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### Guest Editorial

J Biomech Eng. 2009;131(7):070301-070301-1. doi:10.1115/1.3185352.
FREE TO VIEW

Important applications of bioheat and mass transfer include biopreservation (molecular, cellular and tissue) and thermal therapies (high temperature—laser, radiofrequency, microwave, high intensity ultrasound heating of cells and tissues, and low temperature—cryosurgery). These applications in turn support the growing fields of tissue engineering, cell therapeutics, drug stability and delivery, and minimally and non-invasive disease detection and treatment (i.e., cancer, cardiovascular and neural). The frontiers of biopreservation and thermal therapies are increasingly defined at the cellular and molecular level. This special issue focuses on these frontiers in areas of emphasis including thermodynamics (molecular and cellular stability), multi-scale mass transport (sub-cellular to tissue level transport), multi-scale heat transport (nanoparticle to bulk tissue heating), and extreme biology (anhydro and thermal physiology). The papers assembled in this special issue show the breadth of this activity with a series of invited reviews and original contributions in the area.

Topics: Biotransport
Commentary by Dr. Valentin Fuster

### Research Papers

J Biomech Eng. 2009;131(7):071001-071001-10. doi:10.1115/1.3118766.

Skin biothermomechanics is highly interdisciplinary, involving bioheat transfer, burn damage, biomechanics, and physiology. Characterization of the thermomechanical behavior of skin tissue is of great importance and can contribute to a variety of medical applications. However, few quantitative studies have been conducted on the thermally-dependent mechanical properties of skin tissue. The aim of the present study is to experimentally examine the thermally-induced change in the relaxation behavior of skin tissue in both hyperthermal and hypothermic ranges. The results show that temperature has great influence on the stress-relaxation behavior of skin tissue under both hyperthermal and hypothermic temperatures; the quantitative relationship that has been found between temperature and the viscoelastic parameter (the elastic fraction or fractional energy dissipation) was temperature dependent, with greatest dissipation at high temperature levels.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071002-071002-7. doi:10.1115/1.3127259.

Proteins aggregate and precipitate from high concentration solutions in a wide variety of problems of natural and technological interest. Consequently, there is a broad interest in developing new ways to model the thermodynamic and kinetic aspects of protein stability in these crowded cellular or solution environments. We use a coarse-grained modeling approach to study the effects of different crowding agents on the conformational equilibria of proteins and the thermodynamic phase behavior of their solutions. At low to moderate protein concentrations, we find that crowding species can either stabilize or destabilize the native state, depending on the strength of their attractive interaction with the proteins. At high protein concentrations, crowders tend to stabilize the native state due to excluded volume effects, irrespective of the strength of the crowder-protein attraction. Crowding agents reduce the tendency of protein solutions to undergo a liquid-liquid phase separation driven by strong protein-protein attractions. The aforementioned equilibrium trends represent, to our knowledge, the first simulation predictions for how the properties of crowding species impact the global thermodynamic stability of proteins and their solutions.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071003-071003-6. doi:10.1115/1.3118768.

The heat shock (HS) response is a protective mechanism for cells to protect themselves against subsequent lethal stress. HS upregulated heat shock protein (HSP) expression reduced apoptosis following tumor necrosis factor-$α$ ($TNF-α$) stimulation. However, vector-mediated overexpression of HSP70 failed to provide similar protection but rather sensitized cells to $TNF-α$ induced apoptosis. This may be due to the fact that the kinetics of vector-mediated HSP overexpression is totally different from that of HSP upregulation by HS. We hypothesized that the response depends on the timing of $TNF-α$ challenge relative to HSP expression dynamics after HS. Therefore, we investigated the correlation between the dynamic change of HSP expression and the levels of apoptosis induced by $TNF-α$ after HS. Hepatoma cells were subjected to mild heat shock at $42°C$ for 2 h followed by varied recovery times and then treated with $TNF-α$ to induce apoptosis. The results from quantitative apoptosis assays using the TUNEL reaction reveal an optimal HS protection window centered around 5 h post-HS against $TNF-α$ induced apoptosis. In addition, we found a window extending up to 2 h after HS where HS sensitized cells to $TNF-α$ stress. Importantly, the correlation between apoptosis and HSP expression kinetics demonstrates that both high levels of HSPs and proper timing between HS and $TNF-α$ stress were critical for optimal protection. Our study establishes a dynamic experimental model for further investigation of HS as a potential clinical approach to target tissue survival or death.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071004-071004-8. doi:10.1115/1.3147801.

Erbium, chromium: yttrium, scandium, gallium, garnet (Er,Cr:YSGG) lasers are currently being investigated for disinfecting the root canal system. Prior to using laser therapy, it is important to understand the temperature distribution and to assess thermal damage to the surrounding tissue. In this study, a theoretical simulation using the Pennes bioheat equation is conducted to evaluate how heat spreads from the canal surface using an Er,Cr:YSGG laser. Results of the investigation show that some of the proposed treatment protocols for killing bacteria in the deep dentin are ineffective, even for long heating durations. Based on the simulation, an alternative treatment protocol is identified that has improved effectiveness and is less likely to introduce collateral damage to the surrounding tissue. The alternative protocol uses 350 mW laser power with repeating laser tip movement to achieve bacterial disinfection in the deep dentin ($800 μm$ lateral from the canal surface), while avoiding thermal damage to the surrounding tissue $(T<47°C)$. The alternative treatment protocol has the potential to not only achieve bacterial disinfection of deep dentin but also shorten the treatment time, thereby minimizing potential patient discomfort during laser procedures.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071005-071005-7. doi:10.1115/1.3156812.

Insulation reduces heat exchange between a body and the environment. Glabrous (nonhairy) skin surfaces (palms of the hands, soles of the feet, face, and ears) constitute a small percentage of total body surface area but contain specialized vascular structures that facilitate heat loss. We have previously reported that cooling the glabrous skin surfaces is effective in alleviating heat stress and that the application of local subatmospheric pressure enhances the effect. In this paper, we compare the effects of cooling multiple glabrous skin surfaces with and without vacuum on thermal recovery in heavily insulated heat-stressed individuals. Esophageal temperatures $(Tes)$ and heart rates were monitored throughout the trials. Water loss was determined from pre- and post-trial nude weights. Treadmill exercise (5.6 km/h, 9–16% slope, and 25–45 min duration) in a hot environment ($41.5°C$, 20–30% relative humidity) while wearing insulating pants and jackets was used to induce heat stress $(Tes≥39°C)$. For postexercise recovery, the subjects donned additional insulation (a balaclava, winter gloves, and impermeable boot covers) and rested in the hot environment for 60 min. Postexercise cooling treatments included control (no cooling) or the application of a $10°C$ closed water circulating system to (a) the hand(s) with or without application of a local subatmospheric pressure, (b) the face, (c) the feet, or (d) multiple glabrous skin regions. Following exercise induction of heat stress in heavily insulated subjects, the rate of recovery of $Tes$ was $0.4±0.2°C/h(n=12)$, but with application of cooling to one hand, the rate was $0.8±0.3°C/h(n=12)$, and with one hand cooling with subatmospheric pressure, the rate was $1.0±0.2°C/h(n=12)$. Cooling alone yielded two responses, one resembling that of cooling with subatmospheric pressure $(n=8)$ and one resembling that of no cooling $(n=4)$. The effect of treating multiple surfaces was additive (no cooling, $ΔTes=−0.4±0.2°C$; one hand, $−0.9±0.3°C$; face, $−1.0±0.3°C$; two hands, $−1.3±0.1°C$; two feet, $−1.3±0.3°C$; and face, feet, and hands, $−1.6±0.2°C$). Cooling treatments had a similar effect on water loss and final resting heart rate. In heat-stressed resting subjects, cooling the glabrous skin regions was effective in lowering $Tes$. Under this protocol, the application of local subatmospheric pressure did not significantly increase heat transfer per se but, presumably, increased the likelihood of an effect.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071006-071006-12. doi:10.1115/1.3156808.

Nonthermal irreversible electroporation (NTIRE) is a new minimally invasive surgical technique that is part of the emerging field of molecular surgery, which holds the potential to treat diseases with unprecedented accuracy. NTIRE utilizes electrical pulses delivered to a targeted area, producing irreversible damage to the cell membrane. Because NTIRE does not cause thermal damage, the integrity of all other molecules, collagen, and elastin in the targeted area is preserved. Previous theoretical studies have only examined NTIRE in homogeneous tissues; however, biological structures are complex collections of diverse tissues. In order to develop electroporation as a precise treatment in clinical applications, realistic models are necessary. Therefore, the purpose of this study was to refine electroporation as a treatment by examining the effect of NTIRE in heterogeneous tissues of the prostate and breast. This study uses a two-dimensional finite element solution of the Laplace and bioheat equations to examine the effects of heterogeneities on electric field and temperature distribution. Three different heterogeneous structures were taken into account: nerves, blood vessels, and ducts. The results of this study demonstrate that heterogeneities significantly impact both the temperature and electrical field distribution in surrounding tissues, indicating that heterogeneities should not be neglected. The results were promising. While the surrounding tissue experienced a high electrical field, the axon of the nerve, the interior of the blood vessel, and the ducts experienced no electrical field. This indicates that blood vessels, nerves, and lactiferous ducts adjacent to a tumor treated with electroporation will survive, while the cancerous lesion is ablated. This study clearly demonstrates the importance of considering heterogeneity in NTIRE applications.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):071007-071007-8. doi:10.1115/1.3169248.

Direct tissue infusion, e.g., convection-enhanced delivery (CED), is a promising local delivery technique for treating diseases of the central nervous system. Predictive models of spatial drug distribution during and following direct tissue infusion are necessary for treatment optimization and planning of surgery. In this study, a 3D interstitial transport modeling approach in which tissue properties and anatomical boundaries are assigned on a voxel-by-voxel basis using tissue alignment data from diffusion tensor imaging (DTI) is presented. The modeling approach is semi-automatic and utilizes porous media transport theory to estimate interstitial transport in isotropic and anisotropic tissue regions. Rat spinal cord studies compared predicted distributions of albumin tracer (for varying DTI resolution) following infusion into the dorsal horn with tracer distributions measured by Wood in a previous study. Tissue distribution volumes compared favorably for small infusion volumes $(<4 μl)$. The presented DTI-based methodology provides a rapid means of estimating interstitial flows and tracer distributions following CED into the spinal cord. Quantification of these transport fields provides an important step toward development of drug-specific transport models of infusion.

Commentary by Dr. Valentin Fuster

### Technology Reviews

J Biomech Eng. 2009;131(7):074001-074001-5. doi:10.1115/1.3156800.

Advances in nanotechnology are enabling many new diagnostic and therapeutic approaches in cancer. In this review, examples where nanoparticles are employed to induce localized heating within tumors are explored. Approaches to nanoparticle-mediated thermal therapy include absorption of infrared light, radio frequency ablation, and magnetically-induced heating. These approaches have demonstrated high efficacy in animal models, and two are already in human clinical trials.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074002-074002-6. doi:10.1115/1.3184694.

Oxygen is essential to maintaining normal brain function. A large body of evidence suggests that the partial pressure of oxygen $(pO2)$ in brain tissue is physiologically maintained within a narrow range in accordance with region-specific brain activity. Since the transportation of oxygen in the brain tissue is mainly driven by a diffusion process caused by a concentration gradient of oxygen from blood to cells, the spatial organization of the vascular system, in which the oxygen content is higher than in tissue, is a key factor for maintaining effective transportation. In addition, a local mechanism that controls energy demand and blood flow supply plays a critical role in moment-to-moment adjustment of tissue $pO2$ in response to dynamically varying brain activity. In this review, we discuss the spatiotemporal structures of brain tissue oxygen transport in relation to local brain activity based on recent reports of tissue $pO2$ measurements with polarographic oxygen microsensors in combination with simultaneous recordings of neural activity and local cerebral blood flow in anesthetized animal models. Although a physiological mechanism of oxygen level sensing and control of oxygen transport remains largely unknown, theoretical models of oxygen transport are a powerful tool for better understanding the short-term and long-term effects of local changes in oxygen demand and supply. Finally, emerging new techniques for three-dimensional imaging of the spatiotemporal dynamics of $pO2$ map may enable us to provide a whole picture of how the physiological system controls the balance between demand and supply of oxygen during both normal and pathological brain activity.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074003-074003-11. doi:10.1115/1.3156804.

Molecular adjuvants can be used to enhance the natural destructive mechanisms of freezing within tissue.  This review discusses their use in the growing field of combinatorial or adjuvant enhanced cryosurgery for a variety of disease conditions.  Two important motivations for adjuvant use are:  (1) increased control of the local disease in the area of freezing (i.e., reduced local recurrence of disease) and (2) reduced complications due to over-freezing into adjacent tissues (i.e., reduced normal functional tissue destruction near the treatment site).  This review starts with a brief overview of cryosurgical technology including probes and cryogens and major mechanisms of cellular, vascular injury and possible immunological effects due to freeze-thaw treatment in vivo.  The review then focuses on adjuvants to each of these mechanisms that make the tissue more sensitive to freeze-thaw injury. Four broad classes of adjuvants are discussed including:  thermophysical agents (eutectic forming salts and amino acids), chemotherapuetics, vascular agents and immunomodulators.  The key issues of selection, timing, dose and delivery of these adjuvants are then elaborated. Finally, work with a particularly promising vascular adjuvant, TNF-alpha, that shows the ability to destroy all cancer within a cryosurgical iceball is highlighted.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074004-074004-11. doi:10.1115/1.3173281.

Biotransport, by its nature, is concerned with the motions of molecules in biological systems while water remains as the most important and the most commonly studied molecule across all disciplines. In this review, we focus on biopreservation and thermal therapies from the perspective of water, exploring how its molecular motions, properties, kinetic, and thermodynamic transitions govern biotransport phenomena and enable preservation or controlled destruction of biological systems.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074005-074005-20. doi:10.1115/1.3160763.

Targeted delivery of therapeutics is an area of vigorous research, and peptide- and aptamer-functionalized nanovectors are a promising class of targeted delivery vehicles. Both peptide- and aptamer-targeting ligands can be readily designed to bind a target selectively with high affinity, and more importantly are molecules accessible by chemical synthesis and relatively compact compared with antibodies and full proteins. The multitude of peptide ligands that have been used for targeted delivery are covered in this review, with discussion of binding selectivity and targeting performance for these peptide sequences where possible. Aptamers are RNA or DNA strands evolutionarily engineered to specifically bind a chosen target. Although use of aptamers in targeted delivery is a relatively new avenue of research, the current state of the field is covered and promises of future advances in this area are highlighted. Liposomes, the classic drug delivery vector, and polymeric nanovectors functionalized with peptide or aptamer binding ligands will be discussed in this review, with the exclusion of other drug delivery vehicles. Targeted delivery of therapeutics, from DNA to classic small molecule drugs to protein therapeutics, by these targeted nanovectors is reviewed with coverage of both in vitro and in vivo deliveries. This is an exciting and dynamic area of research and this review seeks to discuss its broad scope.

Commentary by Dr. Valentin Fuster

### Technical Briefs

J Biomech Eng. 2009;131(7):074501-074501-4. doi:10.1115/1.3142975.

Lyophilizing frozen pellets, and especially spray freeze-drying, have been receiving growing interest. To design efficient and safe freeze-drying cycles, local temperature and moisture content in the product bed have to be known, but both are difficult to measure in the industry. Mathematical modeling of heat and mass transfer helps to determine local freeze-drying conditions and predict effects of operation policy, and equipment and recipe changes on drying time and product quality. Representative pellets situated at different positions in the product slab were considered. One-dimensional transfer in the slab and radial transfer in the pellets were assumed. Coupled heat and vapor transfer equations between the temperature-controlled shelf, the product bulk, the sublimation front inside the pellets, and the chamber were established and solved numerically. The model was validated based on bulk temperature measurement performed at two different locations in the product slab and on partial vapor pressure measurement in the freeze-drying chamber. Fair agreement between measured and calculated values was found. In contrast, a previously developed model for compact product layer was found inadequate in describing freeze-drying of pellets. The developed model represents a good starting basis for studying freeze-drying of pellets. It has to be further improved and validated for a variety of product types and freeze-drying conditions (shelf temperature, total chamber pressure, pellet size, slab thickness, etc.). It could be used to develop freeze-drying cycles based on product quality criteria such as local moisture content and glass transition temperature.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074502-074502-3. doi:10.1115/1.3142978.

In this article, the microscale ice crystal growth behavior under electrostatic field is investigated via a phase field method, which also incorporates the effects of anisotropy and thermal noise. The multiple ice nuclei’s competitive growth as disclosed in existing experiments is thus successfully predicted. The present approach suggests a highly efficient theoretical tool for probing into the freeze injury mechanisms of biological material due to ice formation during cryosurgery or cryopreservation process when external electric field was involved.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074503-074503-4. doi:10.1115/1.3127254.

The intervertebral disk (IVD) is the largest avascular structure in the human body. Transport of small molecules in IVD is mainly through diffusion from the endplates and the peripheral blood vessels surrounding IVD. Studies have investigated the structure, chemical components, and water content in IVD, but to our knowledge no study has investigated the effect of mechanical loading on oxygen transport in IVD. The objective of this study was to determine the strain-dependent behavior of oxygen diffusivity in IVD tissue. A one-dimensional steady-state diffusion experiment was designed and performed to determine the oxygen diffusivity in bovine annulus fibrosus (AF). The oxygen diffusivity was calculated using equation derived from Fick’s law. A total of 20 AF specimens ($d=6 mm$, $h∼0.5 mm$) from bovine coccygeal IVD were used to determine oxygen diffusivity at three levels of compressive strain. The average oxygen diffusivity $(mean±SD)$ of bovine AF in the axial direction was $1.43±0.242×10−5 cm2/s$$(n=20)$ at $4.68±1.67%$ compressive strain level, $1.05±0.282×10−5 cm2/s$$(n=20)$ at $14.2±1.50%$ strain level, and $7.71±1.63×10−6 cm2/s$$(n=20)$ at $23.7±1.34%$ strain level. There was a significant decrease in oxygen diffusivity with increasing level of compressive strain (ANOVA, $p<0.05$). Oxygen diffusivity of bovine AF in the axial direction has been determined. The mechanical loading has a significant effect on oxygen transport in IVD tissues. This study is important in understanding nutritional transport in IVD tissues and related disk degeneration.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074504-074504-5. doi:10.1115/1.3127255.

The deformation of multiple red blood cells in a capillary flow was studied numerically. The immersed boundary method was used for the fluid red blood cells interaction. The membrane of the red blood cell was modeled as a hyperelastic thin shell. The numerical results show that the apparent viscosity in the capillary flow is more sensitive to the change of shear coefficient of the membrane than the bending coefficient and surface dilation coefficient, and the increase in the shear coefficient results in an increase in the pressure drop in the blood flow in capillary vessels in order to sustain the same flux rate of red blood cells.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074505-074505-4. doi:10.1115/1.3127258.

Grooved structures have been widely studied in particle separation and fluid mixing in microfluidic channel systems. In this brief report, we demonstrate the use of patterning flows produced by two different sorts of grooved surfaces: single slanted groove series (for enrichment patterns) and V-shaped groove series (for focusing patterns), into a microfluidic device to continuously manipulate the flowing particles, including microbeads with $6 μm$, $10 μm$, and $20 μm$ in diameter and mouse dendritic cells of comparable sizes to the depth of the channel. The device with grooved channels was developed and fabricated by soft-lithographic techniques. The particle distributions after passing through the single slanted grooves illustrate the size-dependent enrichment profiles. On the other hand, particles passing through the V-shaped grooves show focusing patterns downstream, for the combination effect from both sides of single slanted grooves setup side-by-side. Compared with devices utilizing sheath flows, the focusing patterns generated in this report are unique without introducing additional flow control. The alignment of the concentrated particles is expected to facilitate the visualization of sizing and counting in cell-based devices. On the other hand, the size-dependent patterns of particle distributions have the potential for the application of size-based separation.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074506-074506-5. doi:10.1115/1.3127260.

A thermal model was needed to predict temperatures in a perfused tissue, which satisfied the following three criteria. One, the model satisfied conservation of energy. Two, the heat transfer rate from blood vessels to tissue was modeled without following a vessel path. Three, the model applied to any unheated and heated tissue. To meet these criteria, a generic bioheat transfer model (BHTM) was derived here by conserving thermal energy in a heated vascularized finite tissue and by making a few simplifying assumptions. Two linear coupled differential equations were obtained with the following two variables: tissue volume averaged temperature and blood volume averaged temperature. The generic model was compared with the widely employed empirical Pennes’ BHTM. The comparison showed that the Pennes’ perfusion term $wCp(1−ε)$ should be interpreted as a local vasculature dependent heat transfer coefficient term. Suggestions are presented for further adaptations of the general BHTM for specific tissues using imaging techniques and numerical simulations.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074507-074507-12. doi:10.1115/1.3128671.

The Arrhenius and thermal isoeffective dose (TID) models are the two most commonly used models for predicting hyperthermic injury. The TID model is essentially derived from the Arrhenius model, but due to a variety of assumptions and simplifications now leads to different predictions, particularly at temperatures higher than $50°C$. In the present study, the two models are compared and their appropriateness tested for predicting hyperthermic injury in both the traditional hyperthermia (usually, $43–50°C$) and thermal surgery (or thermal therapy/thermal ablation, usually, $>50°C$) regime. The kinetic parameters of thermal injury in both models were obtained from the literature (or literature data), tabulated, and analyzed for various prostate and kidney systems. It was found that the kinetic parameters vary widely, and were particularly dependent on the cell or tissue type, injury assay used, and the time when the injury assessment was performed. In order to compare the capability of the two models for thermal injury prediction, thermal thresholds for complete killing (i.e., 99% cell or tissue injury) were predicted using the models in two important urologic systems, viz., the benign prostatic hyperplasia tissue and the normal porcine kidney tissue. The predictions of the two models matched well at temperatures below $50°C$. At higher temperatures, however, the thermal thresholds predicted using the TID model with a constant $R$ value of 0.5, the value commonly used in the traditional hyperthermia literature, are much lower than those predicted using the Arrhenius model. This suggests that traditional use of the TID model (i.e., $R=0.5$) is inappropriate for predicting hyperthermic injury in the thermal surgery regime $(>50°C)$. Finally, the time-temperature relationships for complete killing (i.e., 99% injury) were calculated and analyzed using the Arrhenius model for the various prostate and kidney systems.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074508-074508-4. doi:10.1115/1.3147745.

Effect of formalin fixation on thermal conductivity of the biological tissues is presented. A self-heated thermistor probe was used to measure the tissue thermal conductivity. The thermal conductivity of porcine aorta, fat, heart, and liver was measured before the formalin fixation and then 1 day, 4 days, and 11 days after formalin fixation. The results indicate that the formalin fixation does not cause a significant change in the tissue thermal conductivity of the tissues studied. In the clinical setting, tissues removed surgically are often fixed in formalin for subsequent pathological analysis. These results suggest that, in terms of thermal properties, it is equally appropriate to perform in vitro studies in either fresh tissue or formalin-fixed tissue.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074509-074509-5. doi:10.1115/1.3143027.

Intense but short electrical fields can increase the permeability of the cell membrane in a process referred to as electroporation. Reversible electroporation has become an important tool in biotechnology and medicine. The various applications of reversible electroporation require cells to survive the procedure, and therefore the occurrence of irreversible electroporation (IRE), following which cells die, is obviously undesirable. However, for the past few years, IRE has begun to emerge as an important minimally invasive nonthermal ablation technique in its own right as a method to treat tumors and arrhythmogenic regions in the heart. IRE had been studied primarily to define the upper limit of electrical parameters that induce reversible electroporation. Thus, the delineation of IRE from thermal damage due to Joule heating has not been thoroughly investigated. The goal of this study was to express the upper bound of IRE (onset of thermal damage) theoretically as a function of physical properties and electrical pulse parameters. Electrical pulses were applied to THP-1 human monocyte cells, and the percentage of irreversibly electroporated (dead) cells in the sample was quantified. We also determined the upper bound of IRE (onset of thermal damage) through a theoretical calculation that takes into account the physical properties of the sample and the electric pulse characteristics. Our experimental results were achieved below the theoretical curve for the onset of thermal damage. These results confirm that the region to induce IRE without thermal damage is substantial. We believe that our new theoretical analysis will allow researchers to optimize IRE parameters without inducing deleterious thermal effects.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074510-074510-4. doi:10.1115/1.3143030.

The healing effect of therapeutic hyperthermia induced by widely available heat wrap products is understood to be based on concomitant temperature dependent vasodilation and increase in mass transport. We hypothesize that an additional mechanism of healing associated with increased heat shock protein (HSP) expression is also a contributing factor. HSP expression is controlled by the level and duration of heating and can have a potent effect on healing. We have developed a combined thermal stress and HSP expression model for bioheat transport into the tissues of the back produced by a therapeutic heat wrap. The model predicts temperature distribution in the deep tissues of the back by a modified version of the Pennes (1948, “Analysis of Tissue and Arterial Blood Temperatures in the Resting Human Forearm,” J. Appl. Physiol., 1(2), pp. 93–122) bioheat equation. The model also predicts HSP70/actin concentrations based on existing empirical expression data from our laboratory as a function of heating time and temperature. Thermal boundary conditions were input for a typical heat wrap worn for its functional duration of 8 h or more. Temperatures in the paraspinal muscles of the back increase by a minimum of $1°C$ after 1 h of heating and persist for at least 2 h. HSP70/actin expression is increased 1.7-fold above the control. The model demonstrates that elevated HSP expression may provide an important contribution to the healing process in injured tissue when a therapeutic heat wrap is worn.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074511-074511-5. doi:10.1115/1.3143034.

The freezing step influences lyophilization efficiency and protein stability. The main objective of this work was to investigate the impact on the primary drying stage of an ultrasound controlled ice nucleation technology, compared with usual freezing protocols. Lyophilization cycles involving different freezing protocols (applying a constant shelf cooling rate of $1°C/min$ or $0.2°C/min$, putting vials on a precooled shelf, and controlling nucleation by ultrasounds or by addition of a nucleating agent) were performed in a prototype freeze-dryer. Three protective media including sucrose or maltodextrin and differing by their thermal properties and their ability to preserve a model protein (catalase) were used. The visual aspect of the lyophilized cake, residual water content, and enzymatic activity recovery of catalase were assessed after each lyophilization cycle and after 1 month of storage of the lyophilized product at $4°C$ and $25°C$. The freezing protocols allowing increasing nucleation temperature (precooled shelf and controlled nucleation by using ultrasounds or a nucleating agent) induced a faster sublimation step and higher sublimation rate homogeneity. Whatever the composition of the protective medium, applying the ultrasound technology made it possible to decrease the sublimation time by 14%, compared with the freezing method involving a constant shelf cooling rate of $1°C/min$. Concerning the enzyme activity recovery, the impact of the freezing protocol was observed only for the protective medium involving maltodextrin, a less effective protective agent than sucrose. Higher activity recovery results were obtained after storage when the ultrasound technology or the precooled shelf method was applied. Controlling ice nucleation during the freezing step of the lyophilization process improved the homogeneity of the sublimation rates, which will, in turn, reduce the intervial heterogeneity. The freeze-dryer prototype including the system of controlled nucleation by ultrasounds appears to be a promising tool in accelerating sublimation and improving intrabatch homogeneity.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074512-074512-7. doi:10.1115/1.3147746.

Gold nanoparticles have been found to greatly enhance the polymerase chain reaction (PCR) specificity and yield in recent studies. However, the underlying mechanism is still unclear, though different hypotheses have already been proposed. In this study, a mass-action based model has been developed to investigate the effect of Au nanoparticles on the two-round PCR results. The great affinity of Au nanoparticles to the single-stranded DNA is taken into consideration. Each nanoparticle is treated as a bioreactor and/or a selector, around which, reaction equations are coupled to simulate the particle effect, and to investigate the key parameters that might influence such an effect. It is assumed that there exists a competing mechanism between the specific and nonspecific bindings, both in the solution and on the particle surface during the reactions. The numerical predictions accord well to the experimental results, and can be used to explain the Au nanoparticles’ effect on the enhancement of the PCR specificity and efficiency.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074513-074513-4. doi:10.1115/1.3153325.

The efficacy of chemotherapy is significantly impaired by the multidrug resistance (MDR) of cancer cells. The mechanism of MDR is associated with the overexpression of certain adenosine triphosphate-binding cassette protein transporters in plasma membranes, which actively pump out cytotoxic drugs from the intracellular space. In this study, we tested a hypothesis that freezing and thawing (F/T) may enhance intracellular drug delivery to MDR cancer cells via F/T-induced denaturation of MDR-associated proteins and/or membrane permeabilization. After a human MDR cancer cell line (NCI/ADR-RES) was exposed to several F/T conditions, its cellular drug uptake was quantified by a fluorescent calcein assay using calcein as a model drug. After F/T to $−20°C$, the intracellular uptake of calcein increased by 70.1% ($n=5$, $P=0.0004$). It further increased to 118% as NCI/ADR-RES cells were frozen/thawed to $−40°C$ ($n=3$, $P=0.009$). These results support the hypothesis, and possible mechanisms of F/T-enhanced intracellular drug delivery were proposed and discussed.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074514-074514-4. doi:10.1115/1.3156810.

Ionic liquids (ILs) have shown excellent promise as both solutes and solvents for stabilizing proteins at room temperature. Because many modern drugs are protein-based, these stabilizing characteristics have great potential to provide advances in the field of liquid formulation of therapeutic proteins. However, before these developments can be translated into clinical solutions it is essential to establish data related to the biocompatibility of these ILs. The current work investigates the cytotoxicity of several ILs that were rationally synthesized from natural biomolecules and compounds that have already been approved as excipients for drug formulations. The effect of choline dihydrogen phosphate (choline dhp), choline saccharinate, and 1-butyl 3-methyl imidazolium lactate (bmim lactate) on the metabolic activity of a mouse macrophage cell line (J774) was assessed using the reduction in resazurin as an indicator of activity and, by extension, viability. Two formulations of lysozyme (10 mg/ml and 100 mg/ml) in $80 wt %$ choline dhp (aq) were prepared and the proteins were evaluated for structural stability immediately following formulation and again at 1 month. Equivalent formulations in 0.1 M Na acetate aqueous buffer were evaluated as controls. A differential scanning microcalorimeter (DSC) was used to evaluate the structural stability on the basis of the unfolding temperature and the enthalpy of unfolding, and a micrococcus lysodiekticus activity test was used to evaluate functional activity. All compounds were found to be relatively benign, with toxicity increasing in the order choline $dhp lactate. At 1 month lysozyme that had been stored in choline dhp had a higher activity and folded fraction than lysozyme that had been stored in aqueous buffer. These results suggest that biocompatibility and protein stabilization characteristics can be rationally designed into ionic liquids.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074515-074515-6. doi:10.1115/1.3153326.

A parametric study was performed to understand the effect of preparation parameters on size, morphology, and encapsulation efficiency (i.e., cells/microcapsule) of alginate microcapsules prepared using the electrostatic spray method. The preparation parameters studied include sodium alginate concentration, spray voltage, flow rate, and cell density. It was found that both the flow rate and spray voltage have a significant impact on microcapsule size while the microcapsule morphology is greatly influenced by both the sodium alginate concentration and spray voltage. To obtain small $(∼100 μm)$ cell-loaded microcapsules with good morphology (i.e., round in shape and uniform in size) and high encapsulation efficiency ($>5$ cells/microcapsule), the optimal ranges of spray voltage, flow rate, alginate concentration, and cell density are from 1.6–1.8 kV, 1.5–3 ml/h, >1.5% (w/v), and (3–5)$×$106 cells/ml, respectively. Under optimal preparation conditions, cells were found to survive the microencapsulation process well.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074516-074516-5. doi:10.1115/1.3153310.

Mechanisms governing endothelial cell (EC) injury during arterial gas embolism have been investigated. Such mechanisms involve multiple scales. We have numerically investigated the macroscale flow dynamics due to the motion of a nearly occluding finite-sized air bubble in blood vessels of various sizes. Non-Newtonian behavior due to both the shear-thinning rheology of the blood and the Fahraeus–Lindqvist effect has been considered. The occluding bubble dynamics lends itself for an axisymmetric treatment. The numerical solutions have revealed several hydrodynamic features in the vicinity of the bubble. Large temporal and spatial shear stress gradients occur on the EC surface. The stress variations manifest in the form of a traveling wave. The gradients are accompanied by rapid sign changes. These features are ascribable to the development of a region of recirculation (vortex ring) in the proximity of the bubble. The shear stress gradients together with sign reversals may partially act as potential causes in the disruption of endothelial cell membrane integrity and functionality.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074517-074517-3. doi:10.1115/1.3156802.

Fourier transform infrared spectroscopy (FTIR) provides a unique technique to study membranes and proteins within their native cellular environment. FTIR was used here to study the effects of dimethyl sulfoxide $(Me2SO)$ on membranes and proteins in human pulmonary endothelial cells (HPMECs). Temperature-dependent changes in characteristic lipid and protein vibrational bands were identified to reveal the effects of $Me2SO$ on membrane phase behavior and protein stability. At $Me2SO$ concentrations equal to or below 10% (v/v), $Me2SO$ was found to decrease membrane conformational disorder. At higher $Me2SO$ concentrations (15% v/v), however, membrane conformational disorder was found to be similar to that of cells in the absence of $Me2SO$. This effect was observed over a wide temperature range from $90°C$ down to $−40°C$. $Me2SO$ had no clear effects on cellular proteins during freezing. During heating, however, $Me2SO$ had a destabilizing effect on cellular proteins. In the absence of $Me2SO$, protein denaturation started at an onset temperature of $46°C$, whereas at 15% $Me2SO$ the onset temperature of protein denaturation decreased to $32°C$. This implies that in the presence of $Me2SO$ the onset temperature of protein denaturation is lower than the normal growth temperature of the cells, which could explain the well documented toxic effect of $Me2SO$ at physiological temperatures. $Me2SO$ destabilizes cellular proteins during heating and decreases membrane conformational disorder over a wide temperature range.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074518-074518-6. doi:10.1115/1.3167804.

In the 1990s, there were two experimental studies that sparked a renewed interest in thermal wave behavior at the macroscale level. Both reported thermal relaxation times of 10 s or higher. However, no further experimental evidence of this behavior has been reported. Due to the extreme significance of these findings, the objectives of this study were to try to reproduce these earlier studies and offer an explanation for the outcome. These two previous studies, one using heterogeneous materials and one using bologna, were repeated following the experimental protocol provided in the studies as closely and as practically as possible. In both cases, the temperature response to a specified boundary condition was recorded. The results from the first set of experiments suggested that the thermal relaxation times presented in the previous study were actually the thermal lag expected from applying Fourier’s law, taking into account the uncertainty of the temperature sensor. In the second set of experiments, unlike the distinct jumps in temperature found previously, no indication of wave behavior was found. Here, the explanation for the previous results was more difficult to ascertain. Possible explanations include problems with either the experimental protocol or the temperature sensors used.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074519-074519-5. doi:10.1115/1.3169247.

Melanoma is the deadliest form of skin cancer and has the fastest growth rate of all cancer types. Proper staging of melanoma is required for clinical management. One method of staging melanoma is performed by taking a sentinel node biopsy, in which the first node in the lymphatic drainage path of the primary lesion is removed and tested for the presence of melanoma cells. Current standard of care typically involves taking fewer than ten histologic sections of the node out of the hundreds of possible sections available in the tissue. We have developed a photoacoustic method that probes the entire intact node. We acquired a lymph node from a healthy canine subject. We cultured a malignant human melanoma cell line HS 936. Approximately $1×106$ cells were separated and injected into the lymph node. We also had a healthy lymph node in which no melanoma cells were implanted. We used a tunable laser system set at 532 nm to irradiate the lymph nodes. Three piezoelectric acoustic detectors were positioned near the lymph node to detect photoacoustic pulses generated within the lymph nodes. We also acquired lymph nodes from pigs and repeated the experiments with increased amplification and improved sensors. We detected photoacoustic responses from a lymph node with as few as 500 melanoma cells injected into the tissue, while normal lymph nodes showed no response. Photoacoustic generation can be used to detect melanoma micrometastasis in sentinel lymph nodes. This detection can be used to guide further histologic study of the node, increasing the accuracy of the sentinel lymph node biopsy.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074520-074520-4. doi:10.1115/1.3171565.

Proteins and enzymes can be encapsulated in nanoporous gels to develop novel technologies for biosensing, biocatalysis, and biosynthesis. When encapsulated, certain macromolecules retain high levels of activity and functionality and are more resistant to denaturation when exposed to extremes of $pH$ and temperature. We have utilized intrinsic fluorescence and Fourier transform infrared spectroscopy to determine the structural transitions of encapsulated lysozyme in the range of $−120°C. At cryogenic temperatures encapsulated lysozyme did not show cold denaturation, instead became more structured. However, at high temperatures, the onset of heat denaturation of confined lysozyme was reduced by $15°C$ when compared with lysozyme in solution. Altered dynamics of the solvent and pore size distribution of the nanopores in the matrix appear to be key factors influencing the decrease in the denaturation temperature.

Commentary by Dr. Valentin Fuster
J Biomech Eng. 2009;131(7):074521-074521-8. doi:10.1115/1.3173283.

A major challenge encountered in using electrospun scaffolds for tissue engineering is the non-uniform cellular distribution in the scaffold with increasing depth under normal passive seeding conditions. Because of the small surface pores, typically few microns in diameter, cells tend to congregate and proliferate on the surface much faster compared to penetrating the scaffold interior. In order to overcome this problem, we used a vacuum seeding technique on polycaprolactone electrospun scaffolds while using NIH 3T3 fibroblasts as the model cell system. This serves as a precursor to the bilayer skin model where the fibroblasts would be residing at an intermediate layer and the keratinocytes would be on the top. Vacuum seeding was used in this study to enhance fibroblasts seeding and proliferation at different depths. Our results show that the kinetics of cell attachment and proliferation were a function of varying vacuum pressure as well as fiber diameter. Cell attachment reached a maxima somewhere between 2–8 in. Hg vacuum pressure and fell for lower vacuum pressures presumably because of cell loss through the filtration process. Cell proliferation and collagen secretion over five days indicated that vacuum pressure did not affect cellular function adversely. We also compared the combined impact of scaffold architecture (400 nm versus 1100 nm average diameter fiber scaffolds) and vacuum pressure. At a given pressure, more cells were retained in the 400 nm scaffolds compared to 1100 nm scaffolds. In addition, the cell intensity profile shows cell intensity peak shift from the top to the inner layers of the scaffold by lowering the vacuum pressure from 0 in. Hg to 20 in. Hg. For a given vacuum pressure the cells were seeded deeper within the 1100 nm scaffold. The results indicate that cells can be seeded in electrospun scaffolds at various depths in a controlled manner using a simple vacuum seeding technique. The depth of seeding is a function of pressure and scaffold fiber diameter.

Topics: Vacuum , Pressure
Commentary by Dr. Valentin Fuster

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