Observation of Interfacial Instability of an Ultrathin Water Film.

We observed the instability of a few-nanometer-thick water film encapsulated inside a graphene nanoscroll using transmission electron microscopy. The film, that was left after recession of a meniscus, formed ripples along the length of the nanoscroll with a distance only 20%-44% of that predicted by the classical Plateau-Rayleigh instability theory. The results were explained by a theoretical analysis that incorporates the effect of the van der Waals interactions between the water film and the graphene layers. We derived important insights into the behavior of liquid under nanoscale confinement and in nanofluidic systems.

Feasibility of Concentric Electrodes in Contact Irreversible Electroporation for Superficial Lesion Treatment.

OBJECTIVE: Contact irreversible electroporation (IRE) is a method for ablating cells by applying electric pulses via surface electrodes in contact with a target tissue. To facilitate the application of the contact IRE to superficial lesion treatment, this study further extended the ablation depth, which had been limited to a 400-µm depth in our previous study, by using concentric electrodes. METHODS: A prototype device of concentric electrodes was manufactured using a Teflon-coated copper wire inserted in a copper tube. The ablation area was experimentally determined using a tissue phantom comprising 3D cultured fibroblasts and compared with the electric field distribution obtained using numerical analyses. RESULTS: Experiments showed that cells 540 µm from the surface of the tissue phantom were necrotized by the application of 150 pulses at 100 V. The outline of the ablation area agreed well with the contour line of 0.4 kV/cm acquired by the analyses. The ablation depth predicted for the concentric electrode using this critical electric field was 1.4 times deeper than that for the parallel electrode. For the actual application of treatment, a multiple-electrode device that bundles several pairs of concentric electrodes was developed, and confirmed that to be effective for treating wide areas with a single treatment. CONCLUSION: The electric field estimated by the analyses with the experimentally determined threshold confirmed that concentric electrodes could attain a deeper ablation than parallel electrodes. SIGNIFICANCE: Using the concentric electrodes, we were able to localize ablation to specific target cells with much less damage to neighboring cells.

Application of the Taguchi method to explore a robust condition of tumor-treating field treatment.

Tumor-treating fields have potential as minimally invasive cancer treatment. This study aimed to explore the optimum tumor-treating field conditions that minimize unpredicted variations in therapeutic outcomes resulting from differences in cell size and electrical properties. The electric field concentration that induces a dielectrophoretic force near the division plane of a mitotic cell was calculated by finite element analysis for 144 cases, based on different combinations of six noise factors associated with cells and four controllable factors including frequency, as determined by the Taguchi method. Changing the frequency from 200 to 400 kHz strongly increased robustness in producing a dielectrophoretic force, irrespective of noise factors. However, this frequency change reduced the force magnitude, which can be increased by simply applying a higher voltage. Based on additional simulations that considered this trade-off effect, a frequency of 300 kHz is recommended for a robust TTF treatment with allowable variations. The dielectrophoretic force was almost independent of the angle of applied electric field deviated from the most effective direction by ±20 degrees. Furthermore, increased robustness was observed for extracellular fluid with higher conductivity and permittivity. The Taguchi method was useful for identifying robust tumor-treating field therapy conditions from a considerably small number of replicated simulations.

Atomic-level breakdown of Green-Kubo relations provides new insight into the mechanisms of thermal conduction.

Precise control of thermophysical properties of liquids through tailor-made design of the liquid molecular structure is a goal that, if achieved, could have significant positive impacts on machine design, performance and durability. In this work we show how the breakdown of the Green-Kubo relations down to the atomic level in molecular dynamics simulation can give useful insight into the mechanisms of thermal conduction. Using a group of five small alcohols as a case study, we demonstrate how combining this level of insight with differential-structure analysis reveals the competition for conduction between carbon and hydroxyl group atoms, and show how this competition contributes to the change in thermal conductivity observed in experiment. We hope that this method will become a useful tool in the quest for molecular-structure based thermal design.

Evaluation of Temperature Distribution Around the Probe in Cryoablation of Lipiodol-Mixed-Tissue Phantom.

PURPOSE: To determine whether lipiodol, which has low thermal conductivity, influences ice ball formation during cryoablation of a lipiodol-mixed-tissue phantom. MATERIALS AND METHODS: Lipiodol-mixed-tissue phantoms were created by injecting lipiodol (4-6 ml) into the renal arteries of ex vivo porcine kidneys (lipiodol group). A cryoprobe (CryoHit™ Needle S) with a holder that was set with thermocouples at various positions around the cryoprobe was inserted. After freezing for 300 s, the followings were evaluated: ice ball size on CT, temperature distribution around the cryoprobe, and calculated distances at 0 °C and - 20 °C. Each variable was compared between lipiodol group (n = 6) those obtained in a control group without lipiodol injection (n = 6). RESULTS: Mean ice ball diameter (width/length) on CT was 22.1 ± 2.3/22.9 ± 2.3 mm in the lipiodol group and 21.6 ± 0.7/22.2 ± 1.3 mm in the control group. Mean cryoprobe temperature was - 118 ± 3.0 °C in the lipiodol group and - 117 ± 2.6 °C in the control group. In both groups, temperature at the 3 mm thermocouple reached approximately - 50 °C and was < 0 °C within ~ 10 mm of the cryoprobe. Temperature of 0/- 20 °C occurred at a mean distance from the cryoprobe of 11.1 ± 0.5/6.9 ± 0.4 mm in the lipiodol group and 11.0 ± 0.2/6.9 ± 0.2 mm in the control group. There was no significant difference in any variable between the groups. CONCLUSION: The inclusion of lipiodol in a tissue phantom had no negative effects on ice ball formation that were related to thermal conductivity.

Fundamental Evaluation of Thermophysical Properties of Lipiodol Associated with Cryoablation: Freezing Experiments Using Lipiodol Phantom.

PURPOSE: To elucidate the basic thermophysical properties at low temperatures of lipiodol, which is used as a marker by transarterial injection before CT-guided cryoablation for solid tumors, by fundamental experiments with pure lipiodol phantom. MATERIALS AND METHODS: The freezing point of lipiodol was measured using differential scanning calorimeter (DSC) by detecting differences in the heating rate during heating from - 30 °C. Freezing experiments were conducted using pure lipiodol and a tissue phantom, which were prepared in an acrylic container at 37 °C. The growth of the frozen region was observed for 10 min. Temperatures were monitored at the cryoprobe surface and designated positions around the cryoprobe. RESULTS: The DSC experiment showed that freezing was observed between - 5 and - 30 °C, which indicated that the freezing point was approximately - 5 °C. Freezing experiments revealed that the diameter of frozen region in the lipiodol was smaller than that in the tissue phantom (5 mm vs 24 mm) after 10-min freezing. The temperature at the probe surface was - 130 °C in lipiodol, which was 25 °C lower than that in the tissue phantom. There was a larger temperature gradient near the cryoprobe in lipiodol due to lower thermal conductivity. CONCLUSIONS: The present results suggest that an extremely high concentration of lipiodol (close to pure lipiodol) potentially reduces frozen region because of its lower freezing point and smaller thermal conductivity. However, since lipiodol concentrations in clinical cases differ from the current model, further studies using models that are close to clinical conditions are required. LEVEL OF EVIDENCE: No level of evidence, laboratory investigation.

Low-Voltage Irreversible Electroporation Using a Comb-Shaped Contact Electrode.

OBJECTIVE: Irreversible electroporation (IRE) is a less invasive therapy to ablate tumor cells by delivering short intensive electric pulses more than a few kV via needle-like electrodes. For reducing the required voltage for the IRE, a durable comb-shaped miniature electrode was designed to use in contact with the lesion surface for a new method named contact IRE. METHODS: A miniature electrode was newly fabricated by a fine inkjet patterning and the subsequent etching of a copper-clad polyimide film. A train of 10-µs or 100-µs long electric pulses were applied 90 times at the interval of 1 s to a tissue phantom, and its cross section was observed to measure the necrotized area. RESULTS: Cell experiments showed that the maximum ablation depth increased as a function of the applied voltage and reached 400 µm at 20 V. Furthermore, insulation of the lateral space between electrode teeth with a resin and administration of adjuvants to reduce the IRE threshold of the cell membrane did increase the ablation depth by 26% and the ablation area by 40%. CONCLUSION: The miniature electrode developed in this study successfully necrotized cells in a tissue phantom 400 µm deep from the surface with the electric pulses of only 20 V. SIGNIFICANCE: The contact IRE for the surface of skin and gastrointestinal tract will ablate cutaneous and subcutaneous tumors by applying only several tens of volts.

Experimental study of thermal rectification in suspended monolayer graphene.

Thermal rectification is a fundamental phenomenon for active heat flow control. Significant thermal rectification is expected to exist in the asymmetric nanostructures, such as nanowires and thin films. As a one-atom-thick membrane, graphene has attracted much attention for realizing thermal rectification as shown by many molecular dynamics simulations. Here, we experimentally demonstrate thermal rectification in various asymmetric monolayer graphene nanostructures. A large thermal rectification factor of 26% is achieved in a defect-engineered monolayer graphene with nanopores on one side. A thermal rectification factor of 10% is achieved in a pristine monolayer graphene with nanoparticles deposited on one side or with a tapered width. The results indicate that the monolayer graphene has great potential to be used for designing high-performance thermal rectifiers for heat flow control and energy harvesting.

Assessment of thermal damage in total knee arthroplasty using an osteocyte injury model.

Polymethylmethacrylate bone cement has been widely used for the anchorage of artificial implants in various orthopedic surgeries. Although it is one of the most successful biomaterials in use, excess heat generation intrinsically causes thermal damage to bone cells adjacent to the bone cement. To estimate a risk of thermal injury, a response of bone cells to cement polymerization must be elucidated because of the occurrence of thermal damage. Thermal damage is affected not only by maximal temperature but also by exposure time, temperature history, and cell type. This study aimed at quantifying the thermal tolerance of bone cells for the development of a thermal injury model, and applying this model for the estimation of thermal damage during cement polymerization in total knee arthroplasty. Osteocytes, osteoblasts, and fibroblasts were respectively subjected to steady supraphysiological temperatures ranging from 45 to 50°C. Survival curves of each cell and temperatures were used to formulate the Arrhenius model. A three-dimensional heat conduction analysis for total knee arthroplasty was conducted using the finite element model based on serial CT images of human knee. A maximal temperature rise of 50°C was observed at the interface between the 3-mm thick cement and the tissue immediately beneath the tibial tray of the prosthesis. The probability of thermal damage to the osteocyte, which was calculated using the Arrhenius model, was negligible at a distance of at least 1 mm away from the cement-bone interface. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2799-2807, 2017.

Ultraclean suspended monolayer graphene achieved by in situ current annealing.

Ultraclean graphene is essential for studying its intrinsic transport properties or fabricating high-performance electronic devices. Unfortunately, the contamination on graphene is unavoidable after microelectromechanical system processing. Here, we report an in situ current-annealing method for achieving ultraclean suspended monolayer graphene. The charge mobility of cleaned graphene reached a surprising 3.8 × 105 cm2 V-1 s-1, one of the highest values ever reported. For the first time, the process of current annealing was recorded under a high-resolution electron scanning microscope. It was demonstrated that temperature was the only dominant factor of the current-annealing process. Meanwhile, the mobility of suspended graphene was found to be highly sensitive to structural defects. The mobility decreased by a factor of over 100 after ion irradiation on graphene. The results revealed the underlying mechanism of current annealing on graphene and provided an effective means of preparing ultraclean graphene membranes.

In-situ measurement of the heat transport in defect- engineered free-standing single-layer graphene.

Utilizing nanomachining technologies, it is possible to manipulate the heat transport in graphene by introducing different defects. However, due to the difficulty in suspending large-area single-layer graphene (SLG) and limited temperature sensitivity of the present probing methods, the correlation between the defects and thermal conductivity of SLG is still unclear. In this work, we developed a new method for fabricating micro-sized suspended SLG. Subsequently, a focused ion beam (FIB) was used to create nanohole defects in SLG and tune the heat transport. The thermal conductivity of the same SLG before and after FIB radiation was measured using a novel T-type sensor method on site in a dual-beam system. The nanohole defects decreased the thermal conductivity by about 42%. It was found that the smaller width and edge scrolling also had significant restriction on the thermal conductivity of SLG. Based on the calculation results through a lattice dynamics theory, the increase of edge roughness and stronger scattering on long-wavelength acoustic phonons are the main reasons for the reduction in thermal conductivity. This work provides reliable data for understanding the heat transport in a defective SLG membrane, which could help on the future design of graphene-based electrothermal devices.

Microheterogeneity in frozen protein solutions.

In frozen and lyophilized systems, the biological to be stabilized (e.g. therapeutic protein, biomarker, drug-delivery vesicle) and the cryo-/lyo-protectant should be co-localized for successful stabilization. During freezing and drying, many factors cause physical separation of the biological from the cryo-/lyo-protectant, called microheterogeneity (MH), which may result in poor stabilization efficiency. We have developed a novel technique that utilized confocal Raman microspectroscopy in combination with counter-gradient freezing to evaluate the effect of a wide range of freezing temperatures (-20

The effect of solution nonideality on modeling transmembrane water transport and diffusion-limited intracellular ice formation during cryopreservation.

A new model was developed to predict transmembrane water transport and diffusion-limited ice formation in cells during freezing without the ideal-solution assumption that has been used in previous models. The model was applied to predict cell dehydration and intracellular ice formation (IIF) during cryopreservation of mouse oocytes and bovine carotid artery endothelial cells in aqueous sodium chloride (NaCl) solution with glycerol as the cryoprotectant or cryoprotective agent. A comparison of the predictions between the present model and the previously reported models indicated that the ideal-solution assumption results in under-prediction of the amount of intracellular ice at slow cooling rates (<50 K/min). In addition, the lower critical cooling rates for IIF that is lethal to cells predicted by the present model were much lower than those estimated with the ideal-solution assumption. This study represents the first investigation on how accounting for solution nonideality in modeling water transport across the cell membrane could affect the prediction of diffusion-limited ice formation in biological cells during freezing. Future studies are warranted to look at other assumptions alongside nonideality to further develop the model as a useful tool for optimizing the protocol of cell cryopreservation for practical applications.

In situ spectroscopic quantification of protein-ice interactions.

FTIR and confocal Raman microspectroscopy were used to measure interactions between albumin and ice in situ during quasi-equilibrium freezing in dimethyl sulfoxide (DMSO) solutions. At temperatures of -4 and -6 °C, albumin was found to be preferentially excluded from the ice phase during near-equilibrium freezing. This behavior reversed at lower temperatures. Instead, DMSO was preferentially excluded from the ice phase, resulting in an albumin concentration in the freeze-concentrated liquid phase that was lower than predicted. It is hypothesized that this was caused by the albumin in the freeze-concentrated liquid getting adsorbed onto the ice surface or becoming entrapped in the ice phase. It was observed that, under certain freezing protocols, as much as 20% of the albumin in solutions with starting concentrations of 32-53 mg/mL may be adsorbed onto the ice interface or entrapped in the ice phase.

The effect of low-magnitude, high-frequency vibration stimuli on the bone healing of rat incisor extraction socket.

Effects of small vibration stimuli on bone formation have been reported. In the present study, we used morphological and morphometric procedures to elucidate whether low-magnitude, high-frequency (LMHF) vibration stimuli could enhance the bone healing of rat incisor extraction sockets. After extraction of incisors from six-week-old rats, animals were assigned into a control group and two experimental groups to receive 50 Hz stimuli at either 0.05 mm or 0.2 mm peak-to-peak for an hour/day. LMHF vibration stimuli were generated by placing the mandibles of the animals onto a vibration generator. All groups were subdivided into two, according to the study periods (1 and 3 weeks). After the study period, undecalcified ground sections were taken and morphological and morphometric analyses performed. At both 1 and 3 weeks, newly formed bone was observed mainly in the upper wall of the extraction socket in all groups. Morphometric analyses revealed that the trabecular thickness in both experimental groups at 1 week was significantly greater than that in the control. LMHF vibration stimuli had a positive effect on bone at the early stage of bone healing, particularly in trabecular thickness, at the incisor extraction socket.

Effect of irreversible electroporation on three-dimensional cell culture model.

Irreversible electroporation (IRE) is a new treatment to necrotize abnormal cells by high electric pulses. Electric potential difference over 1 V across the plasma membrane permanently permeabilizes the cell with keeping the extracellular matrix intact if the thermal damage due to the Joule heating effect is avoided. This is the largest advantage of the IRE compared to the other conventional treatment. However, since the IRE has just started to be used in clinical tests, it is important to predict the necrotized region that depends on pulse parameters and electrode arrangement. We therefore examined the numerical solution to the Laplace equation for the static electric field to predict the IRE-induced cell necrosis. Three-dimensionally (3-D) cultured cells in a tissue phantom were experimentally subjected to the electric pulses through a pair of puncture electrodes. The necrotized area was determined as a function of the pulse repetition and compared with the area that was estimated by the numerical analysis.

3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells.

Cell dehydration during freezing results from an elevated concentration of electrolytes in the extracellular medium that is deeply involved in cellular injury. We undertook real-time threedimensional (3-D) observation of osmotic dehydration of cells, motivated by a comparison of cellular responses between isolated cells in suspension and cultured cells adhering to a surface since several studies have suggested a difference in freeze tolerance between cell suspensions and monolayers. A laser confocal scanner was used with a perfusion microscope to capture sectional images of chloromethylbenzamido (DiI)-stained PC-3 cells that were exposed to an increase in NaCl concentration from 0.15 to 0.5M at 23 degrees C. Change in cell volume was determined from reconstructed 3-D images taken every 2.5s. When cells were exposed to an elevated NaCl concentration, isolated cells contracted and markedly distorted from their original spherical shape. In contrast, adhered cells showed only a reduction in height and kept their basal area constant. Apparent membrane hydraulic conductivity did not vary considerably between isolated and adhered cells, suggesting a negligible effect of the cytoskeletal structure on the rate of water transport. The surface area that contributed to water transport in adhered PC-3 cells was nearly equal to or slightly smaller than that present in isolated cells. Therefore, the similarity in properties and dimensions between isolated and adhered cells indicate that there will be similar extents of dehydration, resulting in a similar degree of supercooling during freezing.

In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.

Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions.

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at -10 degrees C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at -10 degrees C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at -20 degrees C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.

Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C.

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.