle mice: utility of a dorsal skin fold chamber for intravital microscopy.

Vascular inflammation, secondary to ischemia-reperfusion injury, may play an essential role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice expressing human alpha and beta(s)/beta(s-Antilles) globin chains. Microvessels in the DSFC were visualized by intravital microscopy at baseline in ambient air and after exposure to hypoxia-reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle mice after hypoxia-reoxygenation but remained constant in normal mice. The mean RBC velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after hypoxia-reoxygenation. None of the venules in normal mice became static at any time during hypoxia-reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation, 11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and 4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous venules in sickle mice remained static (P = 0.01). All of the venules were flowing again after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous venules of sickle mice by tumor necrosis factor alpha (TNF-alpha). Leukocyte rolling flux and firm adhesion, manifestations of vascular inflammation, were significantly higher at baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle (P < 0.01), but not in normal mice, after hypoxia-reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were also seen in subcutaneous venules.

Anemia, Sickle Cell↗

PubMed2004

Direct cell injury associated with eutectic crystallization during freezing.

Freezing induced direct cell injury has been explained by a two-factor hypothesis-intracellular ice formation (IIF) at rapid cooling rates, and solution effects at slow cooling rates. Even though IIF is generally believed to be a major injury mechanism at rapid cooling rates, injury by solution effects is not fully understood and several injury mechanisms have been suggested. Solution effects have generally been considered the result of the elevated electrolyte concentration within the intracellular and extracellular space during freezing. In addition to the injury by this elevated electrolyte concentration, freezing injury associated with eutectic crystallization was investigated. To examine the injury associated with eutectic crystallization, two different experiments were designed and performed. In the first experiment, two groups of AT-1 rat prostate tumor cell suspensions were frozen and thawed on a cryomicroscope in the same way except that eutectic crystallization was initiated in only one group. During the second experiment, AT-1 cells were suspended in several different media, which have different eutectic crystallization temperatures, and exposed to a single cooling-warming cycle with varying end temperature of the protocol on a directional solidification stage. After both experiments, post-thaw viability was evaluated and compared. The post-thaw viability drops significantly upon the occurrence of the eutectic crystallization regardless of suspending media, which suggests direct cell injury associated with eutectic crystallization. Based on these observations, two possible injury mechanisms are anticipated: (i) mechanical damage to the cell membrane due to eutectic crystallization, and (ii) intracellular eutectic formation (IEF). The proposed mechanisms provide a more comprehensive physical explanation of freezing induced cell injury and extend the understanding on solution effects.

Animals↗

PubMed2004

Pre-treatment inflammation induced by TNF-alpha augments cryosurgical injury on human prostate cancer.

Vascular injury is a major mechanism of cryosurgical destruction. The extent of vascular injury may be affected by the addition of molecular adjuvants. This study, in addition to determining the injury mechanism in the LNCaP Pro 5 human prostate cancer subline grown in a nude mouse, examined the effect of cytokine TNF-alpha on cryosurgery of an in vivo microvascular preparation (Dorsal Skin Flap Chamber). A comparison of injury data to a thermal model indicated that the minimum temperature after moderate cooling, thawing, and hold time required for causing necrosis was 3.5+/-6.9 degrees C in TNF-alpha-treated LNCaP Pro 5 tumor tissue (n=4) and -9.8+/-5.8 degrees C in TNF-alpha-treated normal skin of the nude mouse (n=4). Compared to tissues without TNF-alpha treatment, where the minimum temperature required for causing necrosis was -16.5+/-4.3 degrees C in LNCaP Pro 5 tumor tissue (n=8) and -24.4+/-7.0 degrees C in normal skin of the nude mouse (n=9), the results indicate the local use of TNF-alpha can dramatically increase the threshold temperature of cryo-destruction by more than 10 degrees C (p <0.01). These findings were consistent with the hypothesis that vascular-mediated injury is responsible for defining the edge of the cryolesion in microvascular-perfused tissue, and therefore pre-induced inflammation can augment cryoinjury. The local use of TNF-alpha to pre-inflame prostate cancer promises to increase both the ability of freezing to destroy cancer as well as improve the ability of ultrasound or other iceball-monitoring techniques to predict the outcome of the treatment.

Animals↗

PubMed2004

In situ thermal denaturation of proteins in dunning AT-1 prostate cancer cells: implication for hyperthermic cell injury.

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from alpha helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees C min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.

Adenocarcinoma↗

PubMed2004

Thermodynamic nonequilibrium phase change behavior and thermal properties of biological solutions for cryobiology applications.

Understanding the phase change behavior of biomaterials during freezing/thawing including their thermal properties at low temperatures is essential to design and improve cryobiology applications such as cryopreservation and cryosurgery. However, knowledge of phase change behavior and thermal properties of various biomaterials is still incomplete, especially at cryogenic temperatures (< or = -40 degrees C). Moreover, in these applications, chemicals are often added to improve their outcome, which can result in significant variation in the phase change behavior and thermal properties from those of the original biomaterials. These chemical additives include cryoprotective agents (CPAs), antifreeze protein (AFP), or cryosurgical adjuvants like sodium chloride (NaCl). In the present study, phase change behavior and thermal properties of saline solutions--either water-NaCl or phosphate buffered saline (PBS)--with various chemical additives were investigated. The chemical additives studied are glycerol and raffinose as CPAs, an AFP (Type III, molecular weight = 6500), and NaCl as a cryosurgical adjuvant. The phase change behavior was investigated using a differential scanning calorimeter (DSC) and a cryomicroscope. The specific and latent heat of these solutions were also measured with the DSC. The saline solutions have two distinct phase changes--water/ice and eutectic phase changes. During freezing, eutectic solidification of both water-NaCl and PBS are significantly supercooled below their thermodynamic equilibrium eutectic temperatures. However, their melting temperatures are close to thermodynamic equilibrium during thawing. These eutectic phase changes disappear when even a small amount (0.1 M glycerol) of CPA was added, but they are still observed after the addition of an AFP. The specific heats of these solutions are close to that of ice at very low temperatures (< or = -100 degrees C) regardless of the additives, but they increase between -100 degrees C and -30 degrees C with the addition of CPAs. The amount of latent heat, which is evaluated with sample weight, generally decreases with the addition of the additives, but can be normalized to approximately 300 J/g based on the weight of water which participates in the phase change. This illustrates that thermal properties, especially latent heat, of a biomaterial should be evaluated based on the understanding of its phase change behavior. The results of the present study are discussed in the context of the implications for cryobiology applications.

Biomimetic Materials↗

PubMed2004

Improved cryosurgery by use of thermophysical and inflammatory adjuvants.

In the present article, recent research efforts in our laboratory to improve cryosurgery by use of mechanistically derived adjuvants are reviewed. Our research has been focused on enhancing two freezing induced injury mechanisms - i) direct cell injury by use of thermophysical adjuvants, and ii) vascular injury by use of an inflammatory adjuvant. The thermophysical adjuvants are chemicals, usually salts, which can induce secondary crystallization, called eutectic solidification, in a cryolesion; thereby enhancing direct cell injury. The inflammatory adjuvant is a cytokine, tumor necrosis factor-alpha (TNF-alpha), which upregulates inflammation of microvasculature in tumors prior to freezing to promote vascular injury in the cryolesion. Even though the individual mechanism of injury enhancement within the cryolesion of each adjuvant requires further study, both adjuvants are envisioned to enlarge the complete killing zone so that the boundary of the cryolesion matches more closely with the edge of iceball. By bringing the edge of the cryolesion closer to the edge of iceball, the adjuvants hold promise for improvement of image guidance and outcome of cryosurgery.

Adjuvants, Immunologic↗

PubMed2004

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.

Animals↗