Integration of blue light with near-infrared irradiation accelerates the osteogenic differentiation of human dental pulp stem cells.

Blue light is used less in photobiomodulation than red or near-infrared light because of concerns about its high energy. However, some reports have suggested that blue light releases NO from nitrosated proteins, affects cell signal regulation, and promotes stem cell differentiation. Because blue and red lights could have different mechanisms of action, their combination is expected to have new consequences. In this study, human dental pulp stem cells (hDPSCs) were sequentially exposed to blue and near-infrared light to study their effects on proliferation, osteogenic differentiation, and immunomodulation. We found that NIR irradiation applied after blue light can reduce blue light toxicity improving the cell viabiltiy. Delayed luminescence and transmission electron microscopy studies showed that this combination excited hDPSCs and activated mitochondrial biogenesis. Those modulations accelerated hDPSC differentiation, as shown by an increase of about 1.3-fold in alkaline phosphatase activity in vitro and an about 1.5-fold increase in the osteocalcin-positive regions in cells implanted in nude mice compared with mice exposed to near-infrared alone.

Histological Response to 5†kHz Irreversible Electroporation in a Porcine Liver Model.

Unlike necrosis by thermal ablation, irreversible electroporation (IRE) is known to induce apoptosis by disrupting plasma membrane integrity with electric pulses while preserving the structure of blood vessels and bile ducts in liver tissue without a heat sink effect. This study aimed to investigate thermal damage and histopathological effects in the porcine liver by high-frequency electric pulses (5 kHz) which is much higher than the widely used 1 Hz. The electric field and thermal distributions of 5 kHz electric pulses were compared with those of 1 Hz in numerical simulations. 5 kHz-IRE was applied on pigs under ultrasound imaging to guide the electrode placement. The animals underwent computed tomography (CT) examination immediately and 1 day after IRE. After CT, IRE-treated tissues were taken and analyzed histologically. CT revealed that hepatic veins were intact for 1-day post-IRE. Histopathologically, the structure of the portal vein was intact, but endothelial cells were partially removed. In addition, the hepatic artery structure from which endothelial cells were removed were not damaged, while the bile duct structure and cholangiocytes were intact. The thermal injury was observed only in the vicinity of the electrodes as simulated in silico. 5 kHz-IRE generated high heat due to its short pulse interval, but the thermal damage was limited to the tissue around the electrodes. The histopathological damage caused by 5 kHz-IRE was close to that caused by 1 Hz-IRE. If a short-time treatment is required for reasons such as anesthesia, high-frequency IRE treatment is worth considering. Our observations will contribute to a better understanding of the IRE phenomena and search for advanced therapeutic conditions.

Photobiomodulation as an antioxidant substitute in post-thawing trauma of human stem cells from the apical papilla.

Cryopreservation, the most common method of preserving stem cells, requires post-processing because it produces trauma to the cells. Post-thawing trauma typically induces cell death, elevates reactive oxygen species (ROS) concentration, and lowers mitochondrial membrane potential (MMP). Although this trauma has been solved using antioxidants, we attempted to use photobiomodulation (PBM) instead of chemical treatment. We used a 950-nm near-infrared LED to create a PBM device and chose a pulsed-wave mode of 30 Hz and a 30% duty cycle. Near-infrared radiation (NIR) at 950 nm was effective in reducing cell death caused by hydrogen peroxide induced-oxidative stress. Cryodamage also leads to apoptosis of cells, which can be avoided by irradiation at 950 nm NIR. Irradiation as post-processing for cryopreservation had an antioxidant effect that reduced both cellular and mitochondrial ROS. It also increased mitochondrial mass and activated mitochondrial activity, resulting in increased MMP, ATP generation, and increased cytochrome c oxidase activity. In addition, NIR increased alkaline phosphatase (ALP) activity, a biomarker of differentiation. As a result, we identified that 950 nm NIR PBM solves cryodamage in human stem cells from the apical papilla, indicating its potential as an alternative to antioxidants for treatment of post-thawing trauma, and further estimated its mechanism.

Effects of Actin Cytoskeleton Disruption on Electroporation In Vitro.

The role of actin fibers in cellular responses to external electric pulses is not clear yet. In this study, we utilized the blocker of actin polymerization, cytochalasin D (cytoD), and investigated its effects on the electropore generation. Eight 100 µs electric pulses of sub-kilovolt per centimeter voltage with 100 ms intervals were applied to adhered cells in vitro, and the membrane permeability was quantified using membrane-impermeable propidium iodide (PI) dye. With cytoD application, the transfer of PI dye decreased significantly in all the applied voltages. At the same time, the roughness of cells increased, the membrane stiffness decreased, and the transmembrane resting potential decreased. Our result supports that actin fibers have clear effects on electroporation through modulating membrane properties including transmembrane resting potential.

Anticancer Activity of Liquid Treated with Microwave Plasma-Generated Gas through Macrophage Activation.

Reactive nitrogen species (RNS), including nitric oxide (NO·) has been known as one of the key regulatory molecules in the immune system. In this study, we generated RNS-containing water treated with microwave plasma-generated gas in which the major component was nitric oxide (PGNO), and the effect on the macrophage polarization was investigated. The RNS-containing water was diluted in complete cell culture media (PGNO-solution) into the concentration that did not induce cell death in RAW 264.7 murine macrophages. PGNO-solution upregulates M1-type macrophage activation and downregulates the characteristics of M2-type macrophage at the transcriptional level. In addition, the PGNO-solution-treated M2-like macrophages had higher potential in killing melanoma cells. The anticancer potential was also investigated in a syngeneic mouse model. Our results show that PGNO-solution has the potential to convert the fate of macrophages, suggesting PGNO-solution treatment as a supportive method for controlling the function of macrophages under the tumor microenvironment.

Physicochemical factors that affect electroporation of lung cancer and normal cell lines.

Electroporation is used for cancer therapy to efficiently destroy cancer tissues by transferring anticancer drugs into cancer cells or by irreversible tumor ablation without resealing pores. There is growing interest in the electroporation method for the treatment of lung cancer, which has the highest mortality rate among cancers. Improving the cancer cell selectivity has the potential to expand its use. However, the factors that influence the cell selectivity of electroporation are debatable. We aimed to identify the important factors that influence the efficiency of electroporation in lung cells. The electropermeabilization of lung cancer cells (H460, A549, and HCC1588) and normal lung cells (MRC5, WI26 and L132) was evaluated by the transfer of fluorescence dyes. We found that membrane permeabilization increased as cell size, membrane stiffness, resting transmembrane potential, and lipid cholesterol ratio increased. Among them, lipid composition was found to be the most relevant factor in the electroporation of lung cells. Our results provide insight into the differences between lung cancer cells and normal lung cells and provide a basis for enhancing the sensitivity of lung cancers cells to electroporation.

Influence of oxygen on generation of reactive chemicals from nitrogen plasma jet.

A nonthermal plasma jet is operated at atmospheric pressure inside a vacuum chamber filled with nitrogen gas. Various chemical compounds are fabricated from nitrogen and water molecules in plasma jet with varying oxygen content. Detailed theoretical investigation of these chemical compounds is carried out in terms of different oxygen ratio ξ. Experimental measurements are also carried out for comparison with theoretical results. Hydroxyl molecules are mostly generated at surface of water, and some of them can penetrate into water. The density of hydroxyl molecules has its maximum without oxygen, and decreases to zero as ξ increases to 0.25. The density of the ammonia of NH3 also deceases as ξ increases to 0.25. On the other hand, theory and experiment show that the density of the NO3 increases drastically as ξ increases to 0.25. The hydrogen peroxide density in plasma activated water deceases, reaches its minimum value at ξ = 0.05, and then increases again, as ξ increases from a small value to a large value. The pH value of the plasma activated water, which is slightly changed to alkali without oxygen, decreases as ξ increases.

Effects of pulsing of light on the dentinogenesis of dental pulp stem cells in vitro.

Low power light (LPL) treatment has been widely used in various clinical trials, which has been known to reduce pain and inflammation and to promote wound healing. LPL was also shown to enhance differentiation of stem cells into specific lineages. However, most studies have used high power light in mW order, and there was lack of studies about the effects of very low power light in µW. In this study, we applied 810 nm LPL of 128 µW/cm2 energy density in vitro. Upon this value, continuous wave (CW) irradiation did not induce any significant changes for differentiation of human dental pulp stem cells (hDPSCs). However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the pulsed wave (PW) with 30% of duty cycle and 300-3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency.

Pulse frequency dependency of photobiomodulation on the bioenergetic functions of human dental pulp stem cells.

Photobiomodulation (PBM) therapy contributes to pain relief, wound healing, and tissue regeneration. The pulsed wave (PW) mode has been reported to be more effective than the continuous wave (CW) mode when applying PBM to many biological systems. However, the reason for the higher effectiveness of PW-PBM is poorly understood. Herein, we suggest using delayed luminescence (DL) as a reporter of mitochondrial activity after PBM treatment. DL originates mainly from mitochondrial electron transport chain systems, which produce reactive oxygen species (ROS) and adenosine triphosphate (ATP). The decay time of DL depends on the pulse frequencies of applied light, which correlate with the biological responses of human dental pulp stem cells (hDPSCs). Using a low-power light whose wavelength is 810 nm and energy density is 38 mJ/cm2, we find that a 300-Hz pulse frequency prolonged the DL pattern and enhanced alkaline phosphatase activity. In addition, we analyze mitochondrial morphological changes and their volume density and find evidence supporting mitochondrial physiological changes from PBM treatment. Our data suggest a new methodology for determining the effectiveness of PBM and the specific pulse frequency dependency of PBM in the differentiation of hDPSCs.

The Role of Free Radicals in Hemolytic Toxicity Induced by Atmospheric-Pressure Plasma Jet.

Atmospheric-pressure plasma (APP) has received attention due to its generation of various kinds of reactive oxygen/nitrogen species (ROS/RNS). The controllability, as well as the complexity, is one of the strong points of APP in various applications. For biological applications of this novel method, the cytotoxicity should be estimated at various levels. Herein, we suggest red blood cell (RBC) as a good cell model that is simpler than nucleated cells but much more complex than other lipid model systems. Air and N2 gases were compared to verify the main ROS/RNS in cytotoxicity, and microscopic and spectroscopic analyses were performed to estimate the damages induced on RBCs. The results shown here will provide basic information on APP-induced cytotoxicity at cellular and molecular levels.

Histological and Mathematical Analysis of the Irreversibly Electroporated Liver Tissue.

Irreversible electroporation has clinically been used to treat various types of cancer. A plan on how to apply irreversible electroporation before practicing is very important to increase the ablation area and reduce the side effects. Several electrical models have been developed to predict the ablation area with applied electric energy. In this experiment, the static relationship between applied electric energy and ablated area was mathematically and experimentally investigated at 10 hours after applying irreversible electroporation. We performed the irreversible electroporation on the liver tissue of Sprague Dawley rats (male, 8 weeks, weighing 250-350 g). The ablated area was measured based on histological analysis and compared with the mathematical calculation from the electric energy, assuming that the tissue is homogeneous. The ablated area increased with the increase in applied electric energy. The numerically calculated contour lines of electric energy density overlapped well with the apoptotic area induced by the irreversible electroporation. The overlapped area clearly showed that the destructive threshold of apoptosis between electrodes is electric energy density level of 5.9 × 105 J/m3. The results of the present study suggested that the clinical results of the irreversible electroporation on a liver tissue could be predicted through mathematical calculation.

Closed-loop ARS mode for scanning ion conductance microscopy with improved speed and stability for live cell imaging applications.

Scanning ion conductance microscopy (SICM) is an increasingly useful nanotechnology tool for non-contact, high resolution imaging of live biological specimens such as cellular membranes. In particular, approach-retract-scanning (ARS) mode enables fast probing of delicate biological structures by rapid and repeated approach/retraction of a nano-pipette tip. For optimal performance, accurate control of the tip position is a critical issue. Herein, we present a novel closed-loop control strategy for the ARS mode that achieves higher operating speeds with increased stability. The algorithm differs from that of most conventional (i.e., constant velocity) approach schemes as it includes a deceleration phase near the sample surface, which is intended to minimize the possibility of contact with the surface. Analysis of the ion current and tip position demonstrates that the new mode is able to operate at approach speeds of up to 250 µm s(-1). As a result of the improved stability, SICM imaging with the new approach scheme enables significantly improved, high resolution imaging of subtle features of fixed and live cells (e.g., filamentous structures & membrane edges). Taken together, the results suggest that optimization of the tip approach speed can substantially improve SICM imaging performance, further enabling SICM to become widely adopted as a general and versatile research tool for biological studies at the nanoscale level.

Non-thermal plasma with 2-deoxy-D-glucose synergistically induces cell death by targeting glycolysis in blood cancer cells.

In this study, we show the selective and efficient anti-cancer effects of plasma (at a low dose) when cell metabolic modifiers are also included. 2-deoxy-D-glucose (2-DG), a glycolytic inhibitor, was used with effective doses of non-thermal plasma, synergistically attenuating cell metabolic viability and inducing caspase-dependent and independent cell death. The combination treatment decreased the intracellular ATP and lactate production in various types of blood cancer cells in vitro. Taken together, our findings suggest that 2-DG enhances the efficacy and selectivity of plasma and induces the synergistic inhibition of cancer cell growth by targeting glycolysis and apoptosis. Specifically, this treatment strategy demonstrated an enhanced growth inhibitory effect of plasma in the presence of a metabolic modifier that was selective against cancer cells, not non-malignant cells. This is the first study to report the advantage of combining plasma with 2-DG to eradicate blood cancer cells. Finally, we conclude that 2-DG with non-thermal plasma may be used as a combination treatment against blood cancer cells.

Responses of solid tumor cells in DMEM to reactive oxygen species generated by non-thermal plasma and chemically induced ROS systems.

In this study, we assessed the role of different reactive oxygen species (ROS) generated by soft jet plasma and chemical-induced ROS systems with regard to cell death in T98G, A549, HEK293 and MRC5 cell lines. For a comparison with plasma, we generated superoxide anion (O2(-)), hydroxyl radical (HO·), and hydrogen peroxide (H2O2) with chemicals inside an in vitro cell culture. Our data revealed that plasma decreased the viability and intracellular ATP values of cells and increased the apoptotic population via a caspase activation mechanism. Plasma altered the mitochondrial membrane potential and eventually up-regulated the mRNA expression levels of BAX, BAK1 and H2AX gene but simultaneously down-regulated the levels of Bcl-2 in solid tumor cells. Moreover, a western blot analysis confirmed that plasma also altered phosphorylated ERK1/2/MAPK protein levels. At the same time, using ROS scavengers with plasma, we observed that scavengers of HO· (mannitol) and H2O2 (catalase and sodium pyruvate) attenuated the activity of plasma on cells to a large extent. In contrast, radicals generated by specific chemical systems enhanced cell death drastically in cancer as well as normal cell lines in a dose-dependent fashion but not specific with regard to the cell type as compared to plasma.

Influence of hydroxyl group position and temperature on thermophysical properties of tetraalkylammonium hydroxide ionic liquids with alcohols.

In this work, we have explored the thermophysical properties of tetraalkylammonium hydroxide ionic liquids (ILs) such as tetrapropylammonium hydroxide (TPAH) and tetrabutylammonium hydroxide (TBAH) with isomers of butanol (1-butanol, 2-butanol and 2-methyl-2-propanol) within the temperature range 293.15-313.15 K, with interval of 5 K and over the varied concentration range of ILs. The molecular interactions between ILs and butanol isomers are essential for understanding the function of ILs in related measures and excess functions are sensitive probe for the molecular interactions. Therefore, we calculated the excess molar volume (V(E) ) and the deviation in isentropic compressibility (Δκs ) using the experimental values such as densities (ρ) and ultrasonic sound velocities (u) that are measured over the whole compositions range at five different temperatures (293.15, 298.15, 303.15, 308.15 and 313.15 K) and atmospheric pressure. These excess functions were adequately correlated by using the Redlich-Kister polynomial equation. It was observed that for all studied systems, the V(E) and Δκs values are negative for the whole composition range at 293.15 K. And, the excess function follows the sequence: 2-butanol>1-butanol>2-methyl-2-propanol, which reveals that (primary or secondary or tertiary) position of hydroxyl group influence the magnitude of interactions with ILs. The negative values of excess functions are contributions from the ion-dipole interaction, hydrogen bonding and packing efficiency between the ILs and butanol isomers. Hence, the position of hydroxyl group plays an important role in the interactions with ILs. The hydrogen bonding features between ILs and alcohols were analysed using molecular modelling program by using HyperChem 7.

Changes of apoptosis in tumor tissues with time after irreversible electroporation.

Irreversible electroporation is a novel method of ablating living tissues through its non-thermal effects, unlike radiofrequency ablation which has a severe problem of heat sink. It is due to high-energy direct current which leads to permanent disruption of lipid bilayer integrity in terms of exchanges between intra- and extracellular components via nano-sized pores. That finally causes irreversible damage to cellular homeostasis. Irreversibly damaged cells may undergo apoptosis followed by necrosis with time after electroporation. This damage can make it possible to monitor the ablated area with time post-IRE through MR imaging and an ultrasound system. Most previous studies have investigated the immediate response of undesired tissue to IRE. In our study, we showed changes of tumor tissues with time post-IRE by histological analysis and MR imaging. Tissues under IRE ablation showed a peak apoptotic rate at 24 h after IRE ablation with viable tissues at the peripheral rim of treated tissues in histological analysis. This phenomenon was also observed with no enhancement on contrast-enhanced MR images due to devascularization of IRE ablated zones.

Controlling the growth and differentiation of human mesenchymal stem cells by the arrangement of individual carbon nanotubes.

Carbon nanotube (CNT) networks on solid substrates have recently drawn attention as a means to direct the growth and differentiation of stem cells. However, it is still not clear whether cells can recognize individual CNTs with a sub-2 nm diameter, and directional nanostructured substrates such as aligned CNT networks have not been utilized to control cell behaviors. Herein, we report that human mesenchymal stem cells (hMSCs) grown on CNT networks could recognize the arrangement of individual CNTs in the CNT networks, which allowed us to control the growth direction and differentiation of the hMSCs. We achieved the directional growth of hMSCs following the alignment direction of the individual CNTs. Furthermore, hMSCs on aligned CNT networks exhibited enhanced proliferation and osteogenic differentiation compared to those on randomly oriented CNT networks. As a plausible explanation for the enhanced proliferation and osteogenic differentiation, we proposed mechanotransduction pathways triggered by high cytoskeletal tension in the aligned hMSCs. Our findings provide new insights regarding the capability of cells to recognize nanostructures smaller than proteins and indicate their potential applications for regenerative tissue engineering.

Universal parameters for carbon nanotube network-based sensors: can nanotube sensors be reproducible?

Carbon nanotube (CNT) network-based sensors have been often considered unsuitable for practical applications due to their unpredictable characteristics. Herein, we report the study of universal parameters which can be used to characterize CNT network-based sensors and make their response predictable. A theoretical model is proposed to explain these parameters, and sensing experiments for mercury (Hg(2+)) and ammonium (NH(4)(+)) ions using CNT network-based sensors were performed to confirm the validity of our model.