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.

Open-source colorimeter assembled from laser-cut plates and plug-in circuits.

Colorimetric analysis is a fundamental technique for quantifying the concentration of a substance in solution. It is frequently used in primary and secondary education to enhance students' interest in chemistry, biology, life science, and environmental problems. The structure of the colorimeter is quite simple, i.e., a light source, a sample vessel, and a detector are arranged in a straight line. Therefore, a variety of handmade colorimeters have been reported. However, easy-to-make colorimeters lack portability and reproducibility of measurement, whereas high-precision colorimeters require soldering, which is difficult for beginners. To reduce these difficulties, this study proposes a new open-source colorimeter that can be fabricated easily and cheaply without any soldering. Electronic circuits were made by wiring plug-in electronic modules with connectors. The enclosure was designed to be assembled by simply inserting a laser-cut claws into the corresponding claw holes. The colorimeter was used to measure potassium permanganate solutions of different concentrations and its accuracy was verified. The results showed that the absorbance was measurable up to 1.8 for practical use and 1 for reliable use with the resolution of 0.01.

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.

Effect of a Single Injection of Benidipine-Impregnated Biodegradable Microcarriers on Bone and Gingival Healing at the Tooth Extraction Socket.

Objective: A dihydropyridine-type calcium channel blocker, benidipine (BD), is extensively used in hypertension therapy. In vitro study reported BD promoting bone metabolism. We evaluated the effect of sustained release of BD-loaded poly(lactic-co-glycolic acid) (PLGA) microcarriers on the promotion of bone and gingival healing at an extraction socket in vivo. In addition, the effect of BD on osteoblasts, osteocytes, fibroblasts, and epithelial cells was evaluated in vitro. Approach: The maxillary first molar of rats was extracted. Next, PLGA microcarriers containing BD were directly injected into the gingivobuccal fold as a single dose. After injection, bone and soft-tissue healing was histologically evaluated. Effect of BD on proliferation, migration, and gene expression of gingival and bone cell was also examined in vitro. Results: After tooth extraction, BD significantly augmented bone volume and density, and also epithelial wound healing. During in vitro studies, BD promoted significant proliferation and migration of fibroblasts and epithelial cells. Real-time RT-PCR revealed that BD upregulated messenger RNA expression of Ahsg (alpha 2-HS glycoprotein) and Csf2 (colony-stimulating factor 2) in osteoblasts. Innovation: The prevention of bone and soft-tissue reduction associated with tooth extraction has been eagerly anticipated in the field of dentistry. This study first reported the effect of BD on extraction socket healing. Conclusion: A single dose of topically administered BD-loaded PLGA microcarriers promoted bone and soft-tissue healing at the extraction site of tooth.

Mechanical loading induced osteocyte apoptosis and connexin 43 expression in three-dimensional cell culture and dental implant model.

Osteocytes are thought to act as stress sensors, and are known to display a gap junction-mediated stress-transfer mechanism. To demonstrate the stress-related function of osteocytes, cells of an osteocyte-like cell line derived from murine long bone osteocyte Y4 (MLO-Y4) were cultivated in a three-dimensional culture and subjected to cyclic loading from a titanium plate. This application of physiological loading using a titanium plate significantly increased connexin 43 (Cx43) expression, the number of dead and apoptotic cells, and receptor activator of nuclear factor κB ligand expression. Furthermore, the conditioned medium from the loaded osteocytes induced alkaline phosphatase activity in bone marrow cell culture. In addition, we immunohistologically determined whether bone metabolism increased as a result of the occlusal force in the bone surrounding the titanium implants in a rat model. Increased Cx43 expression and apoptotic osteocytes were observed in the loading group as well as a significantly increased number of tartrate-resistant acid phosphatase-positive cells. These findings indicate that stress from the implant adversely affected the osteocytes, which may promote osteoclastic and osteoblastic cell formation around the implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 815-827, 2019.

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.

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

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.

Evidence for osteocyte regulation of bone homeostasis through RANKL expression.

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.

Evidence for the role of osteocytes in the initiation of targeted remodeling.

Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. Even though there is increasing evidence that osteocyte density, microcracks and targeted remodeling are related, there still exist several questions. For example, how osteoclasts are targeted to the specific site of microdamage for repair. It has been proposed that apoptotic osteocytes could secrete a specific signal to target osteoclasts. The other question is the nature of this signal. To elucidate the role of microdamage-induced osteocyte cell death in the initiation of targeted remodelling, this paper discusses the potential use of an in vitro model, in which osteocytes can be three-dimensionally cultured and locally damaged. Furthermore, the method enables one to study the osteocyte-derived soluble interactions with bone marrow cells. It was demonstrated that damaged osteocytes locally affect osteoclast precursors by secreting osteoclastogenic factors, and thus can have a role in the initiation of resorption in bone remodelling. This strongly supports the idea that damage to osteocyte cellular network has the potential to stimulate osteoclastic proliferation and therefore the activation of Basic Multicellular Units (BMUs).

Microdamage detection and repair in bone: fracture mechanics, histology, cell biology.

Bone is an elementary component in the human skeleton. It protects vital organs, regulates calcium levels and allows mobility. As a result of daily activities, bones are cyclically strained causing microdamage. This damage, in the form of numerous microcracks, can cause bones to fracture and therefore poses a threat to mechanical integrity. Bone is able to repair the microcracks through a process called remodelling which is tightly regulated by bone forming and resorbing cells. However, the manner by which microcracks are detected, and repair initiated, has not been elucidated until now. Here we show that microcrack accumulation causes damage to the network of cellular processes, resulting in the release of RANKL which stimulates the differentiation of cells specialising in repair.

Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture.

UNLABELLED: Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling. INTRODUCTION: Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage. MATERIALS AND METHODS: We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP+ cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA. RESULTS: Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP+ cell differentiation on gel surface. The cells with TRACP+ could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP+ cells in bone marrow culture. CONCLUSIONS: These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.

[Bone quantity and quality to its mechanical integrity].

Bone strength is made up of quantity (mass, mineralization), geometry (anatomy, micro architecture, collagen structure), and turnover/damage accumulation. While most of the mechanical behavior can be explained by measures of porosity, several additional descriptors of the geometry have been proposed for better predictions of fracture risk. This review introduces various aspects of these relationships between bone quantity and quality to its mechanical integrity.